Heligmosomoides polygyrus: excretory/secretory antigens released in vitro by exsheathed third-stage larvae

The excretory/secretory antigens released during in vitro culture of infective third-stage Heligmosomoides polygyrus larvae were analyzed by enzyme-linked immunosorbent assay and immunoblotting using sera from repeatedly infected mice. During the first 8-10 hr of culture at 37 C, freshly exsheathed larvae released only one antigen that cosedimented with trypsin (24 kDa) upon ultracentrifugation and was composed of a single 23-kDa polypeptide chain. After 10 hr of culture, the larvae released additional antigens identified by bands equivalent to polypeptides of approximately 18, 25, 26, 32, 58, and 76 kDa on nonreduced Western blots. The release of these molecules was maintained for up to 60 hr. Their staining intensity on blots was in the order 23 much greater than 25 greater than 76 greater than 18 greater than or equal to 58 greater than or equal to 32 greater than or equal to 26 kDa. Velocity sedimentation analysis showed that the 76-kDa component exists as a monomeric 76-kDa "native" antigen. The 32-, 58-, and 76-kDa antigens were specifically adsorbed by concanavalin A (Con A)-Sepharose and the 76-kDa molecule was detected on blots incubated with alkaline phosphatase-conjugated Con A, indicating the presence of mannose-like residues on these molecules. The 18-, 23-, 25-, and 26-kDa antigens did not bind to Con A-Sepharose. Hyperimmune antisera raised against lyophilized larvae had negligible antibody activity against the larval ES antigens, suggesting that the ES antigens are released soon after synthesis rather than being stored in significant quantities within the larvae.