I have some issues with sandwich ELISA that I hope that this forum can help me clarify.. I depend on all of you
Here is my situation: When performing sandwich ELISA I am coating wells with NaHCO3/Na2CO3 buffer and monoclonal mouse antibody. Afterwards I apply blocking buffer followed by the antigen at varying concentrations, rabbit antibody, goat antibody, TMB, stopping reagent.
I prepared a blank on same plate where I place everything in above sequence excluding mouse antibody and antigen.
Results: blank and ALL sample wells with VARYING concentrations of antigen give the same colour no variation in optical density...no gradient in colour. I have repeated experiment more than once yet I get the same results.
An overseas researcher uses the same protocol as me but actually obtains good results using same antigen concentrations, same antibodies, same supplier for reagents and antibodies.
What the heck am I doing wrong? Please Help!!