Hi Pal

Well, at first place this kind of assay is not an Indirect ELISA. You are performing a Sandwich ELISA where you use an Ab as capture an other as detecting Ab. Is there any chance that the secondary ab will bind with the coating ab also? Totally sure that conjugated anti rabbit Ab will bind to both coating and detecting Ab. To avoid this undesirable reaction you may label the detecting Ab maybe directly with enzyme or with other molecule like biotin. Other approach could be to use Fab fraction from Ab 1 or 2 as coating and using a specific conjugated anti rabbit Fc for detecting the Ab 1 or 2.

Regards, Oscar

Hi Pal

Well, at first place this kind of assay is not an Indirect ELISA. You are performing a Sandwich ELISA where you use an Ab as capture an other as detecting Ab. Is there any chance that the secondary ab will bind with the coating ab also? Totally sure that conjugated anti rabbit Ab will bind to both coating and detecting Ab. To avoid this undesirable reaction you may label the detecting Ab maybe directly with enzyme or with other molecule like biotin. Other approach could be to use Fab fraction from Ab 1 or 2 as coating and using a specific conjugated anti rabbit Fc for detecting the Ab 1 or 2.

Regards, Oscar

Yes, an anti-rabbit IgG will bind both layers so the ELISA will not work.

You may label the secondary antibody layer with biotin and develop the sandwich with avidin-enzyme conjugate, for example

First of all, I would test separately, whether your anti-peptide antibodies bind to your antigen. You may coat a microtitration plate with your antigen and test the binding of your Ab 1 and separately of your Ab 2. In this case, you may use your enzyme-conjugated anti-rabbit Ab.