Hello all,
I wish to understand the procedure for generation of polyclonal antibodies against human IgM. How to purify human IgM from serum? How to prepare crude IgM preparation for animal immunization? Is there an alternative to purifying IgM? How to purify animal anti human IgM from anti-serum?
Most methods for purifying IgM are poor compared to methods for IgGs. You can buy kits with immobilized lectins but the purity of IgM will probably be 50-80%. Generally you will need to exploit at least one or two additional chromatographic approaches to get anywhere near to a pure IgM protein. Gel filtration is an obvious complementary method.
You may be able to immunize with an impure IgM fraction, but you still need to be able to isolate a specific anti-igM antibody at the end of the process. In my view, the higher the purity of the immunogen the easier thing are.
I would take this approach. Purchase an anti-IgM monoclonal IgG and conjugate to CNBr Sepharose, and use this resin to isolate IgM antibodies from normal human serum. Immunise your host with h-IgM and purify anti-IgM IgG Abs on an IgM-Sepharose column. This column has to have extremely pure IgM (potentially derived from the IgM used for immunization, but with some extra clean up steps if necessary to ensure ultra high purity.
OR, better still, just buy anti-IgM polyclonal from commercial suppliers - it's much easier!
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