I have been developing mAbs against the native form of a human transmembrane protein. Mice have been immunised using NS0 cells stably transfected with the native form of the protein tagged with GFP. The neat supernatant (contains non-IgG's mAb, possibly IgMs) of the Hybridoma gained from this showing a good binding with the recombinant form of the protein (tagged with GST) in ELISA. Therefore, I've used the GST as a negative control. Surprisingly, the mAbs bind with the GST alone even higher than the recombinant protein!
The mice (Balb/c) are taken with ages 8-12 weeks, and they've never been immunised against any external Ag (so that GST as well).
The hybridomas neat supernatant shows a good to moderate binding with the native form of the proteins (on the cell surface of A549) using FACS.
How we may explain this binding?
Do you think that GST could address a special case of Ag-Ab reactivity/binding?
What you would recommend of assays to see the specificity of the mAbs?
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How about trying to rum western blot with your mAb to native protein, GST and tag-GST? This might give a clue where your mAB binds on and also to confirm the mAb binding.
Thank you I-Hsuan Chen. I'll try this, and it would be interesing to pull-down the protein of interest with the mAbs we generated, this might also give a clue. Still how this non-specific binding with the tag but not the recombinant protein make me confused to explain the binding in ELISA.
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