establishing of a selfmade elisa-help! - (Oct/19/2010 )

dear all,

we need some help from experienced elisa specialists for establishing of a selfmade human granzyme-b elisa from culture supernatant. now, we're using a commercial ready-to-use-kit (with standard protein, dilution buffer, antibodies and so on) but for the future, we need a cheaper way to check our samples (because we have to check a lot of samples for granzyme-b release).
this is not our first elisa and we have established elisa by our own for other projects but now we failed to established this one.
we ordered a recombinant granzyme-b protein (axxora/alexis) but we were not able to create a standard curve! now we have tested different coating conditions (coating anti-granzyme-b antibody from mabtech in pbs or tbs), we have tested different blocking solutions (bsa or milk in diff. dilution (pbs or tbs plus bsa or milk and/or tween), we have tested diff dilution solution for the granzyme-b proteins and we have tested diff concentrations of second biotinylated antibody and third hrp-conjugated antibody. but unfortunately none of our tested conditions gave us a linear standard curve (range between 1000 pg and 30 pg).
does anyone have experiences with selfmade granzyme-b elisa?? or does anyone have an idea regarding the composition of the dilution buffer for the standard protein? unfortunately nothing is mentioned regarding the composition of dilution buffer from the kit and i'm afraid that the protein is the main problem beacuase we have tested two of our undiluted samples and we got an enormous od for both of them ???

thanks in advance for your advices!!

flausch

You indicated you wish to make your own kit to replace one you have purchased and are using in-house. You also state that you do get a response but not linear curve and the concentrated protein yields a very high OD. You have basically replaced all the components of the commercial kit and are getting some strange results. I believe you wish to have your kit perform equally well as the one you purchased.

I would swap out the calibration (antigen) first. Run your dose response curve (dilutions) as unknowns in the commercial kit…do the signals/concentrations match? It could be that you may not be in the same analytical range as the commercial kit. Run your calibrators several fold higher and lower.

You can then swap the biotinylated antibody and test then do the same by testing your coated wells in parallel with those of the kit. It could be that your antibody's specificity is different.

With the liquid components you can check pH and protein concentrations to determine how much BSA or carrier protein to add. I doubt a dilution buffer has a 'magic' ingredient...substitute your diluent for theirs as well (just be sure to match the pH and protein concentration..and test with and without surfactant

I think from your info you are close.