#1                                                                                                                                 

Reprinted from the Indian journal of Biochemistry and Biophysics, Vol.11, No.4, December 1974,pp.338-339

S. L. Govindwar, T. S. Gawande & B. C. Harinath

Biochemistry Division, Mahatma Gandhi Institute of Medical Sciences, Sevagram

Received 27 April 1974; revised received 15 July 2023

Enzymes, present in Wuchereria bancrofti , responsible for human filariasis, have been studied. Activities of amylase (EC 3.2.1.1), alkaline phosphates (EC 3.1.3.1), ATPase (EC 3.6.1.4), aspartate aminotransferase (EC 2.6.1.1), alanine aminotransferase (EC 2.6.1.2) and creatine kinase (EC 2.7.3.2) were determined in the microfilarial homogenate. 

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$2                                                                                                                                  

EFFECT OF DIETHYLCARBAMAZINE ON CERTAIN SERUM AND CELLULAR    

ENZYMES - a Preliminary Study.

Mr. S.L. GOVINDWAR & Dr. B. C. HARINATH

Department of Biochemistry, Mahatma Gandhi Institute of Medical Sciences Sevagram.

SUMMARY

Effect of diethylcarbamazine at two Concentrations (0.05 mg. and 0.5 mg/ml.) was studied on the activity of acetyl cholinesterase, alkaline phosphatase, and aspartate and in alanine aminotransferases in human sera and rabbit liver. Acetyl cholinesterase and alkaline phosphatase were inhibited by the drug whereas no such inhibitory effect was observed on the activities of the transferases. The significant inhibition of the first two enzymes is discussed in the light of the destructive action of the drug on microfilariae.

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%3                                                                                                                                  

Indian J Med Res 64, 11, November 1976, pp 1607-1610

S.L.Govindwar^l, S.N.Ghirnikar² and B.C.Harinath³

Revised article received for publication, April 21, 1976.

Blood samples from normal individuals, microfilaria carriers and chronic filarial cases were studied for leucocyte count as well as various biochemical parameters including protein, lipids, electrolytes, enzymes etc. A significant decrease in amylase activity in patients with microfilaraemia, and a decrease in the level of uric acid along with an increase in alanine and aspartate aminotransferase activities was observed in cases of chronic filariasis.

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$4                                                                                                                                    

Reprinted form "curr.sci",September 5,1977,vol.46,No.17,603-604

DETECTION OF ANTIGENS AND ANTIBODIES IN FILARIAL SERA BY COUNTER CURRENT IMMUNOELECTROPHORESIS

P. KALIRAJ, B. C. HARINATH, S. N. GHIRNIKAR

Dept. of Biochemistry, Mahatma Gandhi Institute of Medical Sciences, Sevagram, and

Filaria Research cum Training Centre,

Wardha, June 27, 1977.

The importance of counter current immunoelectrophoresis (CIE) as a good serological technique is being recognized by various investigators, for its simplicity and sensitivity with added advantage of detection of antigen and antibody in the sample at same time. Desowitz and Una¹ (1976) have shown the presence of antibodies to microfilarial and adult male D. immitis antigens in human and animal filarial sera by counter current immunoelectrophoresis.

W. bancrofti microfilariae were isolated from blood samples from human carriers by dextransedimentation and antisera were raised in rabbits for the microfilarial antigen. Anti microfilarial sera and microfilarial antigen were used for detection of antigen and antibody in the sera of humans infected with W. bancrofti by double diffusion and counter current immunoelectrophoresis.

Sixteen sera from human carriers (microfilarial count ranging from. 1 to 94/20 c.mm. of blood) were analysed. No precipitin band was observed either for the antigen or antisera by double diffusion. However counter current immunoelectrophoresis of human filarial sera showed the presence of precipitin bands (1 to 3) with rabbit antimicrofilarial sera indicating the presence of soluble antigens in all the cases and one precipitin band with microfilarial antigen indicating the presence of antibody in two cases. Use of counter current immunoelectrophoresis in detection of soluble antigens is being investigated further for application as additional confirmatory test in the diagnosis of filariasis.

1.Desowifz, R. S. and Una, S.R.,j. Helminthology, 1976, 50, 53.

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Indian J Med Res 67, January 1978, pp 106-109

Chromatographic separation and colorimetric estimation of

diethylcarbamazine and its metabolites*

B. Chandrasekaran, S.K.B. Patil** and B.C. Harinath

Department of Biochemistry,

Received February 15, 2024; revised article received July 12, 1977.

Diethylcarbamazine (DEC) and related compounds such as piperazine, methylpiperazine and DEC-N-oxide have been separated by paper chromatography using pyridine: amyl alcohol: water and butanol: citric acid solvent systems. Colorimetric method involves the addition of phenol reagent for DEC, methylpiperazine and DEC-N-oxide and the addition of parabenzoquinone reagent for piperazine for colour development. The method can be applied for biological samples.

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Immunization Studies in Rabbits With Human-Filarial

Parasite Wuchereria bancrofti*

P.Kaliraj, K.N.Rao & B.C.Harinath.
Department of Biochemistry, Mahatma Gandhi Institute of Medical Sciences, Sevagram and

S. N. Ghirnikar
Research cum Training Centre, Wardha

Manuscript received 9 December 1977, revised manuscript received 5 May 1978

W. bancrofti microfilariae were isolated from human carriers by dextran sedimentation technique. Rabbits were immunized with whole and soluble W. bancrofti microfilarial (mf) antigens. Rabbit anti whole mf sera showed 1 and rabbit anti soluble mf sera showed 2 specific precipitin bands with the corresponding antigens, in agar gel diffusion. Use of rabbit anti mf sera in the detection of circulating filarial antigen in human filarial cases is discussed.

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$7                                                                                                                                    
Indian Journal of Experimental Biology

Vol. 17, April 1979, pp. 332 335

Indirect Fluorescent Antibody Technique Using Sonicated Wuchereria bancrofti Microfilaria for Immunodiagnosis of Bancroftian Filariasis

P. KALIRAJ, S. N. GHIRNIKAR* & B. C. HARINATH

Department of Biochemistry, Mahatma Gandhi Institute of Medical Sciences,Sevagram 442 102 and

*Research cum Training Centre, Wardha

Manuscript received 16 August 2023

Sonicated W. bancrofti mf was used for the immunodiagnosis of Bancroftian filariasis. Cent per cent of the 21 microfilaraemia sera, 93 % of the 29 clinical filariasis sera, 54 % of the 52 endemic normal sera and none of the 22 nonendemic normal human sera showed positive reactions in indirect fluorescent antibody technique (IFAT). Apparent cross reactions were observed when 20 sera of endemic human cases with proven intestinal helminth infection were tested. Sonicated microfilarial antigens of different filarial parasites were compared for their utility in the diagnosis of bancroftian filariasis by IFAT.

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Indian J Med Res 70, August 1979, pp 305-308

Isolation, characterization and estimation of 

diethylcarbamazine-N-oxide from human urine

B.Chandrasekaran and B.C. Harinath

Department of Biochemistry,Mahatma Gandhi Institute of Medical Sciences, Sevagram.

Received March 14, 1977; revised article received January 5, 1979.

Diethylcarbamazine-N-oxide (DEC-N-oxide), a metabolite of diethylcarbamazine (DEC), has been isolated from human urine and characterized by paper-chromatography, electrophoresis and infra-red spectral-analysis. Dragendroff's reagent followed by 15 per cent acetic acid gave orange colour with DEC-N-oxide and could be measured at 410 mµ.

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$9

Reprinted from the Indian Journal of Experimental Biology 

Vol. 17, No. 10 October 2023 pp. 1143-1149

Detection of Circulating Filarial Antigen in Bancroftian Filariasis

P.Kaliraj, S.N.Ghirnikar* & B.C.Harinath

and

*Research cum Training Centre, Wardha

    Circulating filarial antigen was detected by countercurrent immunoelectrophoresis (CIEP) using specific rabbit antifilarial sera in 6 out of 26 microfilaraemia sera, 2 out of 26 clinical filarial sera, 2 out of 67 endemic normal sera, and in none of the 32 nonendemic normal sera. Rabbit antifilarial sera showed 2 precipitation bands against the Wuchereria-bancrofti soluble mf antigen (mfS) and one identical band against soluble circulating antigen concentrated from microfilaraemia plasma by double diffusion and CIEP.

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10

Cell-Mediated Immune Response to Infection in Human filariasis.

P.N.Raghunath, Amala Cancer Research Centre, Trichur-680 553

and B.C.Harinath. Dept. of Biochemistry,

M.G.Institute of Medical Sciences, Sevagram-442 102.

Status of cell mediated immunity (CMI) in filariasis  has been studied by counting the per­centage of T -lymphocytes in peripheral blood by E-rosette test and the measurement of leukocytes migration inhibition (LMI) of leucocytes in the persence of soluble wuchereria bancrofti microfilariae antigen and purified protein dervative (PPD). peripheral blood samples from low and high microfilariae carriers, filarial patients with clinical manifesta­tions, endemic and nonendemic normals were used for the investigations. Preliminary studies showed that patients high mf count and chronic cases have exhibited depressed cell mediated immune response as indicated by lowered T. cell counts and increased migration index of LMI, where as micro-filariae carriers with low mf count did not show such immune suppression. The possible role of CMI in filarial infection is discussed.

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 11

Reprinted from the Indian Journal of exp. Biology Vol. 18 No. 10, October 1980 pp. 1179-1180

Effect of Diethylcarbamazine & Diethylcarbamazine-

N-oxide on Microfilariae in vitro in the Presence of

Immune Sera & Leukocytes*

B. Chandrasekaran, S. N. Ghirnikar† & B. C. Harinath ‡

Department of Biochemistry, Mahatma Gandhi Institute of

Medical Sciences, Sevagram 442 102

and

†Filaria Research-cum-Training Centre, Wardha

Manuscript received 10 October 1979; revised manuscript received 1 May 1980

Mechanism of action of diethylcarbamazine and diethylcarbamazine-N-oxide have been studied in vitro on Wuchereria bancrofti microfilariae in the presence of immune sera and leukocytes. Diethylcarbamazine at the conc. of 5 μg/ml enhanced adhesion of leukocytes on the surface of microfilariae and cell clump formation with entangled microfilariae whereas at 500 μg/ml conc. inhibited adhesion. Similar effect was observed with diethylcarbamazine-N-oxide but it did not cause cell clumps. Citrate at the conc. of 2.5 μg/ml did not have any effect on adhesion. It is suggested that adhesion and cell clump formation may be some of the mechanisms by which the drug acts upon the circulating microfilariae and removes them from blood.

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12

Reprinted from the Indian Journal of Exp.Biology Vol. 18 No. 7. July 1980 pp. 722-724

Metabolism of Diethylcarbamazine in Mammals*

B. CHANDRASEKARAN & B. C. HARINATH†

Department of Biochemistry, Mahatma Gandhi Institute of Medical Sciences, Sevagram 442 102

Metabolism of diethylcarbamazine was studied in guinea pigs, rabbits and humans by estimating the amount of diethylcarbamazine and its metabolites excreted in urine after administration of the drug. Analysis of urine samples for drug metabolites showed the presence of unchanged drug, ethylcarbamazine. Diethylcarbamazine-N-oxide, methyl piperazine and piperazine. The percentage of the drug and the metabolites excreted at different intervals of time varied with species.

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13

Reprinted from the Indian journal of Experimental Biology Vol. 18 No. 11 November. 1980 pp. 1298-1300

Stimulatory Effect of Diethylcarbamazine on Certain Drug Metabolizing Enzymes

B. Chandrasekaran & B. C. Harinath*

Department of Biochemistry, Mahatma Gandhi Institute of

Medical Sciences, Sevagram 442102

Manuscript received 11 March 2024

Effect of diethylcarbamazine on drug metabolizing enzymes catalyzing demethylation, deethylation and N-oxidation has been studied. Administration of 50 mg diethylcarbamazine once or three times a day for 3 days activated the demethylating and deethylating enzymes but it did not have any effect on N- oxidizing enzyme of diethylcarbamazine.

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14

Indian Journal of Experimental Biology

Vol. 18, November 1980, pp. 1245-1247

In vitro Cultivation of Wuchereria bancrofti Microfilariae

I.KHARAT, U. SATYANARAYANA, S. N. GHIRNIKAR* & B. C. HARINATH†

Department of Biochemistry, Mahatma Gandhi Institute of Medical Sciences,

Sevagram 442102 and *Research-cum-Training Centre, Wardha

Microfilariae of W. bancrofti were cultivated in Medium 199 (A) and in Medium 199 supplemented with organic acids and sugars of Grace's insect medium (B). As many as 30% of microfilariae developed into sausage shaped larvae by 8th day of cultivation in medium B while 10% in medium A by 17th day. Most of the mf in various stages of their development were active and survived for 3 and 2 weeks in medium A and B respectively.

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15

Transactions of The Royal Society of Tropical Medicine and Hygiene, Vol. 75, No.1, 1981

Enzyme linked immunosorbent assay (ELISA) for bancroftian filariasis*

P. KALIRAJ, S. N. GHIRNIKAR** AND B. C. HARINATH 

Dept. of Biochemistry, Mahatma Gandhi Institute of Medical Sciences, Sevagram, India 442 102

and **Research-cum-training Centre, Wardha, India

The indirect fluorescent antibody test (IFAT) using an adult filarial worm (Ambroise-Thomas & Kein troung, 1974) or sonicated microfilarial antigens (Hedge & Ridley, 1977; Kaliraj et al., 1979) has been reported to be sensitive for the immunodiagnosis of filariasis. However, large scale immunofluorescence testing is tedious and needs special equipment. The enzyme-linked immunosorbent assay (ELISA) (test) has been found to be simple, sensitive and suitable for the mass screening of most parasitic infections (Voller et al., 1976). Bartlett et al. (1975) applied a micro-ELISA using the enzyme alkaline phosphatase for the detection of antibody in Onchocerca volvulus infections in which it was not possible to use the homologous antigen as contaminants of host origin reacted with the conjugate. No such problem was encountered (Marcoullis et al., 1978) while using the same antigen after purification. Penicillinase has been used previously in ELISA for the estimation of human chorionic gonadotropin and placental lactogen (Joshi et al., 1978). This communication describes the use of Micro-ELISA employing the enzyme penicillinase (E.C. 3.5.2.6) for the demonstration of antibody in bancroftian filariasis.

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16

Indian Journal of Biochemistry & Biophysics

Vol. 18, April 1981, pp. 139-141

Effect of Diethylcarbamazine on Wuchereria bancrofti Microfilarial Enzymes

B CHANDRASEKARAN, S N GHIRNIKAR* & B C HARINATH†

Effect of diethylcarbamazine on the activities of W. bancrofti microfilarial acetylcholinesterase, alkaline phosphatase, acid phosphatase, urease and inorganic pyrophosphatase was studied. A decrease in the activity of all these enzymes excepting inorganic pyrophosphatase was observed in the presence of diethylcarbamazine (50-10,000,μg/ml).

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17

Am. J. Trop. Med. Hyg . , 30(5), 1991, pp. 982-987

IMMUNODIAGNOSIS OF BANCROFTIAN FILARIASIS:

COMPARATIVE EFFICIENCY OF THE INDIRECT

HEMAGGLUTINATION TEST, INDIRECT FLUORESCENT

ANTIBODY TEST, AND ENZYME-LINKED IMMUNOSORBENT

ASSAY DONE WITH WUCHERERIA BANCROFTI

MICROFILARIAL ANTIGENS*

P. KALIRAJ, S. N. GHIRNIKAR, * AND B. C. HARINATH

Abstract. The Indirect hemagglutination test (IHAT), indirect fluorescent antibody test (IFAT) and enzyme linked immunosorbent assay (ELISA) have been applied to the serodiagnosis of bancroftian filariasis employing Wuchereria bancrofti mf antigens and the efficiency of these tests in the detection of antibody has been compared. Each test was found to be marginally superior to the other two tests with particular group of endemic sera for the detection of filarial antibody .In other words, the IHAT, IFAT and ELISA showed a greater number of positive reactions with endemic normal, microfilaremia and clinical filarial sera, respectively. ELISA is simple, sensitive and can be used in seroepidemiological studies for bancroftian filariasis employing soluble antigens.

