Serum immunoglobulin M in Atlantic halibut ( Hippoglossus hippoglossus): Characterisation of the molecule and its immunoreactivity

• Published in: Fish & shellfish immunology 2006, Vol.20 (1), p.97-112
• Main Authors: Grove, Søren, Tryland, Morten, Press, Charles McL, Reitan, Liv J.
• Format: Article
• Language: English
• Subjects: Animals; Atlantic halibut; Enzyme-Linked Immunosorbent Assay; Flounder - blood; Flounder - immunology; Ig positive cells; Immune Sera - immunology; Immunoblotting; Immunoelectrophoresis; Immunoglobulin; Immunoglobulin M - blood; Immunoglobulin M - chemistry; Immunoglobulin M - immunology; Immunohistochemistry; Polyclonal antiserum; Protein A; Protein G; Redox forms; Substrate Specificity
• ISSN: 1050-4648; 1095-9947
• DOI: 10.1016/j.fsi.2005.05.002

Three preparations of purified immunoglobulin (IgM) were isolated from serum of Atlantic halibut ( Hippoglossus hippoglossus) by means of three different methods, and each of the three IgM preparations was used to produce a polyclonal rabbit anti-halibut IgM antiserum. One of the IgM preparations was employed in the characterisation of halibut serum immunoglobulin. Halibut IgM was shown to consist of two subunits, compatible with heavy (μ) and light (L) chains. A single μ chain at ∼76 kDa, and six possible molecular weight (MW) variants of L chain were found (range ∼25 to ∼28.5 kDa). IgM was glycosylated on the heavy chain and N-linked carbohydrate constituted approximately 10.3% (w/w) of the total MW of IgM. The dominant form of non-reduced IgM had a MW of ∼780 kDa, suggesting a tetrameric structure. Non-reduced IgM also showed a number of minor protein bands. Based on estimated MW, the relative carbohydrate content and the reactivity with all three anti-halibut IgM antisera, mono-, di- and trimeric redox forms of IgM were identified. The three antisera were characterised as to specificity and reactivity by means of enzyme linked immuno-sorbent assay (ELISA), crossed immuno-electrophoresis (CIE), and immunoblotting methods. The antisera showed a considerable diversity in their specificity to the suggested MW variants of halibut Ig light chain. A method for immunohistochemical detection of IgM in tissue was established. Protein A or protein G affinity for the IgM was not detectable.