ELISA For Immunodiagnosis and Immunomonitoring

of Tropical Diseases

B. C. Harinath  &  M. V. R. Reddy  

            Enzyme Linked Immuno Sorbant Assay (ELISA) is a very sensitive and safe technique used in the detection of antigens and antibodies was introduced by Engvall and Perlmann (1). The sensitivity of this technique is comparable with that of Radio immunoassay with an added advantage of safer use of non radioisotopic reagents and longer shelf life of the same. It also eliminates the requirement of sophisticated isotope counters. ELISA can be done in smaller laboratories and is adaptable to field conditions as well.

            The application of ELISA has been well reviewed (2,3). ELISA plays an important role in the laboratory and in the field. The various components involved in ELISA are a solid phase to which specific antigen/antibody is coated, an antigen or antibody enzyme conjugate as probe as the case may be and enzyme substrate. Solid supports used are polystyrene or PVC microtitre plates, tubes or beads. Nitro cellulose paper or Cellulose Acetate Membrane are also used as solid phases. Enzymes used for conjugation to anti immunoglobulins include peroxidase, b-galactosidase, alkaline phosphatase, penicillinase, urease, glucose oxidase. No enzyme fulfills all the criteria for an ideal label in EIA. The influence of the solid phase on the enzyme should be minimal. Conjugation to anti-immunoglobulin should be easy and conjugates should be active and stable. These are the reasons for frequent selection of peroxidase in commercial diagnostics. In our laboratory we have been successful in using penicillinase as marker in EIA for tropical diseases. Penicillinase has also been successfully used as an enzyme marker in hormone assays (4,5). Penicillinase (b-lactamase,    EC has high turnover number of 1,60,000 and has been found to be more sensitive than peroxidase, alkaline phosphatase or b- galactosidase (6). 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 iodometrically. The assay involves decolourisation of blue coloured starch-iodine-penicillin substrate. The result is assessed visually by naked eye. The reaction may be stated as follows.  

  The method depends, like other iodometric procedures, on the reduction of iodine by  penicilloic acid but not by penicillin. Assay procedure was developed for the purpose of measuring the rate of hydrolysis of penicillin to penicilloic acid by penicillinase at substrate concentrations as low as mM. A 1000 fold increase in sensitivity was obtained over existing techniques for penicillinase assay (7). Penicillinase has been used in enzyme immunoassays for detection of microbial antigens (8).


            In our laboratory penicillinase ELISA system has been explored in several ways such as Indirect, Direct, Sandwich, Competitive and Inhibition ELISA for, detection of antibody, antigen and immune complexes in bancroftian filariasis (9,10). Wuchereria bancrofti microfilariae excretory-secretory (Wb mf ES) antigen was biosynthetically labeled using 14C-glucose in the culture medium.  The use of 14C-glucose labeled Wb ES antigen in penicillinase- ELISA and Radio immunopolyethylene glycol precipitation assay (RIPEGA) for detection of antibody showed that penicillinase- ELISA was more sensitive than RIPEGA (11). Similarly inhibition ELISA for detection of antigen was also compared with RIA using biosynthetically labeled ES antigen and observed that ELISA was found to be requiring less antigen was simple and inexpensive than RIA (12).


Indirect ELISA

              Indirect ELISA is useful for the detection of antibody using specific filarial antigen. In this assay, the PVC plate was coated with antigen and the test sample (serum/blood, hydrocele fluid, urine, etc.) was added to the plate. Any antibody specific for the antigen will bind to the available sites. The bound antibody was detected by incubation with an enzyme labeled specific anti-immunoglobulin followed by the enzyme substrate.

            Indirect ELISA using W.bancrofti mf ES antigen, incubated with filarial sera samples followed by anti-human IgG-penicillinase conjugate and starch-iodine penicillin substrate was found to be highly sensitive in detection of filarial infection. As little as 0.35 ng antigen protein per well was found to be sufficient in detecting filarial antibody compared to 0.1-1.5 µg of soluble mf antigen protein per well used earlier (13).