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18

Indian Journal of Experimental Biology

Vol 19, March 1981, pp 287-288

Immune Response to Wuchereria bancrofti Microfilarial Antigen

Department of Biochemistry, M.G. Institute of Medical Sciences, Sevagram 442 102

&

*Research cum Training Centre

Received8September1980

Rabbits were immunized with soluble W. bancrofti microfilarial antigen. Administration by subcutaneous route was found to be more effective than intravenous injection in eliciting better immune response. Six mg antigen protein of booster dose was found to be optimum in producing antisera with maximum antibody titre.

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19

Journal of Helminthology (1981) 55, 133-139

The utility of human filarial serum in the detection of circulating antigen

P. KALIRAJ, INDIRA KHARAT, S. N.GHIRNIKAR* and B. C. HARINATH†

Department of Biochemistry, Mahatma Gandhi Institute Of Medical Sciences,

Sevagram, Wardha, India-442 102

and *Research cum Training Centre, Wardha

ABSTRACT: The utility of human filarial serum immunoglobulin (FSI) in detecting circulating antigen in filarial sera was studied by counter immunoelectrophoresis (CIEP) and the indirect haemagglutination test (IHAT). CIEP was found to be better than IHAT. 23 out of 30 sera from persons with microfilaraemia and one of 30 clinical cases of filariasis, but none of the normal sera or sera from those with helminths other than filariae, showed the presence of circulating filarial antigen in CIEP. FSI was fractionated by DEAE- Sephadex A-50 column chromatography and the antibody active in CIEP was found to be IgG in nature.

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20

Indian Journal of Experimental Biology

Vol 19, June 1981, pp 564-565

Detection & Diagnostic Utility of Exoantigen of Wuchereria bancrofti

Microfilariae

INDIRA KHARAT, P KALIRAJ, S N GHIRNIKAR* & B C HARINATH†

Department of Biochemistry, M G Institute of Medical Sciences, Sevagram, Wardha 442 102

and

*Research cum Training Centre, Wardha

Received 25 October 1980; revised 10 February 2024

Reverse indirect haemagglutination (RIHA) using filarial serum immunoglobulin showed the presence of exoantigen (1:8 titre) in in vitro cultured fluid of W. bancrofti microfilariae.The utility of this exoantigen was studied in indirect haemagglutination (IUA) for the detection of filarial antibody.19 out of 20 microfilaraemia sera, 13 out of 20 clinical filaria sera, 8 out of 30 endemic normal sera and none of the 30 non endemic normal sera showed positive reaction for filarial antibody.

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21

Indian Journal of Experimental Biology

Vol 20, May 1982, pp 378-380

Antibody Analysis in Human Filarial Sera by ELISA Using

Wuchereria bancrofti Microfilariae

Culture Antigen*

INDIRA KHARAT & B C HARINATH†

Department of Biochemistry, M.G. Institute of Medical Sciences, Sevagram, Wardha 442 102

and

S N GHIRNIKAR

Research cum Training Centre, Wardha

Received 21 September 198 1; revised 1 8 February 2024

A total of 120 human sera were screened in enzyme linked immunosorbent assay for detection of filarial antibodies using W.bancrofti microfilariae culture antigen. As little as 0.35 ng antigen protein per well was found to be sufficient in detection of filarial antibody. Four out of the 30 endemic normal, 28 out of the 30 microfilaraemia, all the 30clinical filarial and none of the 30 non endemic normal sera showed the presence of filarial antibody (l:320). The identification of filarial antibody was further studied using class specific anti-immunoglobulin conjugates which revealed the presence of lgM antibody in microfilaraemia and IgG antibody in clinical filarial sera whereas some of the sera contained both IgM and IgG antibodies.

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22

Indian Journal of Experimental Biology

Vol 20, June 1982, pp. 440-444

Fractionation & Evaluation of Wuchereria bancrofti Microfilarial Antigens in

Immunodiagnosis of Bancroftian Filariasis*

P KALIRAJ† & B C HARINATH‡

S N GHIRNIKAR

Received 11 December 198 1; revised 15 April 1982

Soluble antigens (mfS) isolated from W.bancrofti microfilariae were fractionated by SephadexG-15Ogel filtration into 3 antigenic fractions(mfS1,2&3).The mfS3 fraction was weakly reactive in indirect haemagglutination test(IHAT) whereas the same was found to he highly reactive in enzyme linked immunosorbent assay (ELISA). The mfS2 antigen fraction showed cross reaction with nonendemicAncylostoma duodenale sera in IHAT and ELISA similar to the crude soluble antigen (mfS). The antigenic fractions (mfsl and mfS3) were further fractionated by DEAE-Cellulose column chromatography. Analysis by the enzyme linked immunosorbent assay showed that mfSI b and mfS3e antigen fractions were highly active in the detection of filarial antibody from non microfilaraemic (clinical filariasis and endemic normal) and micro filaraemia (mf + ve) sera respectively.

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23

J. Biosci., Vol. 4, Number 4, December 1982, pp. 507-512.

Detection of filarial infection using Wuchereria Bancrofti microfilariae culture antigen and filter paper blood samples in enzyme linked immunosorbent assay

ASHOK MALHOTRA, M. V. R. REDDY, J. N. NAIDU,

S. N. GHIRNIKAR* and B. C. HARINATH

Abstract. Blood collected on filter paper by finger-prick gave results comparable to intravenous serum samples when analysed by enzyme-linked immunosorbent, assay (ELISA). All the 100 microfilaraemia, 5 out of 100 endemic normals and none of the 10 nonendemic normal filter paper blood samples showed the presence of filarial antibody when tested by this method, using culture antigen and anti-immunoglobulins, class G, M and A - penicillinase conjugate. When the same samples were screened for the presence of IgM antibody, 91 out of 1 00 microfilaraemia, 13 out of 100 endemic normal and none of the 10 nonendemic normal samples showed a positive reaction. Enzyme linked immunosorbent assay, using culture antigen and filter paper blood samples, appears to work in large field studies for detection of filarial infection.

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24

Indian J Med Res 77, June 1983, pp 813-816

Immune complexes & immunoglobulins involved

in human filariasis

G.B.K.S. Prasad, M.V.R. Reddy & B.C. Harinath

Department of Biochemistry, Mahatma Gandhi Institute of Medical Sciences, Sevagram

Revised article received January 3, 2024

Circulating immune complexes were determined by 3 per cent PEG precipitation and complement consumption tests, in 40 sera of filarial patients either with microfilaraemia or clinical manifestations such as elephantiasis, hydrocele etc., and 15 healthy persons. Significantly elevated levels of circulating immune complexes were observed in clinical filariasis compared to microfilaraemia and endemic normals. Immunoglobulin classes involved in immune complexes in clinical filariasis were detected by immunodiffusion and immunofluorescence assays; IgG and IgM were mostly found to be present in the immune complexes.

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25

Indian J Med Res 78, September 1983, pp 343-348

Effect of different DEC schedules on microfilaraemia & filarial antibody levels in bancroftian filariasis

Ashok Malhotra, S.N. Ghirnikar* & B.C. Harinath

Revised article received April 20, 2024

Diethylcarbamazine (DEC) was administered to 46 microfilariae (mf) carriers in two schedules. Side reactions, mf count and antimicrofilarial ES antigen antibody levels were monitored in both the groups. While microfilariae were cleared from the circulation of all the 19 individuals on the first schedule, 1 of 27 in the second schedule showed persistent microfilaraemia at the end of DEC treatment. Nine of 10 individuals analysed in each group for IgM antibody levels by enzyme linked immunosorbent assay (ELISA) showed a gradual decrease in reciprocal antibody titre (from pretreatment mean levels of about 1 5,000 to about 4,000 at the end of treatment). Out of 46 mf carriers without clinical manifestation at the start of drug treatment, 2 showed clinical manifestations during chemotherapy.

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26

Indian J Med Res 78, October 1983, pp 497-502

Immunofluorescence studies with Wuchereria bancrofti microfilariae ES antigen

Indira Kharat, S.N. Ghirnikar* & B.C. Harinath

Revised article received May 11, 2024

W. bancrofti mf ES (excretory and secretory) antigen coupled to CNBr-Sepharose 4B was used in indirect fluorescent antibody test (IFAT) for detection of filarial antibody in various human serum samples. Ten of 20 endemic normal, 19 of 20 microfilaraemia, all the 20 clinical filarial sera, none of the 20 non- endemic normal and 8 non-endemic helminthiasis sera showed the presence of filarial antibody when FITC labelled anti- IgG+M+A conjugate was used. Further studies using class specific FITC labelled anti-immunoglobulins detected both IgM and IgG antibodies in filarial sera. IgE antibody against ES antigen was detected in 15 of 21 endemic normal, 22 of 29 microfilaraemia, 12 of 16 clinical filarial and 17 of 26 tropical eosinophilia and none of the 8 non-endemic sera examined by IFAT.

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27

Transactions Of The Royal Society Of Tropical Medicine And Hygiene, VOL.77, No.6, 771-772(1983)

Detection of antimicrofilarial ES antigen-antibody in immune

complexes in Bancroftian filariasis by enzyme immunoassay

G. B. K. S. Prasad, I Kharat and B.C. Harinath

Dept. of Biochemistry, Mahatma Gandhi Institute of Medical Sciences, Sevagram, India 442 102

An enzyme immunoassay using penicillinase conjugated to Wuchereria bancrofti microfilarial ES antigen has been developed to detect specific antibody in circulating immune complexes in Bancroftian filariasis. Immune complexes were prepared by 3% polyethylene glycol (PEG) precipitation. 44 sera belonging to different groups were tested. 16 of 19 clinical filarial and two of 16 endemic normal sera but none of the non-endemic normal sera showed the presence of antimicrofilarial ES antigen-antibody in immune complexes.

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28

Indian J Med Res 78, December 1983, pp 780-783

Detection of filarial antigen in immunecomplexes

in bancroftian filariasis by ELISA

G.B.K.S. Prasad, M.V.R. Reddy & B.C. Harinath

Department of Biochemistry, Mahatma Gandhi Institute Of

Medical Sciences, Sevagram, Wardha

Revised article received June 24, 2023

IgG fraction of filarial serum immunoglobulins (FSI-G) Conjugated to penicillinase has been used to detect by ELISA, filarial antigen in circulating immune complexes (CICS) in bancroftian filariasis. Ninety per cent sera of clinical filariasis patients, showed the presence of antigen. Antigen titre in CICs was determined and compared with the levels of CICS. Wuchereria bancrofti microfilariae ES antigen was found to be present in filarial immune complexes.

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29

Indian J Med Res 79,February 1984,pp 194-198

Detection & monitoring of microfilarial ES antigen

levels by inhibition ELISA during DEC therapy

Ashok Malhotra & B.C. Harinath

Department Of Biochemistry, Mahatma Gandhi Institute of Medical Sciences, Wardha

Revised article received October 3, 2023

Inhibition ELISA for the detection of Wuchereria bancrofti microfilarial ES antigen has been described. Fifteen of 20 microfilaraemic patients, 7 of 20 clinical filariasis, 4 of 20 endemic normals and none of the 7 nonendemic normals showed the presence of filarial antigen by inhibition ELISA. The antigen titres during DEC therapy in 10 mf carriers showed an initial increase followed by a gradual decrease during treatment.

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30

IRCS Medical Science: Cell and Membrane Biology; Clinical Biochemistry; Clinical Medicine; Haematology; Immunology and Allergy; Microbiology, Parasitology and Infectious Diseases. [IRCS Med. Sci., 12, 171-172(1984)]

Ashok Malhotra, G.B.K.S. Prasad and B.C. Harinath

Paper received: 4th November, 1983; amended 24th January, 1984

Abstract:

IgE and IgG+M+A antibodies specific to Wuchereria bancrofti microfilariae excretory-secretory antigen were detected in patients with tropical eosinophilia, microfilaraemia, clinical filariasis and in non-infected individuals in endemic areas by solid phase anti lgE ELISA and Indirect ELISA respectively. While the highest levels of the Specific IgE antibody were observed in the patients with tropical eosinophilia, most of the cases with clinical filariasis (28/30) were negative. A comparison of W.bancrofti mf ES antigen specific lgE antibody versus IgG+M+A antibody showed that specific IgE antibody detection is not useful for immunodiagnosis of human filariasis.

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31

Detection of filarial immune complexes by ELISA using anti C3 and filarial serum

immunoglobulin-G

G.B.K.S. Prasad and B.C. Harinath

Department of biochemistry, Mahatma Gandhi Institute of medical Sciences, Sevagram,

Wardha 442102,India

Paper received: 22nd February. 1 984; amended 12th April, 1 984

Abstract:

Sera from patients with microfilaraemia, clinical filariasis and non infected individuals from endemic regions were examined for filaria specific immune complexes by ELISA using Anti C3 and filarial serum immunoglobulin-G. Twenty-eight out of 32 clinical filariasis, 4 out of 30 microfilaraemia and 3 out of 28 endemic normal sera showed the presence of immune complexes. No correlation could be observed between the levels of circulating immune complexes and clinical manifestations of the patient.

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32

Evaluation of fractionated Wuchereria bancrofti microfilarial

excretory-secretory antigens for diagnosis of bancroftian filariasis

by enzyme linked immunosorbent assay

M. V. R. REDDY, ASHOK MALHOTRA, G. B. K. S. PRASAD and

Department of Biochemistry, M. G. Institute of Medical Sciences, Sevagram, Wardha 442 102,

India

MS received 14 October 2023

Abstract. The Wuchereria bancrofti microfilarial excretory-secretory antigens were fractionated into ESI, ES2, ES3 and ES4 by ultramembrane filtration and evaluated for their diagnostic utility by enzyme linked immunosorbent assay. Three of the four fractions showed antigenic activity (ES2, ES3 and E54). The antigen fractions ES2 and ES4 were highly active in the detection of filarial IgM antibody in clinical filariasis and microfilaraemia respectively. The chemical characterization of the ES2 and ES4 antigen fractions showed that they were glycoproteins.

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33

IRCS Medical Science: Anatomy and Human Biology; Biochemistry; Biomedical Technology; Cell and Membrane Biology; Clinical Biochemistry; Connective Tissue, Skin and Bone; Hematology; Immunology and Allergy; Microbiology, Parasitology and Infectious Diseases; Pathology. [IRCS Med. Sci., 12, 738-739 (1984)]  

Comparative efficiency of penicillinase enzyme-linked immunosorbent assay (ELISA)and radioimmunoprecipitation polyethylene glycol assay (RIPEGA), using ¹⁴C-labelled Wuchereria bancrofti excretory-secretory antigen for the  detection of filarial antibody 

P. Rama Prasad, I.Kharat and B.C. Harinath

Department of biochemistry, Mahatma Gandhi Institute of Medical Sciences, Sevagram,

Wardha 442 102, India

Paper received: 7th June, 1 984; amended 25th July, 1984                                                   

Abstract

Enzyme linked immunosorbent assay (ELISA) using penicillinase and Radio immunoprecipitation polyethylene glycol assay (RIPEGA) were compared for their sensitivity in detecting filarial antibody. When ¹⁴C-labelled Wuchereria bancrofti microfilarial (mf) excretory-secretory (ES) antigen was used, 0.25 ng antigen protein per well was found to be sufficient in ELISA compared to 2.5 μg antigen protein in RIPEGA per assay for detection of antibody. 

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34

Indian Journal of Experimental Biology

Vol 22, October 1984, pp 515-519

Detection of Filarial Antigen in Urine of Humans with Wuchereria bancrofti

Infection by Immunoradiometric Assay

M V R REDDY & BHASKAR C HARINATH*

ROBERT G HAMILTON

Division of Clinical Immunology, Department of Medicine, Johns Hopkins University,

School of Medicine, Baltimore, Maryland 21239, U.S.A.

Human urine samples of different groups were analysed for the presence of filarial antigen by immuno-radiometric assay (IRMA) using¹²⁵ I-Rabbit IgG antibodies to B. malayi antigen. Six out of ten microfilaraemia, One out of five clinical filariasis had detectable antigen, while none of the endemic and non-endemic normals (n = 12) were positive. The effect of heat-acid treatment on the detectability of antigen in the urine and serum was explored. Heat-acid treatment in general did not reduce the level of filarial antigen in the urine samples while there was a measurable reduction in the antigen levels in the sera.

