we are developing an anti spike sars-COV-2 ELISA. we have a high rate of false-positive in BALB/C mice serum. 50% of mice show false-positive results. after check different parameters, we found that the problem is with sample diluent, as with one commercial sample diluent everything is fine and we have 99% specificity. we checked every possible concentration of tween and BSA in PBS for sample diluent but didn't get any result. is there other any specific sample diluent formula for reduction of non-specific binding?
thanks for your kind responses.
Hello
The reason for high false positivity could be because of BSA in the sample diluent.
BSA contains bovine IgG. Many secondary antibodies will react with bovine IgG. Therefore the use of BSA either to dilute samples or in blocking may increase background.
I suggest you use BSA which is IgG free and protease free. You could also use normal serum from the host species of the labelled secondary antibody.
Best Wishes.
You have probably solved this by now but I have used 4% casein hydrolysate in PBS (which is cheap) to block /dilute (dilute + 0.01% tween 20) in what may be a similar ELISA to yours