I have done the first step of optimization of indirect ELISA. It is "optimization of antigen coating". Coating and other procedures are correct. The problem is with evaluating the absorbance values. As it is just a step for selecting the optimum Ag amount for coating, should I consider the linearity of absorbance values? Our ELISA reader have linearity within 0-2 Abs and values read-out range within 0-3.5 Abs. All the values of positive sample show values higher than 2 Abs. But there was a order. When the Ag amount is reduced, absorbance values are going to be lower.
As according to my knowledge, linearity should be optimize with the sera and conjugate concentration which are the other steps of indirect ELISA optimization. Am I correct? Does linearity affect for selecting best concentration of coating antigen?
i think you have to follow ELISA Guidebook chapter 4 (titration of reagents) which discuss in details the chessboard or checkerboard titration of reagents used for both direct or indirect ELISAs and how to deal with poor results and that all are attached with this replay i hope you gonna find that helpful
i think you have to follow ELISA Guidebook chapter 4 (titration of reagents) which discuss in details the chessboard or checkerboard titration of reagents used for both direct or indirect ELISAs and how to deal with poor results and that all are attached with this replay i hope you gonna find that helpful
Be sure of blocking and several washes during ELISA protocol. Also try different pH for coating buffers.
You will have to optimize coating antigen strength and antibody concentration under defined conditions by checker-board method. You may refer any standard Immunology manuals. Lowest amount of coating antigen giving OD value of 1.0 with highest dilution of your antibody are ideal and optimum conditions for detection of ab of interest in your test system, by indirect ELISA. Appropriate blank should be there in every plate which should read OD of 0.00.
I agree with the checkerboard method, this is the first step in optimization. You must find linearity between your antigen and serum concentrations while keeping the conjugate second antibody concentrations constant. Read papers on your specific detection molecule for ballpark concentrations. If it is HRPO, I have had good luck with 1:2500 in TBST (or PBST) when using TMB as a colorimetric indicator. Linearity between antigen and serum concentrations is extremely important because it indicates specificity. If it is not linear, you may be experiencing non-specific binding of your primary antibody to something other than your antigen and this will make further optimization difficult. If you are experiencing non-specific binding, consider different blocking formulas, such as non-mammalian block if you are working on a mammalian pathogen.
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