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Diseases of Aquatic Organisms

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DAO 36:21-28 (1999)  -  doi:10.3354/dao036021

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

M. S. Bryant1,*, R. P. Lee1, R. J. G. Lester1, R. J. Whittington2

1Department of Parasitology, The University of Queensland, Brisbane, 4072, Australia
2Elizabeth Macarthur Agricultural Institute, PMB 8, Camden, 2570, Australia
*Present address: Queensland Museum, PO Box 3300, South Brisbane, 4101, Australia. E-mail:

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.

KEY WORDS: Cryptocaryon irritans · Antibodies · ELISA · Serology · Immunoglobulin · Barramundi · Lates calcarifer · Disease

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