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35

Indian Journal of Experimental Biology

Vol 22, October 1984, pp 520-522

Department of Biochemistry, M G Institute of Medical Sciences,

Sevagram, Wardha 442 102, India

Received 1 June 1984

Comparative efficiency of using W.bancrofti microfilariae (mf) and larval (L₃) excretory-secretory (ES) antigen in ELISA was studied for detection of antibody in tropical oesinophilia and bancroftian filariasis. L₃ ES antigen was found to be more useful in detection of specific IgG+IgM+IgA antibody in tropical oesinophilia whereas mf ES antigen was found to be superior for diagnosis of bancroftian filariasis.

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36

Detection of circulating antigen in bancroftian filariasis by

 sandwich ELISA using filarial serum IgG

M. V. R. REDDY, ASHOK MALHOTRA and B. C. HARINATH 

Department of Biochemistry, Mahatma Gandhi Institute of Medical Sciences, Sevagram,

 Wardha 442 102, India

 ABSTRACT

 The utility of the IgG fraction of human filarial serum immunoglobulin in detecting circulating antigen by sandwich enzyme linked immunosorbent assay (ELISA) was studied. 27 of 33 sera from persons with microfilaraemia, 19 of 30 sera from clinical cases of filariasis, 4 of 30 sera from normal persons from a region endemic for filariasis showed the presence of circulating filarial antigen. All the 20 normal sera from the area where filariasis was not endemic gave negative reaction for filarial antigen. Those sera from persons with microfilaraemia that showed the presence of circulating antigen also showed an apparent positive correlation between the microfilarial density and the antigen titre.

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 37

Bulletin of the World Health Organization,62(6):941 -944(1984)

mf ES antigen for bancroftian filariasis 

B. C. Harinath, ¹ Ashok Malhotra, ² S. N. Ghirnikar, ³ S. D. Annadate ,⁴

V. P. Isaacs, ⁵ & M. S. Bharti, ⁶

 An enzyme-linked immunosorbent assay using Wuchereria bancrofti microfilarial excretory-secretory antigen was used in field studies to screen blood samples collected on filter-paper from persons residing in areas endemic for bancroftian filariasis. This assay system, when compared with examination of night wet blood smears for microfilariae , gave a relative sensitivity of 98% and a relative specificity of 86%. Daytime blood samples can also be used in this test, which can thus replace tedious examination of night blood samples in field surveys in endemic areas. 

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 38

J. Biosci., Vol. 6, Number 5, December 1984, pp.717-722

Monoclonal antibodies against Wuchereria bancrofti microfilarial

excretory-secretory antigens  

M. V. R. REDDY, W. F. PIESSENS* and B. C. HARINATH†

Department of Biochemistry, Mahatma Gandhi Institute of Medical Sciences, Sevagram, Maharashtra 442 102, India

*Department of Tropical Public Health, Harvard School of Public Health, 665, Huntington Avenue, Boston, Massachusetts 021 1 5, U.S.A.

MS received 27 August 2023

 Abstract. A battery of monoclonal antibodies were produced against Wuchereria bancrofti microfilarial excretory-secretory antigens and their specificity was studied using different filarial antigens. Among the 1116 wells plated out, 42 % of the wells developed hybrids and 5 % of the hybrids showed anti Wuchereria bancrofti microfilarial excretory-secretory antigens. Specificity studies on the antibodies produced from 63 cloned and expanded hybrids showed 10 clones which were specifically positive only to Wuchereria bancrofti microfilarial excretory-secretory antigens.

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 39

J. Biosci., Vol. 6, Number 5,. December 1984, pp. 691-699.      

Immunodiagnosis of bancroftian filariasis-Problems and progress

                BHASKAR C. HARINATH

Department of Biochemistry, Mahatma Gandhi Institute of Medical Sciences, Sevagram, Wardha 442102, India

MS received 28 September 1984

Abstract. The immunodiagnosis of bancroftian filariasis is a major challenge to the immunoparasitologist. Significant progress is yet to be made in developing convenient laboratory animal model and in in vitro cultivation of filarial parasites making it very difficult to obtain required amount of parasite material for research. Parasitological examination techniques are not useful in low microfilaraemia, occult or chronicfilarial infections. A precise and accurate immunodiagnostic technique is very much needed for successful filaria control programmes. Such a test will also avoid the need for laborious night blood examination in bancroftian filariasis.

Due to comparatively easy availability, a good amount of work has been done to explore immunodiagnostic potential of heterologous filarial antigens isolated from Litomosoides carinii, Dirofilaria immitis, Brugia malayi, Setaria digitata, Setaria cervi and number of other filarial species. However, there has been limited or no significant success due to number of false negative and false positive reactions.

Extensive study has also been made with antigens isolated from Wuchereria bancrofti microfilariae. Soluble antigens of microfilariae have been used in different immunological techniques such as skin test, counter immuno electrophoresis, indirect haemagglutination test, indirect fluorescent antibody test and enzyme linked immunosorbent assay. Fractionation of Wuchereria bancrofti microfilarial soluble antigens yielded mfS3e antigen fraction which was found to be highly reactive in microfilaraemia by enzyme linked immunosorbent assay, but the yield of the purified antigen was not sufficient enough to make it a practical proposition for large scale isolation of antigen.

Wuchereria bancrofti microfilarial excretory-secretory antigens were found to be specific and highly sensitive requiring as little as 0-35 ng antigen protein per well in penicillinase enzyme linked immunosorbent assay for detection of filarial antibody. One ml of culture fluid was found to be sufficient for 400,000 tests. Field evaluation of this test showed that it can replace laborious night blood examination.

Assay systems have been developed for detection of filarial antigen in serum, urine, hydrocele fluid and immune complexes using immunoglobulins from chronic filarial sera and antisera to excretory filarial antigens. Further purification of excretory-secretory antigens by affinity chromatography and production of monoclonal antibodies should hopefully give suitable reagents for use in sensitive assays such as enzyme immuno assay and immuno radiometric assay, providing an ideal assay system for detection of active filarial infection in the not too distant future.

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 40

Indian Journal of Experimental Biology 

Vol. 23 February 1985, pp. 118-119

 Uptake of ¹⁴C-labelled Sugars and Amino Acids by Wuchereria

bancrofti Microfilariae in vitro

 INDIRA KHARAT & B C HARINATH*

Department of Biochemistry, Mahatma Gandhi Institute of Medical Sciences, Sevagram, Wardha 442102, India

Received 18 June 1984; revised 1 5 November 2023

 Nutritional uptake of certain radiolabelled sugars and amino acids by W. bancrofti microfilariae was studied in vitro. The amount of radioactivity measured in experimental microfilariae when D-[U-¹⁴C] glucose and fructose were added to the medium, was 2311 and 303 d.p.m. in 8 hr while in controls 85 and 87 d.p.m. respectively. Similarly the amount of radioactivity observed in experimental microfilariae was 13440 d.p.m. for L-[U-¹⁴C] glutamic acid, 2645 d.p.m. for L-[U-¹⁴C] proline and 5589 d.p.m. for L-[U- ¹⁴ C] leucine, while in controls it was 1887, 217 and 417 d.p.m. respectively. Comparatively amino acids were well utilized than sugars by W. bancrofti microfilariae in vitro. 

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41

 Indian Journal of Experimental Biology

 Vol. 23 February 1985, pp. 79-82

 Histochemical Distribution of Acid Phosphatase and Acetylcholinesterase

Activity in in vivo and in vitro Larval Stages of Wuchereria bancrofti 

INDIRA KHARAT & B C HARINATH*

Department of Biochemistry, Mahatma Gandhi Institute of Medical Sciences, Sevagram, Wardha 442102, India

Received 18 June 1984; revised 15 November 2023  

 Histochemical distribution of acid phosphatase (EC 3.1.3.2) and acetylcholinesterase (EC 3.1.1.7) in different developing stages of W. bancrofti microfilariae obtained from in vivo and in vitro sources were studied. The acid phosphatase activity was observed in excretory and anal vesicle of mf, in anal vesicle of in vivo and in vitro first stage larvae, in anal plug, intestine, oesophagus and buccal capsule of second stage larvae, and in intestine of in vivo third stage larvae. The acetylcholinesterase activity was observed in amphids and phasmids of microfilariae, in nerve ring of in vivo and in vitro first stage larvae, in vivo second and third stage larvae and also in excretory, anal vesicle, rectum and anus of all the stages. The distribution of the enzymes in vivo and in vitro developmental forms were compared and its significance is discussed. 

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42

Indian J Med Res 81, February 1985, pp 123-128  

Detection of filarial antigen in urine by sandwich ELISA & its use in diagnosis

Ashok Malhotra, M.V.R. Reddy, J.N. Naidu & B.C. Harinath

Department of Biochemistry, Mahatma Gandhi Institute of Medical Sciences Sevagram

Revised article received May 16, 2024

 Filarial antigen was detected in the concentrated urine samples of patients with micro filaraemia by sandwich ELISA. Double antibody sandwich ELISA using anti-rabbit filarial antigen immunoglobulin fraction was found to be more sensitive than using single antibody and neat urine sample could be used for detection of antigen. Urinary filarial antigen was subsequently used in indirect ELISA for the detection of filarial antibody in the sera of filarial patients. Thirty six of 42 patients with microfilaraemia, 17 of 21 with clinical filariasis, 3 of 21 endemic normals, 2 of 7 nonfilarial helminth infected nonendemics and none of the 6 nonendemic normal sera showed the presence of filarial antibody.

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 43
Indian Journal of Experimental Biology,
Vol. 23 April 1985, pp. 180-182.

 Detection and Isolation of Filarial Antigen from Hydrocoele Fluid and Its Use in Diagnosis

ASHOK MALHOTRA, G B K S PRASAD & B C HARINATH* 

Department of Biochemistry, M G Institute of Medical Sciences, Sevagram, 

Wardha 442102, India

Received 18 January 2024

                          Filarial antigen was detected in 19 out of 25 hydrocoele fluid samples screened by sandwich ELISA. Hydrocoele fluid antigen was isolated and filtered on ultrogel ACA-34. Out of the4 protein peaks (HFA1, HFA2, HFA3 and HFA4) obtained, HFAI and HFA2, were reactive in the detection of specific antibody in filarial sera. HFA2 antigen fraction, because of its easy availability and sensitivity, can be another antigen of diagnostic importance.

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 44

Indian J Med Res 82, August 1985, pp 127-132  

In vivo & in vitro development of Wuchereria bancrofti

 microfilariae

Indira Kharat & B.C. Harinath

Department of Biochemistry, Mahatma Gandhi Institute of Medical Sciences, Sevagram

Revised article received March 18, 2024

 The in vivo and in vitro development of W. bancrofti microfilariae was studied in Culex fatigans and in medium 199 supplemented with 20 per cent serum and certain additives respectively. In vivo development was completed within 12-13 days with the presence of third stage infective larvae in the head region of the mosquitoes. The microfilariae from in vitro cultures developed up to sausage stage or first stage (20%) and these sausage forms (2%) further developed to late sausage (first) stage larvae (245-260 μ in length and 26-28 μ in width) and showed body, movement similar to the first stage developing larvae in vivo. 

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45

IRCS Med. Sci. 13: 111 0-1111; 1985.

COMPARISON OF RADIO IMMUNO ASSAY AND INHIBITION ENZYME LINKED IMMUNOSORBENT ASSAY ( ELISA ) USING [¹⁴ C] - LABELLED WUCHERERIA BANCROFT'I MICROFII.ARLAL EXCRETORY SECRETORY ANTIGEN FOR THE DETECTION OF FILARIAL ANTIGEN

P Ram Prasad, MVR Reddy, I Kharat & BC Harinath

Detection of antigens in blood and urine should be much more effective than antibody detection in assessing an individual's infective status. The presence of circulating antigen has been reported in various parasitic infections like schistosomiasis amoebiasls , onechocerciasis and bancreftian filariasis. Qualssi et al. could detect circulating antigen in approximately two thirds of Onchocerca volvulus infected patients using a radio immu­noprecipitation polyethylene glycol assay (RIPEGA) and sandwich radioimmunoassay (RIA). Kaliraj et al. and Reddy et al. have reported the presence of circulating antigen in 77% and 82% of microfilariae carriers using countercurrent immunoelectrophoresis (CIEP) and sandwich enzyme linked immunosorbent assay (ELLSA) respectively, whereas Malhotra and Harinath have observed Wuchereria bancrofti microfilarial excretory secretory (Wb mf ES) antigen in 75% of microfilaraemia sera by Inhibition ELISA. This communication reports the use of [¹⁴C]labelled Wb mf ES antigen in RIA and Inhibition ELISA and compares the efficiency of these techniques in detecting mf ES antigen in sera of filarial patients.

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46

J.of the Indian Academy of  Pediatrics

Vol 23(no.3),169-174 March1986

 TRANSPLACENTAL TRANSFER OF FILARIAL INFECTION

M.Agarwal, G.B.K.S.Prasad, B.C.Harinath, B.D.Bhatia

 Two hundred consecutive mothers and their off springs constituted the study material. Paired maternal and cord sera samples were examined for presence of mixed antifilarial(IgG+IgM+IgA) antibodies and positive cord sera samples were examined for  specific antifilarial IgM and IgE antibodies. Both maternal and cord blood samples were also screened for the presence of microfilaria. Fourteen per cent of the paired maternal and cord sera samples were positive for mixed antifilarial antibodies. Only 10 mothers were positive for microfilariae in their night blood smear. None of the cord blood samples was positive for microfilaria.  Eighteen out of 28 cord sera samples were positive for a Specific antifilarial IgM antibodies. None of the cord sera samples was positive specific antifilarial IgE antibodies. Both mixed (IgG+IgM+IgA) and specific IgM antifilarial anti bodies disappeared after 3 months of follow up. The total maternal anti cord serum IgG and IgM levels were not affected by filarial infection

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  47

J.Com.Dis., 17 (Suppl No. 1) : 113-116,1985

Immunodiagnosis of Filariasis

 BHASKAR C. HARINATH*

  (Received for publication: 17th April, 1985)

 ABSTRACT

 The immunodiagnosis is one of the major challenges to the immuno- parasitologist. Extensive studies have been made to explore the utility of heterologous as well as homologous somatic antigens in immunodiagnosis but with little success. However, Wuchereria bancrofti microfilarial excretory - secretory antigens were found to be specific and highly sensitive requiring as little as 0.35 ng antigen protein per well in penicillinase enzyme linked immunosorbent assay for detection of filarial antibody. Field evaluation of this test showed that it can replace laborious night blood examination.

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 48

Indian J. Pathol. Microbiol 29 : 179-188: 1986 

1solation and Evaluation of Antigens from Microfilaraemia

Plasma and Immune Complexes for Diagnosis of

Bancroftian Filariasis

 M.V.R.Reddy, G.B.K.S Prasad &  B.C.Harinath

Department of Biochemistry, Mahatma Gandhi Institute of Medical Sciences, Sevagram, Maharashtra-442 102, India

The circulating filarial antigen (CFA) isolated by 36-75 percent ammonium sulphate precipitation of microfilaraemia plasma was further fractionated on ultrogel ACA-34 column into four protein fractions (CFA-1, CFA-2, CFA-3, and CFA-4). CFA 1 and CFA 2 fractions showed antigenic activity. Circulating immune complexes (CICs) were Isolated from clinical filariasis sera, dissociated, and fractionated on Sephadex (3-150 column into two protein fractions ICA-1 and ICA-2. ICA 1 showed the presence of antigen and anti­body, while the ICA 2 showed only the antigenic activity. CFA-1 CFA-2 and ICA-2 antigen fractions were further evaluated in ELISA for their diagnostic utility in filariasis. CFA-1 and CFA-2 were sensitive in detecting filarial  IgM antibodies and CFA-2 exhibited greater sensitivity than CFA-1. Studies with ICA-2 antigen fraction showed that all the microfilaraemia sera were positive for filarial IgM antibody and higher number of clinical filariasis sera were positive for filarial IgG antibody. Filarial serum can be another potential source for filarial antigen of diagnostic importance.

