Appendix - II 

Immunological Reagents


1.   B.malayi microfilarial excretory (ES) antigen (Bm mf ES Ag) :


            Collect Brugia malayi microfilariae from infected jird peritoneal lavage, plate on disposable petridish to remove macrophages and wash the mf in RPMI-1640 medium. Purify by density gradient centrifugation using 30 and 40% percoll. Collect the mf  from interphase, wash thoroughly and maintain in RPMI-1640 medium for 48 hr at 370C. Collect the spent medium every 24hr and centrifuge at 13000 g for 15min. Concentrate the supernatant by ultra membrane filtration and dialyse against 0.01 M SPB (pH 7.2).


2.   B.malayi microfilarial PBS soluble Ag (Bm mf S Ag) :


B.malayi  microfilariae were collected in 0.01M PBS pH 7.2 after Es Ag preparation sonicated for 10 min in Vibronic sonicator & extracted overnight at 40C. the sonicate was centrifuged at 25,000 RPM for 30 min. & supernatant was dialysed against 0.01 M SPB pH 7.2 & labeled as Bm mf ' S' Ag.


3.   B.malayi adult antigens:

Collect adult worms from peritoneal cavity of jirds infected 3 months before with B.malayi L3 larvae, freeze dry and pulverize into powder form by manual homogenization. Extract the homogenate in 0.05M SPB at 40C overnight. Centrifuge at 13000 g for 30min. at 40C to remove soluble proteins and label the supernatant as BmA PBS ' S ' Ag. Suspend the insoluble pellet in 0.01M SPB (pH 7.2) containing 5% SDS, 5% 2-mercaptoethanol and 8 M urea, incubate at 1000C for 10 min. and separate the supernatant by centrifugation at 13000 g for 30 min. at 40C. Dialyse extensively against 0.01M SPB (pH 7.2) and label as BmA SDS 'S' Ag.


4.   Rabbit anti Bm mf 'S' antigen antibodies

Rabbit anti Bm mf 'S' antigen antibodies were raised by immunizing 2 male rabbits  of 1 year old with Bm mf 'S' antigen following standard immunization protocol.Bm mf 'S' antigen protein (500 µg in 0.5 ml of 0.05 M PBS) and equal volume of complete Freund's adjuvant were emulsified and injected sub-cutaneously at different sites on shaven back portion. The same amount of antigen emulsified in equal volume of incomplete Freund's adjuvant was given on 7th day, 14th day and on 40th day. After a lapse of 7 days, blood samples were collected thrice at 3 days intervals from ear marginal veins. Further collection of blood samples was carried out after allowing the rabbits to rest for 2 months and followed by administration of one more booster dose.


5.   Circulating filarial antigen (CFA) :

Pool plasma collected from microfilaraemic patients and remove Ig Fraction by precipitating with 35% ammonium sulphate saturation. Increase the ammonium sulphate  saturation of the supernatant from 35% to 75% and centrifuge at 13,000 g for 30 min. at 40C. Collect the pellet, reconstitute in 0.05 M SPB (pH 7.2) and dialyse against the same buffer.


6.   M.tb H37 Ra excretory secretory antigen fraction (ES-31):

The loopful of culture of Mycobacterium tuberculosis H37Ra was inoculated into (8 µg/ ml) thyroxine supplemented Sauton's medium. Exponentially growing tubercle bacilli were isolated from the 7 days old spent culture medium by filtration through Whatman

No.3 filter paper followed by Seitz filter and concentrated by ultrafiltration (Millipore, USA) using a membrane of 10-12 kDa cut off. It was dialyzed extensively against 0.01M PBS & protein content was measured by Lowry's method & labeled as M.tb ES antigen. ES antigen was precipitated with 50% ammonium sulphate. The solubilized fraction was dialyzed, concentrated and labelled as M.tb ESAS antigen. M.tb ESAS antigen was fractionated with 10% non-gradient SDS-Polyacrylamide Gel Electrophoresis. After slicing the gel horizontally at l cm interval, the proteins from 7th fraction with highest seroreactivity (Nair et al, 1998)) were electroeluted from the gel, using an electroeluter and labelled as M.tb ESAS-7 antigen. M.tb ESAS-7 antigen was further fractionated by fast protein liquid chromatography (FPLC) using cation exchange Resource 'S', 1ml column (Pharmacia, Biotech, Sweden). The eluent was monitored at 280nm and 500 µl fractions were collected. The 6th fraction found antigenically active was collected. This protein fraction yielded 31 kDa proteins & labeled as M.tb ES-31 antigen.


7.   Affinity Purified Goat anti ES-31 antibodies:

Antibodies against M.tb H37Ra ES-31 antigen are raised in goat by immunizing with 500 µg of antigen in 4 equal doses with incomplete Fruend's adjuvant on days 0, 10, 40 & 70. The immune sera was collected 10 days after second booster and immunoglobulin fraction was isolated by 33% saturation with ammonium sulphate. Specific antibodies against Es-31 antigen were isolated by affinity chromatography using ES-31 antigen coupled CNBr activated sepharose-4B column.