title: Anti-immunoglobulin antisera used in an ELISA to detect antibodies in barramundi Lates calcarifer to Cryptocaryon irritans.
abstract: 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 (ELISA) before being pooled and characterised by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). Double affinity purification did not improve the purity of the Ig preparation compared to single affinity purification. Barramundi Ig were injected into sheep to produce anti-Ig antisera which were assessed in an indirect ELISA as the secondary antibody to detect serum Ig in barramundi vaccinated with Cryptocaryon irritans theronts. Affinity-purified Ig induced a more specific reagent for use as secondary antibody in ELISA than did normal whole-barramundi sera. The heavy (H) chain 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 conditions 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.
authors.affiliation: Department of Parasitology, University of Queensland, Brisbane, Australia. malb@qm.qld.gov.au
authors.forename: M S
authors.initials: MS
authors.lastname: Bryant
authors.forename: R P
authors.initials: RP
authors.lastname: Lee
authors.forename: R J
authors.initials: RJ
authors.lastname: Lester
authors.forename: R J
authors.initials: RJ
authors.lastname: Whittington
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000913:Antibodies, Protozoan / Q000737:chemistry / Q000276:immunology / Q000302:isolation & purification*; D000953:Antigens, Protozoan / Q000737:chemistry / Q000276:immunology; D001492:Bass; D002846:Chromatography, Affinity / Q000662:veterinary; D016798:Ciliophora / Q000276:immunology*; D016770:Ciliophora Infections / Q000276:immunology / Q000662:veterinary*; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay / Q000662:veterinary; D005393:Fish Diseases / Q000276:immunology*; D007106:Immune Sera / Q000276:immunology; D007136:Immunoglobulins / Q000096:biosynthesis / Q000276:immunology; D008969:Molecular Sequence Data; D017421:Sequence Analysis; D017386:Sequence Homology, Amino Acid; D012710:Serum Albumin, Bovine / Q000276:immunology; D012756:Sheep; D014611:Vaccination / Q000662:veterinary
chemical_list: D000913:Antibodies, Protozoan; D000953:Antigens, Protozoan; D007106:Immune Sera; D007136:Immunoglobulins; D012710:Serum Albumin, Bovine