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49

J. Biosci., Vol. 10, Number 4, December 1986, pp. 461-466.

Diagnostic utility of monoclonal antibodies raised against microfilarial excretory-secretory antigens in bancroftian filariasis  

M. V. R. REDDY, P. RAMA PRASAD, W. F. PIESSENS* and B. C. HARINATH** Department of Biochemistry, Mahatma Gandhi Institute of Medical Sciences, Sevagram, Maharashtra 442 102, India

*Department of Tropical Public Health, Harvard School of Public Health, 665, Huntington Avenue. Boston, Massachusetts 02115, USA

Abstract. Two monoclonal antibodies Wuchereria bancrofti E 33 and Wuchereria bancrofti E 34 raised against Wuchereria bancrofti microfilarial excretory-secretory antigens were studied for their diagnostic utility. Wuchereria bancrofti E 34 monoclonal antibody was found to be relatively specific and sensitive in detection of circulating filarial antigen. When Wuchereria bancrofti E 34 monoclonal antibody was used along with immunoglobulin G fraction of human filarial serum immunoglobulins in double antibody sandwich enzyme linked immunosorbent assay, 68% of microfilaraemic sera (26 out of 38), 12% of clinical filarial sera (3 out of 25), 13% endemic normal sera (2 out of 15) and none of the 20 non-endemic normal sera showed the presence of filarial antigen. The filarial antigen detected by Wuchereria bancrofti E 34 monoclonal antibody in double antibody sandwich enzyme linked immunosorbent assay is possibly associated with the active stage (microfilaraemia) of infection.

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50

Indian Journal of Experimental Biology

Vol. 24, July 1986, pp. 437-439

Disc/Stick ELISA for Diagnosis of Bancroftian Filariasis

ELISA was employed for the detection of antibody to W. bancrofti mf ES antigen using cellulose acetate membrane discs as well as plastic strips with the discs attached. The disc ELISA was found to be equally sensitive and reproducible to plate assay, when 48 sera were screened for filarial antibody. Modification of disc ELISA to stick ELISA made the assay more convenient for washing and simple. The use of cellulose acetate membrane disc in stick ELISA requires as little as 60 pg of antigen per disc thus increasing the utility of ES antigen by 5-fold and also makes the ELISA more economic when compared to the PVC microtitre plate assay.

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51

 G.B.K.S Prasad & B.C. Harinath

Cell mediated immune status was assessed in 14 patients of clinical filariasis by leucocyte migration inhibition test. Suppressed cellular immune response was seen in 10 of 14 patients to Wuchereria bancrofti microfilariae excretory-secretory (mf ES) antigen, in 13 to immune complexed antigen and in 5 to non-specific mitogen Con A. The immune complexed antigen showed more suppressive effect than free mf E5 antigen. A positive correlation was apparent between immune complex levels in the sera and the magnitude of suppression to immune complexed antigen.

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52

Am. J. Trop. Med. Hyg., 36(3), 1987, pp. 554-560

Copyright © 1987 by The American Society of Tropical Medicine and Hygiene

PARASITE ANTIGENS IN SERA AND URINE OF PATIENTS WITH BANCROFTIAN AND BRUGIAN FILARIASIS DETECTED BY SANDWICH ELISA WITH MONOCLONAL ANTIBODIES

ZHENG HUIJUN,* TAO ZHENGHOU,*M. V. R. REDDY,†

BHASKAR C.HARINATH,† AND WILLY F. PIESSENS‡

*Department of Filariasis, Guizhou Provincial Institute of Parasitic Diseases, Guiyang, People's Republic of China, ‡Department of Biochemistry, Mahatma Gandhi Institute of Medical Sciences, Sevagram, India, and ‡Department of Tropical Public Health, Harvard School of Public Health, Boston, Massachusetts 02115

Abstract. We developed a sandwich enzyme-linked immunosorbent assay to detect circulating parasite antigens in human lymphatic filariasis. The assay utilizes a polyclonal rabbit antifilarial antiserum to capture, and a monoclonal antibody to identify, circulating parasite antigen. Using this assay, we found that >95% of sera from microfilaremic donors with bancroftian or brugian filariasis, approximately 60% of sera from amicrofilaremic patients with hydroceles, chyluria, or elephantiasis, and 15%-20% of sera from asymptomatic residents of filariasis-endemic areas evidently contain filarial antigens. Antigen was also detected in the urine of some microfilaremic patients. Serum levels of antigen detected with one monoclonal antibody, ES34, correlated well with microfilarial density in night blood. In contrast, <5% of sera from residents of areas where lymphatic filariasis is not endemic reacted in the assay, even though approximately one-third of the donors whose sera were tested were known to be infected with intestinal nematodes. The assay was designed to be flexible enough to allow the parallel use of multiple monoclonal antibodies with different specificities and simple enough to be applicable in most areas where lymphatic filariasis is endemic.

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53

J. Com. Dis.,18 (4) : 261-266,1986

Detection and Diagnostic Utility of in vitro and in vivo Released Antigens in Bancroftian Filariasis†

BHASKAR C. HARINATH^*

(Received for Publication:17 December, 1986)

ABSTRACT

Recent studies have shown that some of the proteins released in vitro maintenance media are antigenic and are of immunodiagnostic importance. In addition to secreted, excreted or metabolic antigens released by the living parasite, somatic antigens from destruction of microfilariae are also released in the host circulation in vivo. Utility of in vitro released antigens (ES antigens) isolated from culture medium of Wuchereria bancrofti microfilariae was studied in penicillinase ELISA for their immunodiagnostic potential. As little as 60 pg ES antigen protein per test was found to be sufficient in Stick ELISA in detecting filarial antibody.One ml of culture fluid was found to have sufficient ES antigen for 2 million tests. Fractionation of ES antigens by membrane filtration gave ES 2 and ES 4 antigen fractions, which were found to be glycoprotein in nature and were highly reactive with clinical filariasis and microfilaraemia sera respectively. In vitro released filarial antigens in serum, urine and hydrocele fluid of filarial patients, have been demonstrated by immunoelectrophoresis, enzyme linked immunosorbent assay and immunoradiometric assay.The circulating free, immunecomplexed and urinary filarial antigens have been isolated by column chromatography and ultrafiltration and were found to be useful in detecting filarial antibody as well as in monitoring effectiveness of DEC therapy to microfilaraemic patients.

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54

JOURNAL OF IMMUNOASSAY, 8(4), 351-365 (1987)

ANALYSIS OF PAIRED SERUM, URINE AND FILTER PAPER BLOOD SPECIMENS

G.B.K.S Prasad ^a B.C. Harinath^a and R.G. Hamilton^b

 Mahatma Gandhi Inst. Med. Sciences, Dept. Biochem., Sevagram, Maharashtra, 442 102, INDIA, and

b Rheumatology and Clin. Immunogenetics, Dept. Internal Med., Univ. Texas Sch. Med., 5.264 MSMB, 6431 Fannin St., Houston,

Texas 77030, U.S.A.

ABSTRACT

Paired serum, urine, and finger-prick whole blood dried on filter paper were analyzed by immunoradiometric assay (IRMA) for filarial antigen using Brugia malayi-specific rabbit antibody. Nine sera and 6 urines from the 10 paired serum-urine samples obtained from individuals with microfilaraemia contained IRMA detectable filarial antigen. In contrast, all serum and urine specimens from patients with chronic infections, endemic and non-endemic controls were negative. Whole blood eluted, from filter paper spots contained IRMA detectable material; their degree of positivity agreed well with IRMA binding levels obtained with paired urines. Reduced recovery of antigen dried on filter paper was observed at antigen levels <10 ng/ml equivalents, presumably due to irreversible absorption onto the filter paper. Urine and finger-prick filter paper blood specimens can be used in the diagnosis of microfilaremic infections that have been associated with circulating antigen in the blood.

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55

Fractionation and Characterization of Urinary Filarial Antigen in Bancroftian Filariasis

P. Rama Prasad and B.C. Harinath

SUMMARY Urine samples from microfilaraemic patients were concentrated and fractionated by gel chromatography on Ultrogel AcA 44. Four protein fractions labelled as UFA C1, UFA C2, UFA C3 and UFA C4 were tested for filarial antigenicity by sandwich ELISA. UFA C1 and UFA C2 showed antigenic activity. On further analysis by SDS-PAGE, UFA C1 and UFA C2 showed antigenic components with MW ranging from 10.4 K to 123 K. UFA C1-1 and UFA C2-2 showed high antigen titre in ELISA. Urinary albumin was observed as a major component in UFA C2. Absorption of albumin from UFA C2 enhanced its antigenic activity considerably. As little as 0.01 pg antigenic protein per test was found to be sufficient for the detection of filarial antibody in ELISA. Biochemical characterization indicated a glycoprotein nature of UFA C2.

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56

Diagnostic and Clinical Immunology 5:269-275 (1988)

Analysis, Characterization, and Diagnostic

Utility of Filarial Antigen Fractions

Isolated From Immune Complexes in

Bancroftian Filariasis

Circulating immune complexes isolated from clinical filarial patients sera by 3% polyethylene glycol precipitation were analyzed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) on vertical slab gel. Silver staining of the gel offer electrophoresis revealed 20 and 30 protein bands without and with reduction of immune complexes, respectively, in the molecular weight range of 148-18K. Filarial antigen in protein eluates from gel slices was detected by sandwich enzyme-linked immunosorbent essay. The eluates from four gel slices, viz., IC-2, IC-4, IC-7, and IC- 9, showed filarial antigenic activity. These antigen fractions were characterized and explored for their diagnostic use. IC-7 and IC-9 fractions and microfilariae excretory-secretory (mf ES) antigen share common antigenic determinants as revealed by the fact that saturation of immobilized antibodies with IC-7 or IC-9 inhibited the binding of mf ES antigen coupled to penicillinase. IC-9 fraction appears to be useful in serological differentiation of Wuchereria bancrofti infected persons from those with disease manifestations. Biochemical characterization of the IC-9 fraction revealed the protein nature of the antigen. Comparison of the electrophoretic profiles of immune complexes and W. bancrofti mf ES antigen revealed several common protein bands. The 55 K protein band with antigenic activity was observed in both preparations.

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57

Med. Sci. Res., 1988; 16, 635-637

Department of Biochemistry, Mahatma Gandhi Institute of Medical Sciences, Sevagram, Wardha-442 102,India

And *Department of Tropical Public Health, Havard School of Public Health,665,Huntington Avenue,

Boston, Massachusetts 02115,USA

Abstract

SDS-PAGE analysis of Wuchereria bancrofti microfilarial excretory-secretory(ES) antigens has shown 29 protein bands with molecular weights (MWs) ranging from 18K TO 145K .Antigenic analysis by sandwich ELISA using filarial serum immunoglobin-G(FSIgG) showed polydispersed nature of antigen in all the 12 SDS polyacrylamide gel fractions. However using Wb E 34 monoclonal antibody only 7 fractions showed the presence of filarial antigen. Three fractions viz., ESA-6(MW 55-67 K),ESA-11(MW 20-27 K) and ESA-12(MW 18-20 K) were highly antigenic with reciprocal titre of 25600.Determination of specific antibody using ESA-12 fraction showed elevated geometric mean of IgM antibody titre in microfilaraemia sera (3351)compared to clinical filarial sera (396).

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58

Acta Tropica 45, 245-255 (1988) 

Microfilaraemia, filarial antibody, antigen and immune complex levels in human filariasis before, during and after DEC therapy

Department of Biochemistry, Mahatma Gandhi Institute of Medical Sciences,

Sevagram - 442 102, India

A two-year follow-up

P. RAMAPRASAD, G. B. K. S. PRASAD, B. C. HARINATH

Summary

A study on the effect of DEC therapy on microfilaraemia and the immune status in 27 patients with W. bancrofti infection was carried out for two years. Persistence of microfilaraemia was observed in 4 out of 27 cases after one course of DEC therapy and were treated again for one week. On further followup, none was microfilaraemic upto Day 60. The mean filarial antibody titres of IgM and IgG showed a gradual decrease as assessed by enzyme linked immunosorbent assay (ELISA). The mean titres of circulating microfilarial excretory-secretory (ES) antigens and immune complexes (ICs) showed an initial increase during therapy, followed by a gradual fall upto Day 60. Filarial antigen was detected in urine of all the carriers during therapy. Excretion pattern of antigen in urine showed correlation with DEC dose. Reappearance of microfilariae (mf) in circulation in 12 patients after a year showed that DEC had temporary attenuating effect on adult worms or no effect on developing larvae, suggesting further treatment and follow-up of patients. Parasitological and immunoscreening at the end of 2 years showed that the presence of mf ES antigen in blood correlated with the appearance of microfilariae in blood.

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59

J. Biosci., Vol. 13, Number 3, September 1988, pp. 229-233.

Stick enzyme-linked immunosorbent assay using the avidin-biotin

system for detection of circulating antigen in bancroftian filariasis

K. A. PARKHE, P. RAMAPRASAD and B. C. HARINATH*

Mahatma Gandhi Institute of Medical Sciences, Sevagram 442 102, India

MS received 26 November 1987; revised 9 June 2024

Abstract. Detection of filarial antigen in different groups of sera was carried out by sandwich as well as inhibition enzyme-linked immunosorbent assays using antibody-coated sticks. Both systems were found to be equally sensitive in detecting antigen in 90% of microfilariae carriers. Incorporation of avidin-biotin in the sandwich assay system increased the sensitivity of antigen detection from 10^-6 to 10^-16 pg. A 67% decrease in the number of false negative results was observed when the sensitive avidin-biotin inhibition enzyme-linked immunosorbent assay system was used for analysis of filaria blood samples.

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60

Transactions Of The Royal Society & Tropical Medicine And Hygiene (1989) 83, 90-94

Fractionation, characterization and diagnostic potential of filarial antigens isolated

from hydrocoele fluid in bancroftian filariasis.

P. Ramaprasad and B. C. Harinath*

Department of Biochemistyy, Mahatma Gandhi Institute of Medical Sciences,

Sevagram-442 102, India

Abstract

Filarial antigen was isolated from hydrocoele fluid and fractionated on Ultrogel AcA 44. Six protein fractions (HFA Cl to HFA C6) were chromatographically separated from the column. Of the 4 antigenic fractions (HFA Cl, HFA C2, HFA C3 and HFA C5), HFA C3 was a major reactive fraction with filarial serum immunoglobulin G. Analysis of HFA C3 by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) yielded 18 bands of M_r, 18 000-160 000. Two fractions, HFA C3-7 and HFA C3-9 of M_r, 48 000-55 000 and 32 000-39 000 obtained by gel elution, were antigenic in an enzyme-linked immunosorbent assay (ELISA). SDS-PAGE immunoblotting confirmed the ELISA by identifying 3 immunoreactive bands of M_r, 51 000, 38 000 and 35 000. The diagnostic potential of HFA C3-7 and HFA C3-9 was compared in serodiagnosis of active infections and diseased states. HFA C3-9 showed greater sensitivity in detection of filarial specific IgM antibody in cases with microfilaraemia. Physicochemical characterization indicated the protein nature of HFA C3-9.

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61

Indian Journal of Experimental Biology

Vol. 27, August 1989, pp. 681-684

Antigenic analysis of excretory-secretory products of Wuchereria Bancrofti

and Brugia malayi infective larval forms by SDS-PAGE

I Kharat*, K Cheirmaraj, G B K S Prasad†& B C Harinath‡

Received 27 May 1988; revised 2 September 2023

Excretory-secretory (ES) products of W. bancrofti and the closely related B. malayi infective larval forms were analysed for their antigenic activity by SDS-PAGE followed by Western blotting as well as by gel elution-sandwich ELISA using filarial serum immunoglobulin-G (FSIGG) as a capture antibody. In W bancrofti infective larval ES products, the protein molecules of 66, 46, 35, 33, 30 and 14 kDa molecular wt. showed antigenic activity by immunoblotting technique. In sandwich ELISA technique eventhough all SDS-PAGE fractions except ESA 6 (55-47 kDa) showed antigenic positivity, the fractions ESA 8 (37-31 kDa) and ESA 9 (31-25 kDa) showed high reciprocal antigen titre of 262144 and 32768 respectively. In B. malayi infective larval ES products, the protein molecules of 109, 102, 97 and 77 kDa molecular wt. showed reactivity with FSIGG by blotting technique, where as in sandwich ELISA except ESA 7 (47-37kDa), all fractions showed antigenic positivity. However, these fractions failed to show high antigen titre similar to W bancrofti ES products with FSIGG.

