I am trying to screen human B cell culture supernatants for autoreactivity on Hep-2 slides (IFA). The supernatants contain both IgG (~10μg/mL) and IgM (~60μg/mL) antibodies. There are several commercially available kits for IgG reactivity (eg. Inova Diagnostics), but nothing for IgM. Has anyone tried to screen IgM antibodies on Hep-2 cells? What is the secondary antibody used? Any tips? I have tried a couple of polyclonal anti-IgM antibodies (rabbit and goat) but I am getting too much background staining...I also tried a goat polyclonal antibody against light chains that reacts with both IgG and IgM that stains nicely but obviously cannot distinguish between IgM and IgG...
Maybe it would help to clean up the supernatants - removing IgG before testing, and trying a monoclonal anti-human IgM. Another option is to use Luminex or other bead-based technology with anti IgM reagents.
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