Hello,
I am identifying nanobodies for my protein of interest. I have identified several different nanobodies showing specificity towards my target and conducted a validation assay (ELISA titration) to verify specific binding. My nanobodies contain both His tag and HA tag. For the the initial ELISA I used anti-his HRP conjugated antibody and identified the lead candidates. In the ELISA titration I used anti-HA HRP conjugated antibody (THE™ HA Tag Antibody [HRP], mAb, Mouse, Genscript). Unexpectedly, the ELISA signal was inversely correlated with the amount of protein (and thus nanobody) bound to the wells. The same happened using a positive nanobody control.
I replicated the ELISA titration experiment using new wash buffers etc. (nanobody candidates and secondary antibody was the same in both experiments) with the same output - inverse correlation. Can someone help me understand why this is happening? Is the antibody from Genscript (which was procured two days prior to conducting the ELISA) not working properly or am I missing something important?
Thank you in advance!
Hi,
My guess is that the His ELISA kit you are using is a sandwich ELISA, whereas the HA kit is a competitive ELISA. In competitive ELISA the assay signal is opposite to the concentration of the analyte (inversely proportional).
A target analyte that has a detection moiety (eg, a conjugated fluorescent dye) can be added, competing for binding sites on the antibody. A low amount of binding would indicate a high level of target analyte in the sample.
The signal inversely correlates with the amount of analyte such that, if the concentration of the target analyte is high, then the reference signal is diminished through its competitive binding to a limited amount of labelled antibody.
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