Recently I used indirect ELISA and FACS to compare some Abs' binding affinity to a same cell surface expressed antigen. Results showed that some Abs get to saturation fast, which apparently have good binding affinity, but some did not reach the plateau even at the highest concentration. Given that limited antigen presented in both assays, I am confused why the signal generated by unsaturated Abs is higher than other's saturated signal. Could anyone explain this phenomenon to me?
Is there a difference in isotype between the mAbs that might explain what you are seeing owing to different specificities of the secondary (enzyme-labelled) antibody?
Yang Xu , another possible cause is non-specific binding, i.e. the mAb aggregating on the surface of the chip/plate. In those cases you never reach a plateau or reach it only at very high concentrations. Or, depending on the antigen, if the mAbs are directed against different epitopes the effective number of available binding sites on the surface may be different for different mAbs, hence they reach saturation at different concentrations.
- Badges
- Science method
- Similar topics
- Medicine
- Immunology
- Immunology Techniques
- Immunoassay
- ELISA