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62

M.V.R. Reddy,¹ K.A. Parkhe,¹ W.F. Piessens,² and B.C. Harinath¹

1Department of Biochemistry, Mahatma Gandhi Institute of Medical Sciences, Sevagram, Maharashtra, India;

The Wb E34 monoclonal antibody raised against Wuchereria bancrofti microfilarial excretory secretory (mf ES) antigen was reported to be useful in detecting the filarial antigen in W bancrofti and Brugia malayi infected sera. Further studies in this laboratory showed that this monoclonal antibody reacts with a stage-specific antigen of W. bancrofti filarial parasite. Wb E34 identified three antigenic components with molecular weights, 55, 57.5, and 63 kilodaltons (Kd) in western blot analysis. The target antigen of Wb E34 was found to be located in the cytoplasm of microfilariae by indirect immunofluorescence technique. The inhibitory antibodies to Wb E34 were detected in a higher percentage of microfilaraemic sera (81 %) than in clinical filarial (33%) or tropical eosinophilia sera (35%). Thus the inhibition ELISA using W. bancrofti mf ES antigen and Wb E34-penicillinase may be useful in detecting the filarial antibody associated with the active stage of infection.

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63

Indian J Med Res 89, September 1989, pp 322-325

Relationship of C3 & C4 levels with filarial immune complexes

in bancroftian filariasis

G.B.K.S. Prasad* & B.C. Harinath

Department of Biochemistry, Mahatma Gandhi Institute of Medical Sciences, Wardha

Accepted November 8, 2023

C3 and C4 levels were determined by radial immunodiffusion technique and filarial circulating immune-complexes by anti-C3 ELISA in microfilaraemic individuals and patients with clinical filariasis. Decreased levels of C3 and C4 were observed in both groups of filarial patients. Low levels of complement components were associated with low levels of circulating immune complexes.

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64

Indian J Med Res [A] 91, March 1990, pp 133-137

Efficacy of two different DEC regimens in the treatment of human

filarial infection

G.B.K.S. Prasad*, P. Ramaprasad, V. Suryaprakash Rao**, M.S. Bharati †& B.C. Harinath

Accepted October 9, 2023

Two regimens of diethyl carbamazine (DEC) viz., 14 day and 5 day, were compared for microfilaricidal effect and effects, in the treatment of bancroftian filariasis. The rate of successful treatment, cure rate and decrease in mf count were found to he significantly high with the 14 days regimen when assessed immediately after treatment. About 40 per cent of subjects on the 14 days regimen and 66 per cent of patients on 5 days regimen experienced side reactions. The severity of side reactions was more in patients on 5 days regimen. When the effect of DEC was assessed one year after treatment with the 14 days regimen and compared with the results immediately after treatment, the rate of successful treatment, cure rate and decrease in mf count were reduced significantly. The 14 days DEC regimen with initial low dose of DEC along with antipyretics may he better accepted in the control programmes of filariasis.

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65

J.Biosci.,Vol.15,Number 1,March 1990,pp.37-46

Analysis, Characterization and Diagnostic Use of Circulating Filarial

Antigen in Bancroftian Filariasis

K.A.Parkhe,M.V.R.Reddy, K.Cheirmaraj,P.Ramaprasad and B.C.Harinath*

Department of Biochemistry, Mahatma Gandhi Institute of Medical Sciences,Sevagram,Wardha-442102,India

MS Received 16 August 1989;revised 19 February 2024

Abstract:Sodium dodecyl sulphate-polyacrylamide gel electrophoresis of circulating filarial antigen fraction-2 isolated from plasma of microfilaraemia patients with Wuchereria bancrofti infection has shown 21 bands with molecular weight from 12 to ≥120 kDa.The gel(12 cm) was sliced at an interval of 1 cm and the eluates of all the gel slices viz.,CFA2-1to CFA2-12 showed the presence of filarial antigen by sandwich enzyme linked immunosorbant assay.The low molecular weight circulating filarial antigen fractions were found to share a common epitope with Wuchereria bancrofti microfilariae excretory-secretory antigen and urinary filarial antigen. The three antigen fractions CFA2-1,CFA2-9 and CFA2-12 showed higher sensitivity in detecting filarial immunoglobulin M antibodies than immunoglobulin G antibodies.However CFA2-9 fraction was found useful in serological differentiation of microfilaraemics from those with disease manifestations when filarial immunoglobulin G antibodies were detected. The antigenic epitope of CFA2-1 appears to be a carbohydrate, whereas CFA2-9 appears to be protein in nature.

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66

Acta Tropica, 48(1991)305-312

Eisevier

ACTROP 00126

antigen in Brugia malayi infected Mastomys natalensis

K. Cheirmaraj and B.C. Harinath

The sequential changes in the humoral immune response against infective larval antigens during the course of Brugia malayi infection in Mastomys natalensis have been studied using enzyme linked immunosorbent assay. IgM antibody against B. malayi infective larval excretory secretory (ES) antigen was detected in the peripheral circulation within a week of infection, whereas IgM antibody against B. maiayi infective larval somatic antigen and IgG antibody against both somatic and ES antigens were detected on day 20 post-inoculation. Thereafter, the antibody levels showed a steady increase until day 150. A gradual decrease of IgM antibody level was observed upto day 360, whereas IgG antibody level was decreased upto day 250 and then maintained almost the same level upto day 360. Wuchereria bancrofti cross reactive antigen as well as B. malayi infective larval ES antigen were detected in blood circulation on day 20, the level increased upto day 150 and then remained almost the same upto day 360 with slight variations. Studies of antigen and antibody levels in microfilaraemic and amicrofilaraemic animals show that there is no significant difference in antibody level whereas elevated antigen titre was observed in active infection with microfilaraemia.

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67

JOURNAL OF IMMUNOASSAY, 11(4), 429-444(1990)

DIAGNOSTIC USE OF POLYCLONAL ANTIBODIES RAISED IN

MOUSE ASCITIC FLUID IN BANCROF'I'IAN FILARIASIS

K. Cheirmaraj, M.V.R. Reddy and B.C. Harinath

Department of Biochemistry

Mahatma Gandhi Institute of Medical Sciences

Sevagram, Wardha, 442 102. lndia

ABSTRACT

Polyclonal antibodies were produced against Brugia malayi adult antigens (BmA (PBS) SAg and BmA (SDS) SAg) in mouse ascitic fluid by immunising Balb/c mice intraperitoneally with high ratio of adjuvant to immunogen. The diagnostic use of these antibodies in detecting circulating filarial antigen in bancroftian filariasis was studied by sandwich enzyme-linked immunosorbent assay (sandwich ELISA) using stick assay system. Both antibodies raised against PBS and SDS soluble antigens were found to, be equally sensitive and relatively specific in detection of circulating filarial antigen. When anti BmA (PBS) SAg antibody was used in sandwich ELISA. 90% of microfilaraemic sera, 30-40% of acute and sub acute filarial sera, 20% of chronic filarial sera, 7% of endemic normal sera and none of 15 non-endemic normal sera were positive for filarial antigen. Using anti BmA (SDS) SAg antibody, 93% or microfilarial sera, 40% of acute and sub acute filarial sera, 20% of chronic filarial sera and none of 15 endemic and non-endemic normal sera showed the presence of filarial antigen. The filarial antigen detection using anti BmA S Ag antibodies produced in mouse ascitic fluid in sandwich ELISA may be useful in detection of active stage (microfilaraemia) of infection.

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68

Filarial antigen antibody and circulating immune

complexed antigen levels in Bancroftian filariasis by

stick ELISA

A.ALIKHAN, K. A. PARKHE, M. V. R. REDDY, B. C. HARINATH

ABSTRACT

Filarial antigen, antibody and circulating immune complexed antigen (CIC-Ag) profiles were studied by stick ELISA in the sera of patients who were in different stages of Bancroftian filarial infection. The geometric mean titres (GMT), of filarial IgG antibody in patients with microfilaraemia (424), and all three grades of clinical filariasis (1485, 3845 and 4 216 for grades I, II and III respectively), were significantly higher than in normal controls from endemic areas (47). If a filarial antibody titre of greater than 300 is considered positive, the sera of 94% of patients with microfilaraemia and all those with clinical filariasis were positive. The sera of only 11% of normal subjects from endemic areas and none from non-endemic areas were positive for filarial antibody. The antibody titres were significantly higher in patients with grades II and III clinical filariasis than in those with grade I clinical filariasis or microfilaraemia

Analysis or sera for filarial antigen by inhibition ELISA showed higher GMTs in patients with microfilaraemia (5778) and grades I (3917) and II (676) clinical filariasis compared to GRADE III ( 66) clinical filariasis or normal subjects from endemic areas (57). Thus 81% of patients with microfilaraemia, 85% with grade I, 88% with grade II and 20% with grade III clinical filariasis and none of the normal subjects from either endemic or non-endemic areas had a filarial antigen titre of greater than 300. CIC-Ag was found to be present in 8% of patients with grade I, 21% with grade II and 79% with grade III clinical filariasis and in none of those with microfilaraemia or normal controls.

While the detection of filarial antibody is useful for diagnosing microfilaraemia and clinical filariasis, the detection of filarial antigen may be superior for diagnosis of micro filaraemia grade I clinical filarial infection. Assay of CIC–Ag is useful for diagnosing a grade III clinical infection in the absence of microfilariae in the blood.

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69

Suppression of Con A induced lymphocyte transformation by sera from filarial

patients with clinical manifestations

G B K S Prasad*, Indira Kharat †& B C Harinath

Department of Biochemistry, Mahatma Gandhi Institute of   Medical  Sciences,Sevagram, Wardha, India, 442 102

Received 24 April 1990; revised 1 August 2023

Reduced lymphocyte transformation to Wuchereria bancrofti microfilariae excretory-secretory antigen and Con A were observed in clinical filarial patients. Pre-incubation of normal human peripheral blood mononuclear cells with sera from filarial patients with clinical manifestations such as hydrocele and elephantiasis suppressed Con A induced responses. Effect of fractionated clinical filarial serum on Con A induced lymphocyte transformation showed that the inhibitory activity was associated with high molecular weight serum fraction.

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70

J. Biosci., Vol. 16, Number 4, December 1991, pp 199-208.

Differential reactivity of filarial antigens with human sera from

bancroftian filariasis endemic zone

Department of Biochemistry, Mahatma Gandhi Institute of Medical Sciences,

Sevagram 442 102, India

MS received -30 April 1991; revised 4 October 2023

Abstract. The reacting pattern of circulating filarial antigen fraction-2 from Wuchereria bancrofti and soluble antigen from adult Brugia malayi with bancroftian filarial sera were analysed by immunoblotting technique and enzyme linked immunosorbent assay. Microfilaraemic sera reacted specifically with proteins of molecular weight 200, 120, 97, 56, 54, 43, 26 and 17 kDa of circulating filarial antigen fraction-2 and 44, 40, 38, 31, 22 and 18 kDa of Brugia malayi adult soluble antigen. Clinical filarial sera, identified protein molecules of 56, 54 and 42 kDa of circulating filarial antigen fraction-2 an 19, 16 and 4 kDa of Brugia malayi adult soluble antigen. Some components of both the antigen preparation were also identified by endemic normal sera i.e, proteins 120, 97, 62, 43 and 33 kDa of circulating filarial antigen fraction-2 and 170, 120, 43, 31 and 12 kDa of Brugia malayi adult soluble antigen. One of the sodium dodecyl sulphate-polyacrylamide gel electrophoresis fractions of circulating filarial antigen fraction-2 (CFA₂-8) and Brugia malayi adult soluble antigen fraction-6 when used in enzyme linked immunosorbent assay could differentiate microfilaraemic sera from endemic normal and clinical filarial sera. The other antigen fractions (CFA₂-2, 6 and 7 and BmA-2) showed a high geometric mean titre of filarial immunoglobulin G antibodies in endemic normal sera when compared to microfilaraemia and clinical filarial sera. These proteins need to be further studied to assess their involvement in protecting from filarial infection in an endemic area.

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71

J. Biosci,, Vol. 16, Number 4, December 1991, pp 209-216.

Immunoprophylaxis against filarial parasite, Brugia malayi:

potential of excretory-secretory antigens in inducing immunity

K CHEIRMARAJ, V CHENTHAMARAKSHAN, M V R REDDY and B C HARINATH*

Department of Biochemistry, Mahatma Gandhi Institute of Medical Sciences,

Sevagram 442 102, India

MS received 20 June 1991; revised 4 November 2023

Abstract. The role of excretory-secretory antigens in inducing immunity in the host against Brugia malayi microfilariae and infective larvae was studied by in vitro antibody dependent cell-mediated reaction as well as in vivo inoculation of filarial parasites within a microchamber in the host. The immune sera of jirds raised against Brugia malayi microfilarial and infective larval excretory-secretory antigens (Bm Mf ESA and Bm L₃ ESA) promoted the adherence of peritoneal exudate cells to Brugia malayi microfilariae and infective larvae in vitro and induced cytotoxicity to the parasites within 48 h. The anti Bm Mf ESA serum was more effective than anti Bm L₃ ESA serum in inducing cytotoxicity to microfilariae and both antisera had a similar cytotoxic effect on infective larvae. In the microchambers implanted in the immune jirds, host cells could migrate and adhere to the microfilariae and infective larvae and kill them within 48-72 h. Further, Mastomys natalensis immunized against Bm Mf ESA and L₃ ESA generated a high degree of protective response against circulating microfilariae. These results suggest that excretory- secretory antigens are effective in inducing resistance against filarial parasites and thus have potential in immunoprophylaxis.

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72

Journal of Tropical Medicine and Hygiene 1992, 95, 47-51

Evaluation of fractionated circulating filarial antigen indiagnosis of bancroftian filariasis

K. Cheirmaraj, K. A. Parkhe and B. C. Harinath

Department of Biochemistry, Mahatma Gandhi Institute of Medical Sciences,

Sevagram-442102,India

Summary

Circulating filarial antigen (CFA) isolated from the plasma of microfilaraemic patients was fractionated on an Ultrogel ACA 34 column. The second protein peak (CFA2) showing filarial antigen was further fractionated by DEAE cellulose column chromatography into two fractions (CFA₂ DE1 and DE2). CFA2 DE1 fraction, showing antigenic activity, was further evaluated in an ELI SA for its diagnostic use in bancroftian filariasis. Studies with CFA, DE 1and anti-CFA2 DE1 antibody showed that they were highly active in the detection of filarial antibody and antigen in asymptomatic microfilaraemia sera and thus obviate the need for the tedious night blood collection and examination. Fractionated filarial plasma can be another candidate antigen for immunodiagnosis of bancroftian filariasis.

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73

Hindustan. Antibiotic. Bulletin. 34(1-2).24-31;1992.

PEN - ELISA FOR IMMUNODIAGNOSIS AND IMMUNOMONITORING  OF BANCROFTIAN FILARIASlS.

The Enzyme Linked Immunosorbent Assay (ELISA) has attracted considerable attention of the investigators interested in development of immunodiagnostic tests for detection of Infectious diseases. It is comparable with radio-immuno-assay in determining minute con­centrations of proteins, hormones etc (in picogram range) and at the same time needs no sophisticated equipment such as isotope counters. EIA system is easier to handle in smaller laboratories and is adaptable to held laboratories as well. Application of ELISA in differ­ent fields has been well reviewed . Penicillinase has been.sucessfully used as an enzyme marker in hormone assays. The penicillinase (B-Lactamase, EC 3,5,2,6) has high turnover number of 1,60,000 and has been found to be more sensitive than peroxidase, alkaline phos­phatase or B-galactosidase used in enzyme immunoassay system . The substrate (penicillin V) used is not carcinogenic and other advantages of using penicillinase are that the enzyme is quite stable and is not present in biological fluids under normal conditions. Penicillinase activity can be reliably estimated iodomertically. The assay involves decolorization of blue colored starch-iodine-penicillin substrate. The result is assessed visually by naked eye. The reaction may be stated as follows.

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74

DETECTION OF FILARIAL ANTIGEN USING ANTIBODIES

RAISED AGAINST WUCHERERIA BANCROFTI MICROFILARIAL

SDS SOLUBLE ANTIGEN

K Cheirmaraj, MVR Reddy and BC Harinath*

Department of Biochemistry, Mahatma Gandhi Institute of Medical Sciences,

Sevagram 442 102, India.

