The question is, how big are the differences. Commercial ELISAs mostly have CVs in the range of 3-5 %. But anyhow I have some questions/remarks. (i) did you put a standard curve on every plate? Doing so is good practice. (ii) I know you feel that you loos material, but always work in double for all samples, controls and standards. (iii) If you need concentrations, make sure that your ODs fall in the most linear part of your logistic curve. Both in the lower and higher parts of your curve the measuring error becomes larger and larger.

First of all, thanks for responding my question @Freddy Dardenne.

1. I sure did put a standard on every plate.that's why I think my control should fall in to the standard curve so l can interpolated the concentrate.

2. Thanks for the advice once I figure out the plate to plate control deviation, becaus I only have one kit, ill make sure I duplicate.

3. Yes, I do make sure my diluted samples O.D fall into the linear part

Also, I figured out how to avoid dry out plates, I use suction instead of inverting the plates at once. Thanks again for the CV range information and the advice.

You might have given the answer yourself. I suppose that when you say “inverting “ you are talking about the washing steps. It is detrimental that no washing solution is left. So really invert them harsh. I used to actually hit them down on tissues on the lab table. And then obviously immediately piper the next step.