Serum immunoglobulin M in Atlantic halibut (Hippoglossus hippoglossus): characterisation of the molecule and its immunoreactivity.
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 (mu) and light (L) chains. A single mu chain at approximately 76 kDa, and six possible molecular weight (MW) variants of L chain were found (range approximately 25 to approximately 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 approximately 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.
Grove
Søren
S
Section of Immunoprophylaxis, National Veterinary Institute, 0033 Oslo, Norway. soren.rove@vetinst.no
Tryland
Morten
M
Press
Charles McL
CM
Reitan
Liv J
LJ
D000818:Animals; D004797:Enzyme-Linked Immunosorbent Assay; D005432:Flounder / Q000097:blood* / Q000276:immunology; D007106:Immune Sera / Q000276:immunology*; D015151:Immunoblotting; D007122:Immunoelectrophoresis; D007075:Immunoglobulin M / Q000097:blood / Q000737:chemistry* / Q000276:immunology*; D007150:Immunohistochemistry; D013379:Substrate Specificity
D007106:Immune Sera; D007075:Immunoglobulin M