Developing a hybridoma cell line which produces IgM antibodies. Has anyone ever seen problems with non-specific binding in screening these antibodies?
And what kind of IgMs are you producing? Is it something with known specificity or are they naturally occurring IgMs? How was the specificity tested? Are you sure, it's not a problem with the method itself? Did you try to adjust the concentration of IgMs when performing that test? Are your cells growing in the presence of serum? Presence of serum can enhance false positive signal if you were testing the cell culture SN. Or were they purified?
You gave too less information about the experiment setup to get the helpful answer.
Care to share more? :)
Veronika
HI Veronika,
I am developing human IgM monoclonal antibodies against Dengue virus antigens. I got blood from a patient with acute Dengue infection, isolated out PBLs and transformed with EBV. I then fused the transformed cells with a mouse myeloma cell line. I would like to develop a cell line which secretes human IgM antibody against any of the Dengue antigens (1-4, don't mind). So I screened my hybridoma cells with a commerical elisa for Dengue IgM. I used the Panbio elisa. I got some really strong positive hybrids which I then cloned by limiting dilution. I screened my cloning trays with the same Panbio assay and basically through multiple rounds of cloning established a cell line which secretes a human IgM reactivie in the commerical Panbio elisa. However, if I screen this supernatant in another commerical elisa, I don't see reactivity. The Panbio elisa has all 4 dengue antigens in it. The other commerical elisa I used also has all 4 dengue antigens. So I'm not clear why the antibody would not work in both these elisas. Any thoughts? Your help is appreciated.
I presume that you have appropriate positive and negative controls for both ELISA systems that rules out the possibility that one of them is not working properly? If so it sounds as if your selected clone is reacting to something that is specific for just one of the two assay systems, perhaps reacting with a contaminant? My suggestion is that perhaps you should go back to your earlier stages if possible and use both ELISA assays as your selection of hybridomas suitable for cloning.
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