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 (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.
Bryant
M S
MS
Department of Parasitology, University of Queensland, Brisbane, Australia. malb@qm.qld.gov.au
Lee
R P
RP
Lester
R J
RJ
Whittington
R J
RJ
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
D000913:Antibodies, Protozoan; D000953:Antigens, Protozoan; D007106:Immune Sera; D007136:Immunoglobulins; D012710:Serum Albumin, Bovine