Fig. 1- Indirect ELISA


The ELISA plates are to be imported and costly. Also the antigen bound on plate may vary. Hence stick ELISA was developed with cellulose acetate membrane (CAM) square fixed onto a strip of used X-ray sheet. This has become simpler and economic. In indirect ELISA about 100 pg of Wbmf ES antigen protein was found to be sufficient per test and thus one ml of culture fluid can be used for about 2 million tests (14). Brugia malayi microfilarial excretory-secretory antigens have been shown to be very useful in detection of acute, clinical and occult filarial infections by stick indirect ELISA (42). Indirect plate peroxidase ELISA using the same antigen has also been shown to be very useful to detect active filarial infection with high specificity of 95% and sensitivity of 68%. (15). Indirect ELISA using CAM has also been developed to detect tuberculous IgG antibodies against a purified 31kDa antigen (ES-31) from M. tuberculosis H37Ra (16,17). Dot ELISA using nitrocellulose membrane has been used for the diagnosis of visceral leishmaniasis (18) and filariasis (19, 20).    

Direct ELISA

            Direct ELISA was used in our laboratory for detection of antigen in filarial immunecomplexes. Optimal dilution of circulating immunecomplexes (10 µg/ ml) was coated on to the plate. After washing, filarial serum immunoglobulin G-penicillinase (FSIgG-penicillinase) conjugate was added and then assayed. Analysis of immune complexes showed the presence of filarial antigen in 30 out of 33 clinical filarial, 2 out of 15 endemic normal and none of the non endemic normal sera (21).  

  Fig. 2- Direct ELISA

Competitive ELISA

            Competitive ELISA is useful for identification and quantitation of either antigen or antibody. In antigen determination by this method, antigen present in the sample competes for sites on the antibody with labeled antigen added to the medium. The color change will be inversely proportional to the amount of antigen in the sample. Competition principle can be exploited in number of ways.

            To identify the antigen involved in filarial immunecomplexes, circulating immunecomplex (CICs) showing filarial antigen in direct ELISA was coated on to the plates. Then 50 µl of culture fluid containing W. bancrofti mf ES antigen (7ng/ml) was added together with 50 µl of FSIgG  penicillinase conjugate. After incubation and washing, the enzyme activity was assayed. In this assay system, freely added W.bancrofti mf ES Ag competes with the immune-complexed antigen bound to the plate, for binding sites on FSIgG conjugated with penicillinase. Presence of W.bancrofti mf ES antigen in CICs was confirmed by the persistence of blue color (21).  

Fig. 3- Competitive ELISA

Sandwich ELISA

            Double antibody sandwich ELISA is useful for detection of antigen. In the assay system the antigen to be detected is sandwiched between two similar or different antibodies of which one is labeled with an enzyme. IgG fraction of human filarial serum immunoglobulin (FSIgG) has been successfully used for detection of circulating filarial antigen by sandwich ELISA (22). When analysed using FSIgG  sandwich ELISA, 27 out of 33 microfilaraemia, 19 out of 30 clinical filarial and none of the 20 non-endemic normal sera showed the presence of filarial antigen. Detection of filarial antigen using FSIgG in sandwich ELISA showed an apparent positive correlation between microfilarial density and antigen titer. When Wb E34 monoclonal antibody was used along with FSIgG in double antibody sandwich ELISA 68% of microfilaraemic sera showed the presence of filarial antigen (23). Detection of filarial antigen in urine and hydrocele fluid samples by sandwich ELISA using FSIgG was also reported (24,25). Stick sandwich ELISA was also developed for detection of circulating free antigen and CIC-antigen in tuberculosis sera (26).  

  Fig. 4- Sandwich ELISA  

Inhibition ELISA

            Inhibition ELISA works similar to competitive ELISA but in this system the two antigens (antigen in test sample and enzyme labeled antigen) are added one after another. This is useful especially when test serum contains both antigen and antibody of interest in the immune reaction. Inhibition ELISA was also useful in determining the identity of specific antigen or antibody (27,28).  

Fig- 5. Inhibition ELISA  

Inhibition ELISA was found to be better than sandwich ELISA in detection of active filarial infection (27). FSIgG was coated to the plate followed by addition of test serum containing antigen. After washing Wb mf ES antigen conjugated with penicillinase was added. Positive reaction for ES antigen was indicated by the persistence of the blue color. By inhibition ELISA using Wb mf ES Ag-penicillinase conjugate, filarial antigen was detected in about 90% of microfilaraemic sera and 60% of clinical filarial sera. Incorporation of avidin-biotin system in inhibition ELISA increased the sensitivity of the assay system by 67% decrease in the false negative results (29). Stick inhibition ELISA using B. malayi mf ES antigen- penicillinase and FSIgG was found to be useful to detect filarial antigen in acute, chronic and occult filarial cases where mf cannot be detected in peripheral circulation. While the filarial antigen positivity ranged between 43-67% in lymphadenopathy cases, as many as 70% of epididymoorchitis cases, 61-62% of TPE & tenosynovitis cases and 80% of retroperitoneal lymphadenitis cases showed antigenemia (30). Filarial sera after acid-heat treatment for dissociation of immunecomplexes have been analysed in inhibition ELISA to detect immunecomplexed filarial antigen (31).