Abstract. Polyclonal antibodies raised in mouse ascitic fluid against Wuchereria bancrofti microfilarial antigens (Wb Mf SDS S Ag) were studied for their diagnostic use in bancroftian filariasis using a dip stick,enzyme-linked immunosorbent assay. In sandwich ELISA, 100% of microfilaremic sera (30 out of 30) 53% of acute filarial sera (7/13), 40% of subacute filarial sera (6 out of 15), 13% of chronic filarial sera (2/15) and 20% of endemic area normal sera (3/15) showed the presence of filarial antigen. Determination of filarial antigen titer in microfilaremic sera showed an apparent positive correlation between microfilarial density and antigen titer. The antibody raised against Wb Mf SDS S Ag was found to be cross reactive with phosphorylcholine epitopes. The filarial antigen detected by anti Wb Mf SDS S Ag antibodies in sandwich ELISA is possibly associated with the active stage (microfilaremia) of infection.

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75

Diagnostic use of filarial antibody and antigen isolated from hydrocele fluid for

human filariasis

T A Singh, K Cheirmaraj, M V R Reddy, R Narang* & B C Harinath†

Departments of Biochemistry and Surgery*, Mahatma Gandhi Institute of Medical Sciences 

Sevagram. Wardha 442 102. India

Received 31 December 1991; revised 31 July 2023

Paired samples of serum and hydrocele fluid of filarial patients associated with hydrocele were analysed for filarial antibody and antigen. Sera samples showed higher titers of filarial antibody and antigen compared to their corresponding hydrocele fluid samples. HFIgG isolated from hydrocele fluid was equally useful as FSIgG isolated from serum and detected filarial antigen in 23 out of 26 microfilaraemic sera, 7 out of 10 chronic sera and 3 out of 18 endemic normal sera by inhibition ELISA. Filarial antigen was isolated from hydrocele fluid. In inhibition ELISA antigen fraction, HFA-9 (20-25 kDa) isolated by sodium-dodecyl sulphate-polyacrylamide gel electrophoresis showed high reciprocal antigen titer of 2048. This active antigen fraction was evaluated for its diagnostic utility in comparison with Wuchereria bancrofti microfilariae excretory-secretory antigen (Wb mf ES Ag) in inhibition ELISA. Both antigen preparations detected filarial antigen in about 80% of microfilaraemic sera, 60% chronic sera and 20-30% of endemic normal sera. This study showed that antibody and antigen isolated from hydrocele fluid were equally sensitive as FSIgG and Wb mf ES Ag in the detection of filarial antigen by inhibition ELISA. Thus hydrocele fluid may be used as an alternative source for the isolation of antibody and antigen of immunodignostic importance.

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76

Indian Journal of Clinical Biochemistry 1992, 7(2) 101-107

Awadesh Saran Memorial Oration 1992

B.C. HARINATH*, K. CHEIRMARAJ & M.V.R. REDDY

Department of Biochemistry. Mahatma Gandhi Institute of Medical Sciences

Sevagram - 442 102, Wardha, India.

introduction

Lymphatic filariasis is an important public health problem in the developing countries affecting about 100 million people in the world, more than 90% of whom suffer from bancroftian filariasis caused by the nematode Wuchereria bancrofti (1). Over one third of the world population who are to risk of lymphatic filarial infection live in India with the estimated 374 million population exposed to the risk of infection, of which 25 million are mf carriers and 19 million suffer from disease manifestations (2). Because of the continuous spread of the disease and the protracted sufferings and disability caused in the affected population. Filariasis has been recognized as a disease of national importance. The early diagnosis of filarial Infection is one of the prerequisite for the effective control of the disease. It requires a highly sensitive method to detect very low levels of infection in population and it should be simple to perform, economic costwise and should be easily adaptable to field conditions in endemic areas. The classical diagnostic test i.e., parasitological examination of thick smear collected at night is not sensitive enough to detect carriers with low mf count, prepatent and occult filarial Infection. An estimated number of 30-35% microfilaraemics are missed by this method (3). A daytime test of infection is needed to obviate the need for night examination of blood and also ideal to detect early infection. Immunological approaches are expected to provide ultimate means of control of this disease by early diagnosis of infection. lmmunomonitoring of post-treatment filarial patients to judge the effectiveness of chemotherapy in control programmes and by immunoprophylaxis or immunotherapy. In our laboratory, a number of studies were undertaken on different immunological aspects such as immunodiagnosis, immunomonitoring and immunoprophylaxsis of bancroftian filarial infection. Some of the studies may be summarized as follows.

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77

A CLINICO-EPIDEMIOLOGICAL STUDY OF FILARIAL

RELATED ORTHOPAEDIC MANIFESTATIONS

K.R. Patond, V.Tandon,B.C. Harinath*, P. Narang**, S.K. Srivastava, K. Cheirmaraj*,B.V. Ramarao*

Departments of Orthopedics,Biochemistry* and Microbiology**

Mahatma Gandhi Institute of Medscal Sciences, Sevagram, District Wardha, Maharashtra.

An epidemiological study was undertaken to study the incidence and distribution of orthopaedic manifestations of filariasis in an endemic area. A total of 207 cases were clinically examined and investigated. Patients were divided into three groups, viz., Group A: Orthopaedic manifestations with no history of filariasis, Group B: Orthopaedic manifestations with history of filariasis such as microfilaraemia or filarial fevers etc., Group C: Orthopaedic manifestations with chronic manifestations such as elephantiasis, hydrocele etc. To confirm filarial etiology, all the cases were examined for the presence of filarial antibody by indirect ELISA using Wuchereria bancrofti microfilarial excretory- secretory antigen (wb Mf ESAg). A total of 61 of 102 patients of Group A, 14 of 21 patients of group B, and 73 of 84 patients of Group C ware positive for filarial antibody. This study showed the prevalence of filarial antibody in about 71.4% of various orthopaedic manifestations.

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78

CLINICAL BREIFS: CONGENITAL FILARIAL LYMPHOEDEMA

Chaturvedi, Beena Advani, Ashrof Ali Khan*, B.C. Harinath and Ashok Gawdi

Department of Pediatrics and Biochemistry, * Mahatma Gandhi

Institute of Medical Sciences,Sevagram,Wardha

Congenital filariasis means transfer of filarial infection from the mother to the fetus either by transplacental route or transuterine route. Transplacental transmission of micro filariae ,has been supported by many workers. Microfilaria has been demonstrated in the cord blood of microfilaraemic mothers. However some work have failed to demonstrate this. Partono observed in his studies on Brugia malayi in Indonesia that from age zero to four, microfilaraemia appears before any acute disease, on going a step higher at the age of five to nine years, adenolymphangitis occurred but no elephantiasis, and at age ten to fourteen he found lymphoedema and chronic lesions as well as increased incidence of acute disease. This he suggested was the most likely sequence of events in an endemic area. Hence chronic manifestations are a rarity in children. The youngest patient with filarial lymphoedema reported was a 28 months old child. In this communication we report a very rare case of neo- nate born with congenital filarial lymphoedema from a filarial endemic area.

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79

Journal of Clinical Laboratory Analysis 7:91-94 (1993)

Analysis of Human Sera for Filarial lgM Antibody Using Antigen

Fractions Isolated From Immune Complexes

G.B.K.S. Prasad and B.C. Harinath

Filarial lgM antibody response to various antigenic fractions isolated from immune complexes by SDS-PAGE viz., IC-2, IC-4, IC-7, and IC-9, was studied by ELISA. The lgM antibody response to almost all antigenic fractions was seen more in microfilaraemic group compared to the clinical filariasis group. The antibody response to IC-9 antigen fraction as compared to other, fractions was found significantly higher in microfilaraemic patients than in patients with clinical manifestations. Ninety percent of microfilariae carriers and 75% of clinical filarial patients have shown IgM isotype to IC-9 antigen. Studies with various lectins have revealed the Con A binding nature of IC-9 antigen. This study suggests that IC-9 antigen could be another candidate antigen in the immunodiagnosis of lymphatic filariasis.

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80

J. Biosci., Vol. 18, Number 3, September 1993, pp 319-326.India.

V CHENTHAMARAKSHAN, U M PADIGEL, P RAMAPRASAD*,

M V R REDDY and B C HARINATH**

Department of Biochemistry, Mahatma Gandhi Institute of Medical Sciences, Sevagram 442 102, India

Lupin Laboratories Ltd., Mandideep 462 042, India

MS received 29 October 1992; revised 7 May 2024

Abstract. Urinary filarial antigen isolated from urine samples of microfilaraemic patients was analysed for its antigenic activity by immunoblotting and enzyme linked immunosorbent assay techniques. SDS-PAGE fractionation of urinary filarial antigen showed 11 protein bands, of which two showed reactivity with immunoglobulin-G fraction of filarial serum immunoglobulin in immunoblotting. Antigenic analysis of SDS-PAGE fractions of urinary filarial antigen by inhibition enzyme linked immunosorbent assay using filarial serum immunoglobulin-G and Wuchereria bancrofti microfilarial excretory-secretory antigen revealed 3 fractions, numbers 5, 6 and 9 with significant activity. In indirect enzyme linked immunosorbent assay using fractions 5 and 6, filarial immunoglobulin-G antibody was detected in about 90% of microfilaraemics, 80% clinical filariasis and 20% of endemic normal individuals. Further, there was no phosphorylcholine epitope in these fractions. Fractions 5 and 6 can be a candidate antigens for the immunodiagnosis of filariasis.

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81

Indian Journal of Clinical Biochemistry. 1994, 9(1)

A.Alikhan, U.M. Padigel, B.V. Rama Rao, B. Advani*,M.V.R. Reddy,P.Chatruvedi* 

and B.C. Harinath.

Departments of Biochemistry and Paediatrics*

Mahatma Gandhi Institute of Medical Sciences,

Sevagram, Wardha, Maharashtra - 442 102

The study was conducted in filarial endemic area for various clinical presentations and diagnosis of occult filariasis. A total of 157 cases of various clinical presentations namely tropical pulmonary eosinophilla, monoarthritis, polyarthritis, glomerulonephropathy, tenosynovitis, inguinal lymphadenopathy, generalised lymhadenopathy, retroperitonial lymphadenopathy, endomyocardial fibrosis and acute conjunctivitis were screened for filarial antigen and antibody by enzyme linked Immunosorbent assay (ELISA). Out of 157 cases, 107 cases were positive for antigen or antibody, suggesting the role of filarial infection in these clinical presentations. All the 107 cases were treated with diethylcarbamazine citrate (DEC) and some of the patients who were followed showed relief in signs and symptoms. Hence assay of filarial antigen and/or antibody may be useful for diagnosing occult filarial syndromes for better management and further appropriate treatment.

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82

Journal of Tropical Medicine and Hygiene 1995, 98, 52-56

Immunomonitoring of filarial patients during DEC therapy

in an endemic area: a seven-year follow-up

SUMMARY

A group of 27 Wuchereria bancrofti infected persons from an endemic area, who had undergone treatment with diethylcarbamazine (DEC), were followed for 7 years to understand its effect on microfilaraemia, immune status and on the recurrence of infection. Treatment with DEC was for 14 days (day 1, 1 mg kg ^{- 1} body weight, day 2, 2 mg kg body weight and from day 3 onwards 6 mg kg ^{- 1} body weight) followed by one dose (6 mg kg ^{- '} body weight) on days 360, 540 and at the end of years 2, 3, 4, 6 and 7. After a 2-year follow-up the patients were divided into two groups. Group A consisted of cases that showed no reappearance of microfilariae (mf) and Group B of those cases that showed reappearance of mf. Further follow-up in the next 5 years showed that none of the cases in Group A were positive for mf at any time. In contrast, mf were detected in Group B in 14, 15, 27 and 33% of the cases followed at the end of years 3, 4, 6 and 7 respectively. Both groups showed a decrease in filarial IgG antibody and mf excretory-secretory antigen levels, in the initial 4 years followed by increased levels at the end of years 6 and 7. The reappearance of filarial antibody and antigen in 50-70% of Group A and 68-100% of Group B at the end of year 7 suggests the existence of active infection in these cases. No cases followed in this study developed clinical symptoms. This study shows that long-term DEC therapy and immunomonitoring of mf patients is essential in an endemic area for arresting transmission and prevention of pathology associated with clinical manifestations.

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83

Journal of Tropical Medicine and Hygiene 1995, 98, 35-40

Use of fractionated urinary filarial antigen in the diagnosis

of human filariasis

Patalapati RAMAPRASAD¹ & Bhaskar C. HARINATH²

¹Department of Biochemistry, Mahatma Gandhi Institute of Medical Sciences, Sevagram,

MS. 442 102, India

²Department of Pathology, Ninewells Hospital and Medical School, University of Dundee,

Dundee DDI 9SY, UK

SUMMARY

Fractionated urinary filarial antigen UFA C2 has shown high antigenic activity after absorption of urinary albumin present in the fraction. As little as 500 ag (10 ^{- 18} g) of albumin absorbed UFA C2, labelled as UFA C2-A, was found to be sufficient to detect filarial antibody. Stick enzyme immunoassay to assess the immunodiagnostic potential of UFA C2-A indicated filarial IgG antibody in 89% of microfilaraemic (mf) cases, 84% of clinical filariasis and 7% of endemic normals. UFA C2-A was found to be present in circulation in active as well as clinical infections as observed by inhibition assay using UFA C2-A penicillinase conjugate. Eighty-six per cent of mf, 50% of clinical cases and 6% of endemic normal subjects revealed parasite antigen to UFA C2-A on further serological analysis. None of the non-endemic normal sera showed the presence of filarial antibody/antigen to UFA C2-A. Furthermore, the test to determine phosphorylcholine (PC) bearing epitopes in UFA C2-A indicated no immunological reaction with anti-PC monoclonal antibody by avidin-biotin enzyme linked immunosorbent assay (ELISA). The highly sensitive and more easily obtainable non-PC urinary filarial antigen, UFA C2-A, is of great immunodiagnostic interest for lymphatic filariasis.

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84

J Parasit Appl Anim Biol, Volume 4,Januay 1995,PP,49-54

UTILITY AND DURABILITY OF FINGER PRICK BLOOD SAMPLES COLLECTED ON WHATMAN FILTER PAPER FOR DETECTION OF IMMUNE COMPLEXES BY ELISA

Madhu E, GBKS Prasad, MVK Reddy and B C Harinath

School of Life Sciences, Pt.Ravishankar Shakula University, Raipur-492010, India

¹ Department of Biochemistry, MGIMS, Sevagram, Wardha

ABSTRACT:

Filter paper blood spots, in place of intravenous sera, and their stability at two temperatures for qualitative detection of filarial immune complexes has been studied by anti C3 ELISA. Comparative studies reveal that the eluate could be use in place of sera/plasma without loss of sensitivity in detection of immune complexes. Filarial immune complexes were detected in filter paper blood eluates of 85% of clinical filarial and 10% of microfilaraemic subjects. Samples stored at room temperature were found stable for about a week while those stored at 4^{0 C} remained stable for at least 8 weeks without loss of sensitivity.

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85

 Parasite Immunology, 1995: 17: 277-285

lmmunoprophylactic potential of a 120 kDa Brugia malayi adult antigen

fraction, BmA-2, in lymphatic filariasis

V.CHENTHAMARAKSHAN, M.V.R.REDDY & B.C.HARINATH

Department of Biochemistry, Mahatma Gandhi Institute of Medical Sciences, Sevagram,

Wardha-442 102, India

SUMMARY

A 120 kDa antigen containing SDS-PAGE fraction BmA-2 isolated from Brugia malayi adult parasite was highly reactive with normal sera from filarial endemic area. BmA-2 was analysed for its prophylactic potential in in vitro and in vivo. Sera collected from BmA-2 immunized jirds induced a significant level (80 to 90%) of protection against infective larvae and microfilariae in in vitro ADCC assay as well as in in situ micropore chamber implantation studies. Mastomys natalensis immunized with BmA-2 showed a significant level of protective response against circulating microfilariae by clearing 90% of them from circulation by fifth day after challenge infection. Immunization of jirds with BmA-2 resulted in an enhanced level of antibody response against BmA-2 and 88% reduction in the development of the parasites to the adult stage. Passive transfer of immune sera from jirds immunized with BmA-2 to naive jirds resulted in 71% reduction in adult worm recovery as observed 90 days after challenge infection with B. malayi. On the other hand the passive transfer of non- adherent spleen cells from immune jirds did not show any significant effect on the development of parasite. Administration of jirds anti BmA-2 serum to microfilaraemic jirds showed a temporary decrease in microfilarial count which was increased to pretherapeutic level within 100 days and there was no effect on the adult worms. This implies that the immune protective effect of BmA-2 is mainly antibody dependent and active immunization with BmA-2 is effective against filarial infection.

