I am using a Gen 5 plate reader system and I am trying to set up a protocol to read a checkerboard titration for an indirect sandwich ELISA I am trying to develop. Would it be correct to define all my values as samples since the protein I am working with (Factor V) does not have a known concentration in plasma in mice and I am just doing dilutions of the antigen against dilutions with known concentrations of capture antibody and constant primary and detecting antibody concentration? I included a photo of how I am planning to run it.
you can start the dilution horizontally, (1 to 11). It would be either in form of 1:10 or 1:2 serial dilution in triplicate ( suppose A to C 1:2 serial dilution ), ( D to F 1: 10 dilution), keep two wells empty without Ag. Then read the plate against primary Ab and 2' Ab, If read is range ELISA reader in one set of wells, Thus find the dilution of that Ag. If require further dilution, you can do it.
In this way you can also find dilution of 1' Ab against known dilution of Ag