Anti C3 ELISA:

            Anti C3 ELISA is useful to detect disease specific circulating immunecomplexes or specific antigen present in circulating immune-complexes. Test sera are incubated in microtitre plate wells prior sensitized with rabbit/goat anti human C3 antibody. The filarial antigen in bound immunecomplexes is detected by further incubating the wells with enzyme labeled specific antibody. In our laboratory we have coated anti C3 antibody (0.1 µg/100 µl/well), blocked with 3% BSA and further incubated with optimally diluted (1:150) test sera. FSIgG penicillinase conjugate was used as a probe to detect the filarial specific antigen in bound immunecomplexes. Sera from clinical filarial patients (10 years or more duration) were negative for free circulating antigen but showed high level of immunecomplexed antigen (32)

Fig 6. Anti C3 ELISA  

Immunodiagnosis of filarial infection

            Immunodiagnosis  of  filarial  infection  still  presents  a  major  challenge  to  the immunoparasitologist  because  of  the  complexities  of  the  disease  and  the  high  degree  of cross-reactivity between filaria and other nematode species. The sequential changes in the humoral immune response against filarial antigens during the course of Brugia malayi infection in Mastomys natalensis have been studied using penicillinase ELISA which was useful in understanding immune recognition and in assessing the status of host immunity to invading parasite(33).Stick ELISA using penicillinase system was used for antigen, antibody and immunecomplex detection in sera of patients who were in different stages of bancroftian filarial infection and found to be useful in understanding disease status and its relevance to immunopathogenesis. The diagnostic assay system based on PEN-ELISA using Wb mf ES antigen for the detection of filarial antigen and antibody was found to be one of the three promising assay systems for diagnosis of filariasis in the multi-centre trial held world wide by WHO-TDR programme (W.F. Piessens, personal communication). Blood collected on filter paper by finger prick can also be used in ELISA making it easily adaptable for field studies (34). Field evaluation of PEN-ELISA using Wb mf ES Ag for the detection of filarial antibody in filter paper blood samples showed the sensitivity and specificity of about 80-90 % in diagnosis of filarial infection (35). It was observed in our laboratory that 0.5 femtogram of antigen isolated from filarial patients urine is sufficient per test for detection of antibody (36). Twenty four hours urine from a carrier can give sufficient antigen for 600 billion tests. Thus it has been proved in our hands and in this laboratory, that the more you sharpen penicillinase ELISA with specific reagent the more efficient it works. B. malayi mf  ES antigens have been shown to be very useful in detection of acute, clinical and occult filarial infections. Filarial IgG antibodies against Bm mf ES antigen were detected in 54-72% of acute lymphadenopathy cases, 67-84% of chronic filarial cases and 44-66% of occult filarial cases that included cases of tropical pulmonary eosinophilia  (TPE), mono or poly arthritis etc.(30). Recently, B.malayi microfilarial ES antigen and Filarial serum Immunoglobulins have been used to pick up 2/7 and 5/7 of hematuria cases with filarial etiology respectively (37). While a number of immunoassays such as the ICT kit and Og4C3 ELISA have been used, developed based on filarial antigen detection using monoclonal antibodies (38,39), these assays are capable of detecting microfilaraemic cases and failed to detect acute and early filarial cases. A study evaluating Seva-Fila Chek and the ICT kit showed a much greater sensitivity of the former in detecting clinical and occult cases (40).


