Weird Reuslts with Sandwich ELISA

[dave2006]

Yoodles People!!
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!!
:mf_surrend er:

[tgd]

Hello,

I think we need a few more experimental details here. What do you block with and is the blocking reagent included in all antibody-containing solutions for example?

However, one thought springs to mind immediately. Is your 'goat antibody' anti-rabbit or (by any chance) anti-mouse? It should of course be anti-rabbit, for obvious reasons. I am presuming that by 'goat antibody' you mean goat (anti-rabbit) peroxidase conjugate or goat (anti-rabbit) alkaline phosphatase conjugate, or the equivalent. I am probably wrong, but the fact that you colleague gets the ELISA to work suggests some subtle mistake?

Good luck,

tgd