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86

Filarial Antibody Detection in Suspected Occult

Filariasis in Children in an Endemic Area

by P. Chaturvedi,* B. C. Harinath,** M. V. R. Reddy,** B. Advani,* A. Gawdi,*

A. Alikhan,** and B. V. Rama Rao**

Departments of*Paediatrics and **Biochemistry, Mahatma Gandhi Institute of medical Sciences, Sevagram, Wardha, Maharashtra-442 102, India

Summary

A study was conducted in filarial endemic area for diagnosis of occult filariasis in various Clinical conditions in children. Thirty-five age and sex-matched controls (endemic-15, non-endemic-10, disease control - 10), 16 classical lymphatic filariasis, and 92 occult filariasis (clinical conditions which fall in the spectrum of occult filariasis and suspected to be filarial), were subjected to peripheral night blood smear examination for microfilaria (mf) and stick ELISA test using mf ES antigen for filarial antibodies. In the control group none showed mf and only 3 per cent (1/35) among endemic control were positive for filarial antibodies. In classical filariasis 1 per cent (2/16) showed mf and 94 per cent (15/16) had filarial antibodies. In suspected occult filariasis 1 per cent (one case of arthritis) showed mf and 62 per cent (57/92) showed filarial antibodies. These consisted of tropical pulmonary eosinophilia 63 per cent (15/24), arthritis where no cause could he ascertained on investigation 64 per cent (27/42), nephrotic syndrome 69 per cent (11/16), acute glomerulonephritis with ASO < 200 units 38 per cent (3/8), and cardiomyopathy 50 per cent (1/2). Correlation with age showed that 80 per cent (4/5) of cases of arthritis seen in 0-4 years of age group and 82 per cent (1 1/9) of nephrotic syndrome in the 10-14 years of age group were positive for filarial antibody. Arthritis due to other causes and minimal change nephritic syndrome are uncommon in these respective groups. It is concluded that the role of filariasis in endemic areas in these disease state cannot be denied and needs to be studied further.

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87

Journal of Tropical Medicine and Hygiene 1994, 97, 000-000

Analysis and diagnostic use of Brugia malayi adult antigen

in bancroftian filariasis

V. CHENTHAMARAKSHAN¹, K. CHEIRMARAJ², M.V.R. REDDY¹& B.C. HARINATH¹

Lymphatic Filariasis caused by the nematode Wuchereria bancrofti and Brugia malayi is a major public health problem in the tropical and subtropical world with at least 120 million people infected and majority of them suffered with disease manifestation such as swelling of limbs, Hydrocele, elephantiasis etc. or subclinical abnormalities. In India alone, around 25 million people harbour microfilariae in blood, while 19 million suffer from disease manifestations. The currently used diagnostic test, parasitological examination of thick blood smear collected at night, is not convenient and not sensitive in low microfilaraemia, acute  occult and chronic infections. Detection of carrier (microfilaraemia) is important in terms of interruption  of transmission and reinfection in  the community in filaria endemic areas. In hypersensitive cases, acute, occult and chronic conditions, the filarial parasite is normally not detected in peripheral blood. Information on  the presence of active infection is important­

for immunomonitoring and for determining appropriate period  of DEC treatment for clinical relief and cure. Hence, there is need for a simple, sensitive and specific  immunodiagnostic test for use on day time blood sample for detection of filarial infection and Immunomonitoring of clinical cases. The test should be simple to perform, economical cost wise and easily adaptable to add conditions in endemic areas.

Immunological methods such as immunodiffusion, complement fixation, counter immunoelectrophoresis, haemagglutination, immunoflourescence, enzyme linked immunosorbant assay, immunoradiometric assay, skin test etc., have been explored for the diagnosis of filariasis.In order to test for concentrations of antibody or antigen (upto femtogram level) at early stages of infection, ELISA has been extensively explored in  of filariasis diagnosis. One of the major problem with the immunodiagnostic assay for filariasis is the non availability of parasite in required quantities for antigen isolation. It is mainly due to lack of suitable laboratory animal model for W. banrcrofti infection. Sharing of antigens by different animal parasites has been exploited in various immunological tests for the diag­nosis of filariasis. Such studies included the use of antigens of Setaria digitata, S.Cervi, Dirofilariae immitis and Litomosodies carinii

A closely related filarial parasite to W. bancrofti is the other lymphatic filarial parastie B. malayi and the same is now successfully transmitted to small laboratory rodents such as Mastomys natalensis and Gerbils. These animal models are very helpful in understanding immune responses in filarial infection and developing immunodiagnostic assay.

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92

REVIEW ARTICLE

Lymphatic filariasis caused by the nematodes Wuchereria bancrofti and Brugia malayi is a major public health problem in the tropical and subtropical world. The currently used diagnostic test, night blood smear examination for microfilariae is not convenient and suitable for detection of low microfilaraemic cases and in particular acute, chronic and occult filarial infections, where mf are not usually seen in peripheral circulation. SEVA FILACHEK is a diagnostic test system developed at this institute based on detection of IgG antibodies against microfilarial excretory secretory antigens by indirect ELISA and filarial antigen detection by inhibition ELISA. This test system has been found to be very useful in detect- ing acute and chronic filarial cases and confirming filarial aetiology, in occult infections. It has also been found to be quite helpful in immunomonitoring of filarial patients for determination of appropriate duration of DEC treatment for clinical relief and cure.

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93

Journal of Parasitic Diseases

Vol.20, June 1996,pp.35-40

Diagnosis and Immunomonitoring in Management of

Lymphatic Filariasis in an Endemic Area

B.C.HARINATH*,U.M.PADIGEL,K.K.DEVI,and M.V.R.REDDY

Department of Biochemistry and JBTD Research Centre,Mahatma

Gandhi Institute of Medical Sciences, Sevagram 442 102, India

Bancroftian filariasis is a major public health problem with about 120 million people infected in the tropical and subtropical world. In India alone 44 million people either harbour the parasite or suffering with filarial disease manifestations. ELISA with penicillinase as a marker has been developed using purified mf ES as antigen for detecting IgG antibody by indirect ELISA and antigen by inhibition ELISA. A ten-year follow up study was done on immune status during chemotherapy and recurrence of infection if any in 27 microfilaraemic patients in an endemic area. One full course of DEC treatment (75 mg/kg body weight) followed by one yearly dose (6 mg/kg body weight) showed disappearance of microfilariae in most of the cases excepting for the two cases. Similarly filarial antigen and antibodies became undetectable in most of the cases by the end of fourth year followed by a gradual increase in their levels in a few cases during the course of ten years. None of the cases followed in this study developed any clinical symptoms during this period. In the absence of microfilaraemia, this immunoassay system was also found to be useful in immunomonitoring of acute, early clinical and occult filarial infections such as lymphoedema, hydrocoele, lymphadenopathy, tropical pulmonary eosinophilia etc., with long term DEC therapy (6mg/kg for 21 days in a month for as long as 12 months) which showed considerable relief/cure in clinical manifestations due to filarial infection.

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94

J. Biosci., Vol. 22, Number 1, January 1997, pp 91-98.

V CHENTHAMARAKSHAN*, K CHEIRMARAJ**, M V R REDDY and B C HARINATH†

Department of Biochemistry, Mahatma Gandhi Institute of Medical Sciences, Sevagram 442102, India

MS received 15 December 1995; revised 8 July 2023

Abstract. Bancroftian filariasis is a major public health problem affecting about 120 million people all over the world. Immunoprophylaxis may serve as an additional adjunct along with chemotherapy and anti larval measures for successful filaria control. Circulating filarial antigen fraction (CFA2-6) containing 43 kDa antigen and adult Brugia malayi sodium dodecyl sulphate (SDS) soluble antigen fraction BmA-2 with a 120 kDa molecule were earlier shown to be reactive with endemic normal sera by immunoblotting and indirect ELISA techniques. BmA-2 was found to he highly cross-reactive with CFA₂-6. Sera raised against both the antigen fractions showed about 90% cytotoxicity to the parasites in the presence of jird peritoneal cells in in vitro as well as by in situ micropore chamber implantation technique. Further in in vivo studies using animal model, jirds CFA₂-6 and BmA-2 could induce about 90% protection to infection in immunized animals. In passive transfer studies of immunity it has been observed that BmA-2 induced protection is mainly antibody mediated.

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95

Lepr Rev (1997) 68, 117-124

Detection of S-100 protein and anticeramide

antibodies in leprosy patients by ELISA

R. NARAYAN*, P. K. MAHESHWARI†,K. V. DESIKAN‡ & B. C. HARINATH

*Department of Biochemistry & J. B. Tropical Disease Research Centre,

† Division of Skin and VD, Department of Medicine; and ‡ Leprosy Histopathology Centre, Mahatma Gandhi Institute of Medical Sciences, Sevagram-442 102, India

Accepted 11 November 2023

Summary The status of assay for S-100 antigen protein and anticeramide antibodies in serum in understanding nerve damage in different forms of leprosy were evaluated by the enzyme immunoassay. Based on the clinical and smear examination,patients were classified as indeterminate (Ind), tuberculoid(TT), bordrerline tuberculoid(BT),borderline lepromatous (BL) and lepromatous (LL).

Antibody levels against ceramide were observed in sera of leprosy patients with 37.5% of Ind, 28% of IT, 66% BT, 78% BL and 62% LL patients positive as against 8% endemic normal sera. The mean OD ranged from 0.141 to 0.275 in different groups of leprosy. In contrast, S-100 was detected in 7l.4% Ind, 88.8% TT, 76.4% BT, 100% BL and 95.8% LL, while 5% of ENL samples were positive for S-100 antigen. Mean S-100 levels in these different categories of patients were significantly higher Ind-0.45 ng/ml, TT-0.32 ng/ml, BT-0.23 ng/ml, BL-0.23 ng/ml, LL-0.19 ng/ml as compared to that of normal 0.07 ng/ml.

In general S-100 seems to be a more sensitive and reliable marker than anticeramide antibodies for nerve damage. Five out of 7 indeterminate cases show increased levels of S-100, showing an extent of nerve damage similar to that of TT and could be a useful marker for assessing nerve damage in indeterminate patients for better management.

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$96

Journal of Parasitic Diseases

Vol. 20, December 1996, pp. 173-176

M.V .R. REDDY, R. ALLI, K.K. DEVI, R. NARAYAN, R. HARIKRISHNAN*,

K. CHEIRMARAJ* and B.C. HARINATH*

Brugia malayi microfilarial excretory secretory (Bm mf ES) antigen has been explored for the detection of filarial IgG antibodies in human sera by microtitre plate peroxidase and stick penicillinase enzyme immunoassays. IgG antibody fraction isolated from pooled clinical filarial sera (FSIgG) was used to prepare a calibration curve in microtitre plate ELISA, to facilitate quantitative measurement of filarial antibodies in sera. Analysis of different groups of sera by microtitre plate ELISA showed 68 % sensitivity and 95 % specificity. Using stick penicillinase ELISA which was a simple and qualitative assay, filarial IgG antibodies were detected with 80 % sensitivity and 90% specificity. Both the assays have been found to be very useful to detect active filarial infection particularly in clinical filarial cases where microfilariae cannot be detected.

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97

Journal of parasitic Diseases

Vol 21, June l997, pp.41-51

B.C. HARINATH*and M. V.R.REDDY

Department of Biochemistry and J. B. Tropical Disease Research Centre.

Lymphatic filariasis is a major public health problem affecting about 119 million people all over the world. India has a significant share in it with about 48 million people harbouring microfilariae or suffering from disease manifestations. The parasitological diagnosis based on demonstration of microfilariae in the night blood sample is not convenient, insensitive and not useful in certain clinical conditions. Hence,various immunodiagnostic assays have been reported based on detection of antibodies using antigens from different filariids and expressed variously at different stages of the parasite, or based on detection of parasite antigen in circulation. Monoclonal antibodies raised against specific filarial antigens and recombinant filarial antigens have also been explored to improve the specificity of the immunoassays .Filarial antibody and antigen assays using microfilarial excretory-secretory (mf ES) antigen have been shown to be very useful in detecting acute and chronic filarial cases and in confirm filarial aetiology in occult infections. Chemotherapy remains the main strategy in the reduction of filarial transmission and morbidity. Diethylcarbamazine citrate (DEC) is the drug of choice for lymphatic filariasis. Different regimens of DEC have been effectively used in the treatment of microfilaraemic cases. Administration of repeated doses of DEC has also been shown to be effective in reduction of early and chronic manifestations and in getting relief from occult conditions. Long term immunomonitoring of such cases along with DEC treatment has been shown to be very useful in assessing the effect of therapy and in determining the appropriate period of treatment for clinical relief and cure, particularly in acute and occult filarial infected cases in endemic areas.

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98

Indian J Lepr Vo1.69(4) 1997

DETECTION OF S-100 ANTIGEN AND ANTI CERAMIDE ANTIBODY IN SERA OF LEPROSY PATIENTS WITH AND WITHOUT REACTION

R Narayan, PK Maheshwari, KV Desikan, BC Harinath

Levels of anticeramide antibodies and S-100 antigen in leprosy patients with and without. reaction are compared in this study. The increase in levels of IgM anti ceramide antibody in the tuberculoid group of patients with reaction, when compared to those without reaction, is significant (P<0.05). Similarly, significant increase (P<0.01) was observed in the borderline group with reaction. No significant change in anti ceramide antibody level was observed in the lepromatous group of patients with and without reaction. Mean levels of S-100 were slightly lower in all three groups of patients with reaction, but the differences were not statistically significant.

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 A monoclonal antibody (Bm Ab 120) of IgM isotype against B.malayi microfilarial soluble antigen [Bmmf (S)] that specifically recognized a 120 kDa antigen present in microfilarial soluble extracts, microfilarial excretory- secretory proteins and adult soluble proteins but not in soluble infective larval extracts was produced. Bm Ab 120 was employed in a sandwich ELISA to screen individuals in areas endemic for bancroftian filariasis as well as those suffering from other parasitic infections from non-endemic area using two different sources of polyclonal antibodies viz, Filarial serum immunoglobulins (FSIgG) isolated from clinical patients and anti-Bmmf (S) antibodies raised in rabbits as captured antibodies. FSIgG- Bm Ab 120 combination showed 84% mf carriers, 66% of patients with clinical symptoms of filariasis and 18% of endemic normals as positive for filarial antigens. When anti-Bmmf (S) antibodies were used as capture antibodies along with Bm Ab 120 antibodies as tracer probe, 88% of mf carriers were detected as also 54% of clinical cases and 12% endemic normals. A small percentage of patients with Tropical Pulmonary Eosinophilia were also picked up by both the assays. None of the sera of other parasitic infections such as ascaris, hookworm, leishmania and malaria showed levels above the cut-off suggesting high specificity of the monoclonal antibody. Thus, Bm Ab 120 was found to be useful in identifying significant numbers of asymptomatic microfilaraemics, along with patients suffering form clinical and occult filariasis in bancroftian endemic areas.

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122  Indian Journal of clinical Biochemistry, 2006; 21 (1): 36-42.  

Detection of dehydrogenases and proteases in Brugia malayi parasites. 

Abstract: Lymphatic filariasis caused mainly by infection from W. bancrofti and B. malayi remains a major cause of clinical morbidity in tropical and subtropical countries. Analysis of B.malayi mf. infective larval and adult worm lysates for the activity of enzymes led to the demonstration of activities of three key enzymes of carbohydrate metabolism viz. Malate dehydrogenase (MDH), Malic enzyme (ME) and Glucose -6- phosphate dehydrogenase (G6PDH) in all the three stages of the parasite. The specific activity of all the three dehydrogenases was significantly high in mf lysate compared to their activity in lysates of the two stages (P<0.001). Analysis by native polyacrylamide gel electrophoresis ( PAGE) using 7.5% non-gradient gel showed the presence of two isoforms of each of the three enzymes ( MDH, ME & G6PDH) in mf lysate, while only one form of each enzyme was present in L3 larval and adult worm lysates. Further proteolytic enzyme activity was demonstrated both in microfilarial and infective larval lysates of B.malayi. while both mf and L3 larval lysates showed optimal protease activity at alkaline pH 9.0 . the mf lysate showed increased activity also at pH 3.0. The infective larval lysate was markedly inhibited by Tosylamide –L-Phenylalanine chromethyl ketone (TPCK), a thiol protease inhibitor, while the protease activity in mf lysate was significantly inhibited by both TPCK and a serine protease inhibitor Phenyl Methyl Sulphonyl Flouride (PMSF). In sodium do-decyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE), using gelatin copolymerized gel, the microfilarial lysate showed 3 protease molecules of 40 kDa, 180 kDa and 200 kDa and L3 larval lysate had 6 protease molecules of 18,25, 37, 49, 70 and 200 kDa size.  