Immunomonitoring of filarial infection

            Although mass or targeted administration of diethylcarbamazine (DEC) has been the regular procedure of filariasis control programmes, the logistics of such programme are greatly complicated by the haphazard result in total clearance of microfilariae. A long term study on the effect of chemotherapy in microfilaraemic patients and   immunomonitoring of filarial patients in an endemic area will be useful in developing an immunological parameter in place of parasite detection, determining appropriate duration of treatment and to judge the effectiveness of chemotherapy and relapse of infection or reinfection in control programme in an endemic area. A group of 27 W. bancrofti microfilaraemic cases were treated with DEC and followed up for 10 years (41).  Treatment with DEC was for 14 days (day 1, 1 mg Kg-1 body weight; day 2, 2 mg Kg-1 body weight and from day 3 onwards 6 mg Kg-1 body weight) followed by one dose (6 mg Kg-1 body weight) each year. Out of the 27 carriers followed, 13 cases never showed reappearance of mf while 14 cases showed reappearance of mf in the peripheral blood during the study period. Immunomonitoring showed decrease in antigen and antibody levels during therapy followed by increased titres with recurrence or re-infection in few cases in an endemic area suggesting use of antigen/antibody assay as a marker for infection. None of the cases under DEC treatment developed any clinical symptoms during the study.

            Further a total of 38 filarial cases with acute, chronic and occult clinical symptoms were given a long term treatment with DEC (6 mg Kg-1 body weight per day for 21 days in a month repeated for 3 to 12 months) and were monitored by immunological tests for antigen and antibody (42). A significant number of filarial patients showed a reversal or reduction in the clinical symptoms. The immunomonitoring thus helped in deciding the appropriate period of treatment in the absence of microfilaraemia in these clinical conditions. Immunomonitoring followed by opDEC (optimal DEC) therapy was also found to be very effective for clinical relief and cure (43) in such cases. In another report from this laboratory (44) number of filaria associated clinical manifestations have been shown in children in an endemic area. These cases with manifestations like lymphadenopathy, asthmatic bronchitis, pulmonary eosinophilia, monoarthritis, recurrent URI, pneumonia, pain in abdomen etc. were effectively managed by immunomonitoring and optimal DEC (op DEC) therapy.

The PEN-ELISA thus has been explored in a variety of ways to detect, identify and  quantitate antibodies, free and immunecomplexed antigen of interest in diagnosis, protection, immunomonitoring and immunosurveillance of filariasis.  

Immunodiagnosis of tuberculosis  

            Tuberculosis continues to be a major public health problem in India and other developing countries. Operational difficulties in control programmes, and coexistence with HIV infection have contributed to the increased burden of the disease. Early diagnosis of tuberculosis is an essential requirement for initiating prompt treatment and containment of the disease. Presently available methods like radiological and bacteriological examination are insensitive and even intradermal tuberculin test cannot differentiate active and past infection. In our laboratory Mycobacterium  tuberculosis H37Ra excretory-secretory (M.tb ES) antigen was isolated from logarithmic growth phase of the bacilli in synthetic Sauton medium and explored for detection of tuberculous antibodies by Penicillinase ELISA (45). This assay was also shown to be useful for detection of pulmonary tuberculosis and extrapulmonary tuberculosis in children (46). A comparative analysis of M.tb ES antigen and other tubercular antigens Viz., phosphate buffer saline soluble (PBS-S) antigen, SDS soluble antigen (SDS-S), and PPD showed M.tb ES antigen to be superior to other antigens in diagnosis of tuberculosis (47). Two purified antigens from M.tb ES antigen i.e. ES-31 (a 31 kDa protein obtained by ammonium sulphate, SDS-PAGE and FPLC fractionation using Resource ‘S’ column) and ES-41 (a 41 kDa protein isolated by trichloroacetate solubilization followed by FPLC fractionation using Superdex HR 10/30 gel filtration and Resource ‘S’ columns) were shown to be of immunodiagnostic importance in tuberculosis (16,17,48,49). ES-31 antigen was useful to detect tuberculous IgG antibodies in 92% of pulmonary and 88% tuberculous lymphadenopathy and 90% of tuberculosis meningitis cases, while the ES-41 antigen detected antibodies in 82% of abdominal tuberculosis and 85% osteoarticular tuberculosis cases. A process of isolation and purification of M.tb ES-31 protein and monitoring of M. tuberculosis infection has been patented.  A sandwich ELISA using affinity purified goat anti ES-31 antibodies was found to be useful to detect tubercular free antigen and immunecomplexed antigen in 80% and 72% of pulmonary tuberculosis cases respectively (26).  

Immunodiagnosis of Leprosy

            Leprosy is another widely distributed Tropical disease. Studies have been initiated in our laboratory to develop suitable immunoassays for the early diagnosis of leprosy and for early detection of nerve damage in affected cases. Anti ceramide antibodies and S-100 protein levels were estimated in leprosy patients (50, 51). S-100 antigen protein was reported as more sensitive and reliable marker than anti ceramide antibodies for nerve damage (50). 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 (51).  


