high background in the ELISA - (May/25/2011 )

Hi,

I am new for the ELISA. I have some problems for my Elisa assay and i hope i can get good suggestions from all of you.

I use the Ni plate to do the ELISA. I use the 2ug/ml protein (in PBS) to coat the plates; PBS/0.5% BSA as a blocking buffer, PBS/0.05%Tween as a wash solution, 2nd ab for human serum is peroxidase labeled goat anti-human IgG. My problem is : 1, i almost got the same result for the coated wells and non coated wells, high read. Is it because the non-specific binding or other reason? do i need to change the blocking buffer? 2, what is the good plate for the ELISA? 3, any other suggestions for the new ELISA people? Thanks.

I assume from your post you are detecting IgG in human serum. What dilution of serum are you using? What signal do you get in ELISA? Which substrate are you using? Human serum has a high tendency of giving a high background. If you can, dilute it more. Otherwise you can use special dilution buffers to reduce background but I have never had very good results with that.