Corticosterone antigen as standard for Direct ELISA - (Jan/07/2008 )

Hi

Hope someone can help.....I'm trying to use a Corticosterone antigen for a Direct ELISA and I've tried reconstituting it in both 1xPBS with 4% ethanol (does not go into solution well at all) as well as 85% ethanol (goes into solution immediately). I've tried binding it to the ELISA plates at 2 and 4hrs as well as overnight at both room temperature and at 4 degrees. My plates are blocked using 1% BSA and washed using PBS with 0.05% Tween. After addition of my TMB solution I do get a signal produced however it is extremely weak and I am not seeing a change in concentration for my standard wells as I should. Any suggestions would be greatly appreciated!

Thanxs:)

Are you doing this using an ELISA kit for Corticosterone or you are trying to make your own?

Using a kit would have been so much easier however, in this case I am trying to make my own. With respect to that I am using a monoclonal Corticosterone antibody and mouse HRP conjugated secondary both diluted in 1% BSA.

This is why I asked you. Small ligands dont stick to the plate tight enough and the detergent-containing wash buffer can strip them off. Also, if the ligand absorbs to the surface, how can an antibody recorgnize it? I believe the correct way to do so is using an indirect, competition method with a plate coated with ligand conjugated to BSA or other carrier protein, then co-incubate your samples containing the ligand with antibody in the wells, followed by washing and HRP-secondary antibody incubation. I am not an expert on this and you may need to research it with relavent refs.

Thank you so much, that is very helpful!