I have been developing a mAb against the native form of a mammalian trasmembrane protein. Mice immunised using cells stably transfected with the native form of the protein. The neat supernatant of the Hybridoma gained from this showing a good binding with the recombinant form of the protein in ELISA. This recombinant protein is tagged with GST - therefore, I've used the GST as a 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 never been immunised against any external Ag (so that GST as well).
The immunisations done using NS0 cell line transfected with the gene of interest tagged with GFP.
The hybridoma neat supernatant shows a good to moderate binding with the native form of the protein (on the cell surface) using FACS.
Dear Scientists, how you 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?
Any contribution/answer is much appreciated!