APPLICATION OF INDIRECT PLATE ELISA IN EARLY DIAGNOSIS OF PARAMPHISTOMOSIS USING PURIFIED POLYPEPTIDES OF SOMATIC ANTIGEN OF PARAMPHISTOMUM EPICLATUM

An indirect Plate ELISA was standardized using purified polypeptides of somatic antigen of Paramphistomum epiclitum, sera of experimentally infected sheep (n=4) at different time intervals post infection and rabbit hyper immune sera. Plate ELISA was standardized using 2 g/ml of antigen concentration with 1:200 and 1:1000 of sera and conjugate dilution, respectively. Whole somatic antigen of P. epiclitum was fractionated by gel filtration using seralose-6B gel filtration media for large molecular weight molecules, equilibrated with 50 mM Tris Hydrochloric acid (Hcl) - 20 mM and sodium chloride (NaCl) buffer. Out of total 19 peaks obtained as per the value of protein concentration in purified fractions of P. epiclitum antigen, 8 peaks were found to be broader. It was found that heterogeneous nature of whole somatic antigen of P. epiclitum contains large number of polypeptides of different molecular weights. These fractions were characterized for immunogenicity by indirect plate-ELISA using four experimentally P. epiclitum infected sheep sera. A pattern of antibody response assessed at weekly interval till the end of experiment (25th week). Antibody response showed peak values of responses at different time intervals (5th, 8th, 9th and 11th week post infection) in the four sheep.