Affinity purification and partial characterisation of systemic immunoglobulin of the snapper (
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Morrison, RN and Nowak, BF, Affinity purification and partial characterisation of systemic immunoglobulin of the snapper (
Pagrus auratus), Aquaculture, 201, (1) pp. 1-17. ISSN 0044-8486 (2001) [Refereed Article]
Immunoglobulins (Ig) from tank housed wild caught snapper (Pagrus auratus, Bloch and Schneider) were single step purified from serum using staphylococcal protein A (SpA) affinity chromatography. Purified proteins were analysed using SDS-PAGE under reducing, non-reducing denaturing and PAGE under native conditions. Under native conditions, a single population of Ig was identified and using gel filtration chromatography was found to have an approximate molecular weight of 766 kDa. Further, using SDS-PAGE under non-denaturing reducing conditions the single population of Ig was found to be heterogeneous in subunit linkages. Ig subjected to fully reducing conditions dissociated into heavy (H) and light (L) chain polypeptides. Two H and two L chain variants based differences in electrophoretic mobility were detected by SDS-PAGE, however evidence of an isotypic disparity was not proven. The L chains were shown to be approximately 30.2 and 29.0 kDa in molecular weight while the H chains were 71.8 and 67.7 kDa, suggesting that the native molecule was likely to be tetrameric in structure. Polyclonal antisera against snapper Ig were produced and screened by indirect ELISA, Western blot and flow cytometry. Specificity of the antisera was demonstrated by probing against purified Ig, whole snapper serum and heterologous serum in Western blots. Antisera reacted predominantly with the H chains of purified Ig however antisera reacted with both H and L chain variants in reduced serum. A lack of cross-reactivity with five of six heterologous sera tested, demonstrated a high degree of specificity of both antiserum. In flow cytometry, both antiserum bound to the putative B cell population, reacting with 40.8% (rabbit 1) and 29.5% (rabbit 2) of the gated lymphocytes in the peripheral blood. © 2001 Elsevier Science B.V. All rights reserved.
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