Optimisation of immunoaffinity purification of Wuchereria bancrofti specific antibodies from human sera.

Immunoaffinity column using Setaria digitata antigens coupled to cyanogen bromide activated Sepharose 4B beads were developed to purify antibodies from sera of filarial patients. Chaotropic (KSCN) ion elution was more efficient for purifying specific antibodies from the column in comparison to ]c elution. Dot blot analysis indicated that purified antibodies showed a high degree of reactivity with cattle filarial antigen and recombinant filarial protein but not with bacterial proteins of E. coli suggesting that the antibodies are specific.

Purification and characterization of E. histolytica antigen for diagnosis of amoebiasis.

Entamoeba histolytica soluble crude antigen was fractionated by gel filtration on Sephacryl S-300 into four fractions, viz. F1(669 kDa); F2(51.2 kDa); F3(25.1 kDa) and F4(10.5 kDa). F1 fraction was observed to be more sensitive and specific for the detection of antibody in amoebiasis than the crude and other fractions of purified antigens employing IHAT and ELISA. ELISA was found to be better than IHAT since it could detect antibody in the sera (3/6) of asymptomatic cyst passers. The cross reaction of crude antigen with toxocariasis (1/4) and toxoplasmosis (2/5) sera were associated with F4 fraction. F3 and F4 were having low molecular weight and were not sensitive in detection of antibody in amoebiasis. Biochemical characterization revealed glycoprotein nature of the specific (F1) antigen fraction.

Development of simple and stable ELISA for detection of copro antigen in intestinal amoebiasis.

A simple, sensitive and stable ELISA (enzyme linked immunosorbent assay) was developed using rabbit antibody to fractionated Entamoeba histolytica antigen for the detection of copro antigen in the faeces of individuals with intestinal amoebiasis. In this test none of the healthy individuals, all trophozoite positive, 40% cyst passers and 6% individuals living in endemic area showed the presence of copro antigen. ELISA using polyclonal rabbit antibody could detect 1-5 trophozoites/well and 20-50 ng protein per well of NIH-200 strain of E. histolytica and the sensitivity of the test was comparable with that using monoclonal antibody. Cross reaction was observed only with E. invadens when faeces having other parasites were screened. The reagents of ELISA were stabilized and found to be stable for more than 6 months when stored at 4 degrees C.