I've got a humanized bi-specific monoclonal antibody targeting to PD-1 and CTL-4. how can i verify the function of it? what experiment should be taken?
What is your purpose? First, you could test whether the antibody binds both targets, but further experiments depend on what you want to do with the antibody at the end.
What is your purpose? First, you could test whether the antibody binds both targets, but further experiments depend on what you want to do with the antibody at the end.
I'm presuming you mean CTLA-4, right? You will need 2 cell lines - one CTLA-4 positive and one PD-1 positive. Presuming this is human, you can check ATCC for potential target lines for each antigen.
First check that your antibody binds each of the target lines individually. You'll need a secondary antibody to bind the bi-specific mAb. If the antibody does bind both target lines, you next add the antibody to a 50:50 of the two cells and see if it aggregates them. By flow cytometry, this could be evaluated by looking for doublets that light for other antigens on both cell lines. Alternately, examining the cells under microscopy to see aggregate formation may suffice for a first round test. If the CTLA-4 cell line has CTL function, you can look for killing of the PD-1 target cell via a classic Cr51 release test.
Thanks for your suggestion @Gary Lee Gilmore.@Abel Tan @Gertrudis Rojas. that's very useful for me. Binding activity of antibody with the target antigen and 293T-PD1 and 293T-CTLA4 (cells were transfected with plasmids containing PD1 or CTLA4 gene, and cells could high express the target gene on the surface of the 293T cell) cell have been conducted by ELISA and FASC method.
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