1.         Engvall & Perlmann P. (1971). Enzyme Linked immunosorbant assay (ELISA): Quantitative assay for immunogobulin. Immunochemistry. 8. 871.  

2.         Voller A, Bidwell DE and Bartlett A (1979) The enzyme linked immunosorbent assay (ELISA). A guide with abstracts of microplate applications. Dynatech Europe publication.  

3.         Talwar G.P (1983) Non-isotopic immunoassays and their applications. Vikas Publishing House Pvt. Ltd., New Delhi.  

4.         Desai N., KhatKhatay I, Sankolli G, Neherji P and Joshi U (1989) Enzyme labeled  immunoassay for urinary gonadotropins using penicllinase, Clinica Chemica Acta. 184,315.  

5.         Shrivastav TG, Kumari GL and Rao PN (1988) Enzyme immunoassay of cortisol in human plasma using penicillinase label. Clinica Chemica Acta, 174,83.  

6.         Sauar NJ, Foulkes JA and O’Neill PM (1989) A comparison of alkaline phosphatase,  ß-galactosidase, penicillinase and peroxidase used as labels for progesterone determination in milk by heterologous microtiter plate enzyme immunoassays. J. Steroid Biochem. 33,423.   

7.         Novick RP (1962) Micro-iodometric assay for penicillinase, Biochem. J., 83, 236.  

8.         Yolken RH. Wee SB, Regenmortel MV (1984) The use of B-lactamase in enzyme   immunoassays for detection of microbial antigens J. Immunol. Methods, 73, 109.  

9.         Harinath BC (1984) Immunodiagnosis of bancroftian filariasis-problems and progress. J.Biosci., 6. 691.  

10.       Harinath BC (1986) Detection and diagnostic utility of in vitro and in vivo released antigens in bancroftian filariasis. J. Comm. Dis., 18, 261.  

11.       Ramaprasad P, Kharat I and Harinath BC (1984) Comparative efficiency of penicillinase ELISA and RIPEGA using 14C -labeled Wuchereria bancrofti  excretory-secretory antigen for the detection of filarial antibody. IRCS Med. Sci, 12, 738.  

12.       Ramaprasad P, Reddy MVR, Kharat I and Harinath BC (1985). Comparison of  radio immunoassay and inhibition enzyme linked immunosorbent assay (ELISA) using 14­C-labeled Wuchereria bancrofti microfilarial excretory-secretory antigen for the detection of filarial antigen. IRCS Med. Sci, 13, 1110.  

13.       Kharat I, Ghirnikar SN and Harinath BC (1982) Antibody analysis in human filarial sera by ELISA using Wuchereria bancrofti microfilariae culture antigen. Indian J.Exp.Biol, 20, 378.  

14.       Parkhe KA, Prasad GBKS, Das A, Harinath BC, Roebber N and Hamilton RG (1986) Dics/stick ELISA for diagnosis of bancroftian filariasis, Indian J. Exp. Biol, 24, 437.  

15.       MVR Reddy, R Alli, KK Devi, R Narayan, R Harikrishnan, K Cheirmaraj and BC Harinath. Comparative evaluation of microtitre  plate peroxidase and Stick Penicillinase enzyme immunoassays for detection of filarial antibodies using Brugia malayi microfilarial excretory- secretory antigen. J. Parasitic. Dis. 1996, 20, 173-176.

16.       Nair ER, Kumar S, Reddy MVR and Harinath BC (1998) Mycobacterium tuberculosis H37 Ra ESAS-7 an excretory - secretory antigen fraction of immunodiagnostic potential in pulmonary tuberculosis; Indian J Clin. Biochem 13 (2), 98-105.  

17.       Nair ER, Banerjee S, Kumar S, and Harinath BC, 2000. Isolation and characterization of a 31 kDa mycobacterial antigen from tuberculosis sera and its identification with in vitro  released culture filtrate antigen of M.tb H37 Ra bacilli; Scand J Infect Dis. 32: 551-556. 

18.       Pappas MG, Hajkowshi R and Hockmeyer WT (1983) Dot enzyme linked immunosorbent assay (Dot-ELISA): a micro technique for the rapid diagnosis of visceral leishmaniasis. J. Immunol. Methods, 64, 205.  