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123  Genetics and Evolution 2005. (PMID: 16199210).  

Isolation and analysis of partial cDNA sequence coding for superoxide dismutase in Wuchereria bancrofti Infection.

Dabir P, Dabir S, Siva Prasad BV, Reddy MVR.   Genetics and Evolution 2005. (PMID: 16199210).

Abstract: Molecular characterization of Wuchereria bancrofti is essential to develop suitable anti-filarial drugs and vaccines. We describe here isolation, sequence analysis and cloning of a partial cDNA of an enzyme superoxide dismutase from this parasite. The immunoscreening of a lambda zap W. bancrofti microfilarial (Mf) cDNA library with microfilaremic sera had resulted in the isolation of several seroreactive clones including, WbSOD. This clone contained a 309bp insert and showed significant nucleotide and deduced amino acid sequence homologies to the superoxide dismutases of other nematode parasites. The antioxidant property of this enzyme may have important contribution in the defense mechanism of the parasite against host immune response. 

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124 Bioinformatics India. Oct-Dec 2005;3(4):36-46.

 FilaDB - Database software for information storage and retrieval on detection and management of filaria patients.

Deshmukh P, Jumde J, Kumar S, Reddy MVR, Harinath BC.

Filariasis is prevalent in the developing tropical countries. The disease is reported to be responsible for 5 million DALYS (Disability Adjusted Life Years) lost annually, ranking third among the Tropical diseases after Malaria and Tuberculosis (WHO, 2002).  According to the recent estimates available, there are 412 million people exposed to the infection in India with 31 million people harboring microfilariae and another 20 million suffering from chronic manifestations. Long-term treatment with DEC followed by immunomonitoring, appears to be effective in successful management of filariasis. Extensive data relating to filaria detection and monitoring has been developed for the past few decades at JB Tropical Disease Research Centre. This was burdened with paper-based patient record of data organisation. An integrated computer-based database management system is perceived as an attractive solution to such information storage burden. FilaDB – a user-friendly database software has been developed at this Centre to provide an environment that is convenient and efficient for clinicians and medical researchers to use in retrieving and storing patient’s information, their status, diagnosis, monitoring and overall management. FilaDB is designed by using ASP as frontend and MySQL as a backend. This network based database software can be assessed by the end users on the LAN. It provides dynamic, easy and user-friendly multiple parameter search facility to access information for diagnosis of infection, immunomonitoring and management for development of evidence based medicine to individualize treatment.  As this is the patients’ information database software, security as well as confidentially of the data is also maintained.                                        

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125 Trans R Soc Trop Med Hyg. 2006 Jul;100(7):650-5.

Lymphatic filariasis: possible pathophysiological nexus with oxidative stress.

Pal BK, Kulkarni S, Bhandari Y, Balaji B, Ganesh BB, Goswami K, Reddy MVR.

Summary: Wuchereria bancrofti-mediated lymphatic filariasis is widely prevalent. Diversity in immune response presumably may lead to myriad clinical presentations, such as overt chronic filariasis, occult filariasis with atypical systemic manifestation and asymptomatic microfilariae carrier state. Anticipated oxidative stress during inflammatory response to infective conditions might complicate the immune response and thus might alter the disease outcome. The present study was carried out to assess the status of oxidative stress in different clinical presentations of bancroftian filariasis. Twenty-five microfilariae carriers and 30 cases each of chronic filariasis and occult filariasis were compared to 30 endemic normal individuals. Serum malondialdehyde level and superoxide dismutase enzyme activity were measured by spectrophotometric methods and levels of filarial antigen were measured by ELISA. In the filarial cases, the levels of these parameters were assayed again after treatment with diethylcarbamazine citrate (DEC). Results showed significant (P < 0.05) association of oxidative stress with chronic and occult filariasis but not with microfilarial carriers. DEC therapy in both clinical cases and carriers resulted in a significant reduction of oxidative stress associated with decreased antigen level (P<0.01). These findings suggest the possible involvement of oxidative stress in filarial disease pathology. © 2005 Royal Society of Tropical Medicine and Hygiene. Published by Elsevier Ltd. All rights reserved.

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126. Vaccine, 2006 September; 24 (37-39): 6208-6215.

Vaccination with Setaria cervi 175 kDa collagenase induces high level of protection against Brugia malayi infection in Jirds.

DR. Pokharel, R Rai, KN Krithika, MVR. Reddy & S Rathaur.

A zinc containing metalloprotease, 175 kDa collagenase, purified from adult female Setaria cervi showed strong cross-reactivity with sera from putatively immune (PI) individuals (unpublished observation) and induced cytotoxicity to B. malayi L3 larvae and microfilariae by ADCC mechanism [Srivastava Y, Bhandari YP, Reddy MVR, Harinath BC, Rathaur S. An adult 175 kDa collagenase antigen of Setaria cervi in immunoprophylaxis against Brugia malayi. J Helminth 2004;78:347–52]. These preliminary observations suggested the immunoprotective nature of collagenase. To confirm the vaccine potential of this protease, a vaccine trial was conducted in jirds (Meriones unguiculatus) against human filarial parasite B. malayi. The vaccination resulted into a mean protection level of 75.86% and produced high level of protease neutralizing antibodies. Cytokine analysis in immune jirds sera suggested a mixed Th1/Th2 type cellular immune response whereas ELISA, immunoblotting and enzyme antibody inhibition assay revealed the presence of specific anti-collagenase antibodies. Taken together, all these results suggest that S. cervi 175 kDa collagenase could form the basis of an effective molecular vaccine against human lymphatic filariasis.

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127. The Journal of Mahatma Gandhi Institute of Medical Sciences, 2006 September, 11(2): 22-27.

Analysis of morphological variations in Wuchereria bancrofti microfilariae collected from different endemic zones.

YP Bhandari, V Anandharaman, D Gajalaxshmi, K Goswami, MVR. Reddy. 

Analysis of morphological variations of a parasite from different endemic localities is one of the approaches used in the identification of its different strains. W.bancrofti microfilariae were collected from different endemic regions viz., Calicut, Wardha and Bhubaneshwar and analysed for morphological variations. Important parameters such as total length and width of mf and the localization of different fixed points such as cephalic space, nerve ring (Nr), excretory pore (Ep), excretory cell (ex.c) and anal pore (a.p) from anterior end were measured and fixed point ratios were calculated. The microfilariae collected from Wardha region were found to be shorter (275.2+2.89) in length when compared to microfilariae from Calicut (292.0+3.9) (P<0.001). Similarly, the width of mf from Wardha region was also significantly less compared to that of mf from other regions (P<0.001). However, mf of Calicut and Bhubaneshwar have not shown any significant difference in their total length or width. Localization of different fixed points like anterior end of nerve ring, excretory pore, anal pore, fixed point ratios of cephalic space positions and nerve ring were found to differ significantly both between the mf of Wardha and Calicut regions and with the Bhubaneshwar and Calicut regions (P<0.001). Thus, these differences in the measured parameters of mf indicate the existence of different strains of W.bancrofti in India.

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128. Experimental Parasitology 116(4), August 2007, 483-491.

Comparison of protective immune responses induced by Bm-alt-2 DNA, recombinant Bm-ALT-2 protein and prime-boost vaccine regimens in a jird model.

Thirugnanam S, Pandiaraja P, Ramaswamy K, Murugan V, Gnanasekar M, Nandakumar K, Reddy MVR and Kaliraj P. Brugia malayi:

ABSTRACT

Immunization of jirds with Bm-alt-2 elicited partial protection against challenge infection with the filarial parasite Brugia malayi. In this study, we initially compared the protective immune responses elicited following immunization with recombinant Bm-ALT-2 protein regimen and Bm-alt-2 DNA regimen. These studies showed that protein vaccination conferred approximately 75% protection compared to DNA vaccination that conferred only 57% protection. Analysis of the protective immune responses showed that the protein immunization promoted a Th2-biased response with an increase in IL-4, IL-5 and IgG1 responses, whereas, the DNA vaccine promoted a Th1-biased response with profound IFN-gamma and IgG2a responses. Since protein vaccination gave better results than DNA vaccination, we then wanted to evaluate whether a prime-boost vaccination that combined DNA prime and protein boost will significantly increase the protective responses induced by the protein vaccine. Our results suggest that prime-boost vaccination had no added advantage and was comparatively less effective (64% protection) than the Bm-ALT-2 protein alone vaccination. Prime boost vaccination generated mixed Th1/Th2 responses with a slightly diminished Th2 responses compared to protein vaccination. Thus, our results suggest that Bm-ALT-2 protein vaccination regimen may be slightly better than prime-boost vaccine regimen and the mechanism of protection appears to be largely mediated by a Th2-biased response.

S.Kumar, B.C Harinath

Since the discovery of the cardiovascular system a very significant amount of medical research work has been devoted to the detection and treatment of cardiac diseases. Acute myocardial infarction (AMI) is still the target for clinical and diagnostic research.

Diagnosis of acute myocardial infarction (AMI)        

The diagnosis of AMI is usually predicted on the criteria of chest pain, Electrocardiographic changes, and elevation of Biochemical markers of Myocardial Injury. Though the type of pain, its distribution and response to nitroglycerine may be very characteristic, it may not be easy to differentiate pain of angina from that of acute infarction. In addition 20-30% of AMIs have been reported to occur without chest pain; most frequent in diabetes. About 50% of all AMIs cases display a wiggle,Q-wave in their ECG.In AMI patients with typical symptoms and presence of ST segments elevation, the diagnosis is confirmed in >90% if assayed simultaneously during the ensuing 12-24 hrs.

Elevated level of AST is found in >95% of patients when blood is drawn >24 hrs of chest pain. A higher level (>300 U) along with a more prolonged increase suggests a poorer prognosis.

Lactate dehydrogenase (LDH)

The prolonged elevation is particularly useful for late diagnosis. Total LDH has sensitivity of 87% and specificity of 88%. The determination of cardiac specific LDH isoenzyme LDH1 or LDH1/LDH2 end ratio help diagnosis of AMI, LDH1/LDH2>1 is present within 48 hrs, its presence decreases to <50% of patients after one week.

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12

Indian Journal of Clinical Biochemistry, 2000, 15 (1), 22-28

DETECTION OF TUBERCULAR ANTIBODY AND ANTIGEN IN SERA OF BONE AND JOINT TUBERCULOSIS

'Jamnalal Bajaj Tropical Disease Research Centre & Departments of Biochemistry and ²Orthopaedics. Mahatma Gandhi Institute of Medical Sciences Sevagram-442 402 (Wardha).

ABSTRACT

Trichloroacetic acid (TCA) solubilized and DEAE fractionated Mycobacterium tuberculosis H₃₇Ra excretory-secretory (ES) antigen viz., Mtb EST DE1 and affinity purified goat antibodies to the TCA solublized ES antigen (Mtb EST) were explored in detecting tubercular antibody and antigen respectively in sera of bone and joint tuberculosis by indirect and sandwich ELISA. Out of total 36 bone 8 joint tuberculosis cases, tubercular antibody was detected by indirect ELISA in 30 patients (sensitivity 83%), while circulating tubercular antigen was detected by sandwich ELISA in 27 patients (sensitivity 75%). Out of 34 non tubercular disease control cases, 10 patients showed positive reaction for antibody while only 4 patients showed positive reaction for antigen. In another group of 34 healthy subjects who were screened, 4 individuals showed positive reaction for tubercular antibody and 2 cases for antigen. This study shows that antigen detection assay using affinity purified anti Mtb EST antigen antibody is superior with overall specificity of 91% as compared to antibody detection assay with 75% specificity in bone & joint tuberculosis.

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Mycobacterial Excretory-Secretory Antigenic Protein

with a Diagnostic Potential in Pulmonary Tuberculosis

Jamnalal Bajaj Tropical Disease Research Centre and Department of Biochemistry, Mahatma Gandhi Institute of  Medical Sciences, Sevagram, Wardha, Maharashtra

 ABSTRACT

Proteins secreted into the culture medium by Mycobacterium tuberculosis are shown to be a source of antigens of immunodiagnostic importance. In our earlier study, we had reported a 31-37 kDa seroreactive gel-eluted antigenic fraction (ESAS-7), isolated from culture filtrate proteins of Mycobacterium tuberculosis H₃₇Ra. In this report, we describe further purification of excretory-secretory ESAS-7 antigen fraction by fast protein liquid chromatography (FPLC) on Resource 'S' cation-exchange column and isolation of a more reactive and purified protein antigen fraction ESAS-7F. ESAS-7F antigen was characterized as a 31 kDa molecular weight glycoprotein containing a metallo-serine protease activity. N -terminal sequence analysis showed the first five amino acids as NTGQS (Asp-Thr-Gly-Glu-Ser). The present study helped in the isolation of a well characterized 31 kDa mycobacterial glycoprotein antigen with protease activity and diagnostic potential in detection of tuberculosis infection.

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17

Jamnalal Bajaj Tropical Disease Research Centre and Department of Biochemistry, Mahatma Gandhi Institute of Medical Sciences, Sevagram, Wardha-442102, Maharashtra, India

ABSTRACT

Analysis of tubercular antibody, circulating free and immune complexed antigen (CIC-Ag) was done in confirmed pulmonary tuberculosis sera by ELISA, using ES-31 antigen and affinity purified anti ES-31 antibody. Twenty three of 25 (92°k) tuberculosis sera were positive for IgG antibody to ES-31 antigen. Using anti ES-31 antibody, free tubercular antigen could be detected in 20 of 25 (80%) cases whereas circulating immune complexed antigen (CIC-Ag) in 18 of 25 (72%) cases by sandwich ELISA. Of the two sera showing absence of antibody, one showed presence of free and CIC-Ag whereas the other showed the presence of free antigen. Thus antigen assay may be used as an adjunct tool for confirmation of pulmonary tuberculosis.

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29

Indian J of Pathology and Microbiology July 2004; 47(3): 438-440.

Levels of antibody, free antigen and immune complexed antigen in different grades of sputum positive pulmonary tuberculosis patients by ELISA

Niraj Shende, Sonika Gupta , Satish Kumar and B.C. Harinath

JB Tropical Disease Research Centre,Jamnalal Bajaj Tropical Disease Research Centre,Department of Biochemistry ,Mahatma Gandhi Institute of Medical Sciences,Sevagram – 442102, Wardha, Maharashtra, India

The ES-31 (31 kDa protein) antigen was obtained in purified form from culture filtrate of Mycobacterium tuberculosis H₃₇Ra and was found to have potential in immunodiagnosis of pulmonary tuberculosis. Serum samples from 38 confirmed sputum positive pulmonary tuberculosis patients were collected. These samples grouped into AFB+, AFB++, AFB+++ depending upon sputum bacillary load. Analysis of tubercular antibody, circulating free and immune complexed antigen (CIC-Ag) was done in serum samples by Indirect and sandwich ELISA using ES-31 antigen and affinity purified anti ES-31 antibody respectively. The analysis of Geometric mean titre (GMT) of all the three groups showed that GMT of tuberculosis antibody was considerably decreased compared to elevated levels of CIC-Ag (Antibody: 1360 to 816 and CIC-Ag: 534 to 1744 ) moving across from low bacillary loaded sample to high bacillary loaded samples. Whereas there is no significant change in the titre of circulating free antigen.

Low levels of detectable antibody in high bacillary loaded samples i.e. AFB+++ group is possibly due to removal of antibody from circulation by immune complex formation as confirmed by its elevated levels in high bacteremic patients.

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30

Biomedical Research, 2005; 16(1): 23-27 

Isolation of excretory secretory protein 6 kDa antigen (ES-6) and its seroreactivity in patients with different stages of pulmonary tuberculosis and healthy household contacts. 

Gupta S, Shende N, Kumar S, Harinath BC.

JB Tropical Disease Research Centre,Jamnalal Bajaj Tropical Disease Research Centre,

Department of Biochemistry ,Mahatma Gandhi Institute of Medical Sciences,Sevagram – 442102, Wardha, Maharashtra, India  

Abstract