Anti-immunoglobulin antisera used in an ELISA to detect antibodies in barramundi Lates calcarifer to Cryptocaryon irritans

Immunoglobulins (Ig) in serum from barramundi vaccinated with bovine serum albumin (BSA) were purified by ammonium sulphate precipitation and affinity chromatography using BSA as the ligand. The BSA-binding activity of eluted putative Ig fractions was assessed by enzyme-linked immunosorbent assay (E LISA) before being pooled and characterised by sodium dodecyl sulphate poly acrylamide gel electrophoresis (SDS-PAGE). Double affinity purification did not improve the purity of the Ig preparation compared to single affinity p urification. Barramundi Ig were injected into sheep to produce anti-Ig anti sera which were assessed in an indirect ELISA as the secondary antibody to detect serum Ig in barramundi vaccinated with Cryptocaryon irritans theront s. Affinity-purified Ig induced a more specific reagent for use as secondar y antibody in ELISA than did normal whole-barramundi sera. The heavy (H) ch ain of barramundi Ig had an apparent molecular weight of 70 kDa while that of the light (L) chain was 27 kDa in SDS-PAGE studies. Under non-reducing c onditions 2 putative populations of Ig were identified, at 768 and 210 kDa. The N-terminal sequence of the barramundi Ig H chain showed 78 % homology with channel catfish Ictalurus punctatus Ig H chain sequence.