19.       Tandon A, Murthy PK, Saxena RP, Sen AB and Saxena KC (1988) Dot-ELISA for               diagnosis of lymphatic filariasis. Indian J. Med. Res. 87, 429.


20.       Balaji Ganesh B, Kader AM, Agarwal GS, Reddy MVR and Harinath BC (2001) A simple & inexpensive dot blot assay using a 66 kDa B. malayi microfilarial antigen protein for diagnosis of filarial infection in an endemic area. Trans. Roy. Soc. Trop. Med. Hyg. 2001; 95: 1-2.


21.       Prasad GBKS, Reddy MVR and Harinath BC (1983) Detection of filarial antigen in immunecomplexes in bancroftian filariasis by ELISA. Indian J. Med. Res, 78, 780.  

22.       Reddy MVR, Malhotra A and Harinath BC (1984) Detection of circulating antigen in bancroftian filariasis by sandwich ELISA using filarial serum IgG. J.  Helminthol, 58, 259.  

23.       Reddy MVR, Ramaprasad P, Piessens WF and Harinath BC (1986) Diagnostic utility of monoclonal antibodies raised against microfilarial excretory-secretory antigens in bancroftian filariasis. J. Biosci, 10, 461.  

24.       Malhotra A, Reddy MVR and Harinath BC (1985) Detection of filarial    antigen in urine by sandwich ELISA and its use in diagnosis. Indian J. Med. Res, 81, 123.      

25.       Malhotra A, Prasad GBKS and Harinath BC (1985) Detection and isolation of filarial antigen from hydrocele fluid and its use in diagnosis.  Indian J. Exp. Biol, 23, 180.  

26.       S Banerjee, E Nair, S Kumar, MVR Reddy & BC Harinath. Assay of tubercular antibody, circulating free and immune complexed antigen in the diagnosis of pulmonary  tuberculosis. Ind. J. Clin. Biochem, 2001, 16(2), 203-206.  

27.       Malhotra A and Harinath BC (1984) Detection and monitoring of   Microfilarial ES antigen level by inhibition ELISA during DEC therapy. Indian J. Med. Res, 79, 194.  

28.       Reddy MVR, Parkhe KA, Piessens WF and Harinath BC (1989) Wb E34   monoclonal antibody: Further characterization and diagnostic use in bancroftian filariasis. J.Clin. Lab. Analysis, 3, 277.  

29.       Parkhe KA, Ramaprasad P and Harinath BC (1988) Stick enzyme linked immunosorbent assay using the avidin-biotin system for detection of circulating antigen in bancroftian filariasis. J. Biosci, 13, 229.   

30.       Harinath BC & Reddy MVR. (1997) Diagnosis & Immunomonitoring in successful  management of bancroftian filariasis. J. of Parasitic Diseases, 21, 41-51. 31.       B Bhunia, S Kulkarni, M V R Reddy & B C Harinath. Diagnostic evaluation of circulating filarial antigen assay using sera samples with and without acid- heat treatment. Biomedical Research, 2002, 13(1), 39-42.  

32.       Alilkhan A, Parkhe KA, Reddy MVR and Harinath BC (1990) Filarial antigen, antibody and circulating immunecomplexed antigen level in bancroftian filariasis by Stick ELISA. Natl. Med. J. India, 3, 265.  

33.       Cheirmaraj K and Harinath BC (1991) Humoral immune response to infective larval antigen in Brugia malayi infected Mastomys natalensis. Acta Tropica, 48, 305.  

34.       Malhotra A, Reddy MVR, Naidu JN, Ghirnikar SN and Harinath BC (1982) Detection of filarial infection using Wuchereria bancrofti microfilariae culture antigen and filter paper blood samples in enzyme linked immunosorbent assay. J. Blosci., 4. 507.  

35.       Harinath BC, Malhotra A, Ghirnikar SN, Annadate SD, Issacs VP and Bharati KS (1984) Field evaluation of ELISA using Wuchereria bancrofti Mf ES antigen for bancroftian filariasis. Bull. WHO, 62, 941.  

36.       Ramaprasad P, Kharat I and Harinath BC (1987) Fractionation and characterization of     urinary filarial antigen. Asian Pacific J. Aller Immunol, 5, 173.   

