I recently developed a few mAbs that are able to neutralize SARS2. By ELISA and WB, I found that they all bind to SARS2 RBD. I wanted to map their RBD epitopes so I ordered pre-coated plates with each well containing a 15-mer peptide spanning SARS2 RBD. There was a 5aa overlap between each peptide/well. I wanted to make sure I was doing everything right so I just tried it with one of my mAbs and found that the mAb bound to the first 8 wells, i.e spanning the first 44 amino acids of SARS2 RBD. I was expecting just one single well to light up, not 8 of them. Anyone can tell me what I could do to improve this? Or how this can be explained? I don't want to test the rest of the mAbs until I know what's going on to not waste the plates/peptides.
Hi,
Are the peptides just randomly coated onto the plates you are using?
Recognition of epitopes occurs through interaction between structured domains: the complementarity-determining regions of the antibody on the one hand and the epitope in the antigen on the other.
The higher order structure of the epitope is probably largely, if not completely, lost in the plate you are using, which could lead to false positive, or false negative, results.
An(other) example of a difficult antigen to target in an ELISA plate is CD20, which also needs to be in a certain 3D configuration to be recognized by antibodies like rituximab and ocrelizumab: linear peptides containing the known epitope do not work.
Hope this helps!
Eef Dirksen The peptides are biotinylated coated on streptavidin plates. They are not randomly coated but are coated in their amino acid sequence. I did consider that they may only bind to 3D/conformational epitopes but I did Western Blots first under denatured conditions and the mAbs bound to the linearized/denatured RBD which means that they would/should bind to linear epitopes and ideally bind to an individual peptide (which is linear). This is why I'm not sure why one mAb is binding to 44 consecutive amino acids.
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