USE OF CLONED EXCRETORY SECRETORY LOW-MOLECULAR-WEIGHT PROTEINS OF COOPERIA-ONCOPHORA IN A SEROLOGICAL ASSAY/

The potential of Cooperia oncophora excretory/secretory (ES) proteins as antigens in a serological assay which aims to establish exposure le vels in cattle was assessed, ES proteins were analyzed by one- and two -dimensional polyacrylamide gel electrophoresis and immunoblotting. Th e N-terminal domains of two ES proteins were sequenced, and the corres ponding cDNAs were cloned, Two cDNAs, designated CoES14.0 and CoES14.2 , were expressed in Escherichia coli. The recombinant proteins were te sted in an indirect enzyme-linked immunosorbent assay (ELISA) in which crude worm antigen (CWA) was used as a reference standard, In total, 67 reference serum samples were used: 27 negative serum samples, 29 C. oncaphora-specific serum samples, 7 Dictyocaulus viviparus-specific s erum samples, and 4 Ostertagia ostertagi-specific serum samples, This showed respective sensitivities and specificities of 17 and 84%, 0 and 100%, and 100 and 100% by the ELISAs with the three different types o f proteins (CWA, CoES14.0, and CoES14.2, respectively), Since the CoES 14.2 ELISA had the best sensitivity and specificity with reference ser a, its specificity was further validated in an antigen inhibition ELIS A. In this assay CoES14.2 and CWA preparations of C. oncophora, Cooper ia curticei, O. ostertagi, Nematodirus helvetianus, Fasciola hepatica, D. viviparus, Haemonchus placei, and Trichostronglyus colubriformus w ere used as competitor antigens, This experiment showed that only the homologous antigens C. oncophora CWA and CoEs14.2 resulted in 100% inh ibition, The CWA preparations of all other nematodes did not affect th e ELISA, even if concentrations of 250 times the 50% inhibitory concen tration of C. oncophora CWA were used, These results indicate that CoE S14.2 does not share cross-reactive epitopes with heterologous CWAs. F inally, we tested the CoES14.2 ELISA with sequential serum samples fro m naturally infected groups of animals, The optical density values tha t mere obtained correlated well with exposure levels based on cumulati ve egg excretion. Thus, the CoES14.2 ELISA seems to be a very sensitiv e tool for estimating exposure levels in cattle.