37.       R.Alli, B. Bhunia, M.V.R.Reddy & B.C. Harinath. Microscopic haematuria as an occult filarial infection in an endemic area for bancroftian filariasis. Ind. J. Clin. Biochem. (2002). (In Communication).  

38.       G J Weil, D C Santhanam, A Malhotra , H Kumar, K V P Sethumadhavan, F Liftis and TK Ghosh. A monoclonal antibody based enzyme immunoassay for detecting parasite antigenemia in bancroftian filariasis. J. Inf. Dis., 1987, 156: 35- 355.  

39.       SJ Moore and DB Copeman. A highly specific and sensitive monoclonal antibody based ELISA for the detection of circulating antigen in bancroftian filariasis. Trop. Med. Parasitol., 1990, 41, 403-406 .  

40.       R Alli, S Kulkarni, MVR Reddy and BC Harinath. Evaluation of Seva Fila Chek  immunoassays and rapid ICT- filariasis test for detection of bancroftian filariasis Ind. J. Clin. Biochem, 2001, 16(2), 207-210.  

41.       Padigel U M, Reddy M V R, Ali Khan A, and Harinath B C (1995). Immunomonitoring of filarial patients during DEC therapy in an endemic area: a seven year follow up. J. Trop Med Hyg. 98.52.  

42.       Harinath BC, Padigel UM, Reddy MVR & Devi KK (1996). Diagnosis and immunomonitoring in management of lymphatic filariasis in an endemic area. J. Pars. Dis. 20, 35-40.  

43.       Harinath BC, Reddy MVR, Alli R, Mehta VK, Chaturvedi P, Patond KR, Kalantri SP & Gupta RKC (1999) Immunomonitoring followed by optimal DEC therapy for successful management of clinical filariasis in an endemic area. Ind. J. Clin. Bio. Chem. 14 (2), 100-108.  

44.       Harinath BC, Reddy MVR, Bhunia B, Bhandari YP, Mehta VK, Chaturvedi P, Prajapati N C & Gupta RKC (2000). Filaria associated clinical manifestations in children in an endemic area & morbidity control by immunomonitoring & optimal DEC therapy: Sevagram experience. Ind. J. of clin. Bio. Chem. 15 (Suppl): 118-126.  

45.       Basak A, Sinha Choudhary S, Lodam A.N, Gupta OP, Narang P, and Harinath B C. (1994). Detection of IgG using Mycobacterium tuberculosis H37 Ra ES antigen in tuberculosis by Penicillinase stick ELISA. Proceedings of CSIR Golden Jubilee symposium on tropical diseases “Molecular Biology and control strategies”. 558.  

46.       Bhaskar A, Pradan P, Chaturvedi P, Basak A, Lodam A N, Narang P, and Harinath BC, (1994). Immunodiagnosis of childhood pulmonary and extrapulmonary tuberculosis using mycobacterium ES antigen by Penicillinase ELISA. Annals of Trop Pead. 14, 25.  

47.       Satish Kumar, Chenthamarakshan V, Reddy M V R, Narang P, Gupta OP and Harinath B C., (1994) Detection of tuberculous IgG antibodies using Mycobacterium tuberculosis H37 Ra Excretory Secretory antigen and tuberculous purified protein derivative. Ind. J. Exptl Biol  32. 163.  

48.       S Banerjee, S Gupta, S Kumar, A J Shrikhande, M V R Reddy & BC Harinath. Seroreactivity of 31 kDa and 41 kDa mycobacterial secretory proteins isolated from culture filtrate in extrapulmonary tuberculosis. Ind. J. Pathol Microbiol. ( In press)  

49.       S Banerjee, E Nair, S Kumar & BC Harinath. Isolation and characterization of in vivo              released 41kDa  mycobacterial antigen in pulmonary and bone and joint tuberculosis and its identification with H37Ra in vitro released antigen. Int. J. Tuberculosis and lung disease (In Press).  

50.       Narayan R, Maheshwari PK, Desikan KV and Harinath BC (1997) Detection of S-100  protein and anticeramides antibodies in leprosy patients by ELISA. Lepra Rev 68.  

51.       Narayan R, Maheshwari PK, Desikan KV and Harinath BC. Detection of S-100 antigen and anticeramides antibody in sera of leprosy patients with and without reaction. Ind. J. of Lepr. 69 (4), 347-352.  


This review is broadly based on the work done at the

 Department of Biochemistry and

Jamnalal Bajaj Tropical Disease Research Centre,

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