I'm having some problems in Indirect ELISA Developement.
In my case , the antigen is c-erbB 2 (Human recombinant C-ERBb2 , GST fused ,Calbiochem).
The antibody is not available in pure form commercilly. So I'm using Protein G based IgG purification kit to purify Mouse IgG1 520C9 from hybridoma culture sup.
Detection antibody Rabbit antimouse IgG -HRP
Substrate =OPD, Read @ 492 nm
Antibody range 100 ng/ml to 1 pg/ml
detection antibody 1000ng/ml
1% bsa for blocking
Problems are following:
Control ( without antigen ) is around 0.3
though I'm varying the concentration of antibody (100 ng/ml -1pg/ml) in different wells, i'm getting more or less same absorbance values in all the wells. I'm not getting any trend, i mean I' m not getting higher value for higher concentration of antibody. even i'm getting low valeu where high antibody concentration has been used.
besides, i would like to mention that, in a well , coated with antigen, but without antibody, when detection antibody is added...that control is always giving higher value( around 2.2).
I'm really confused and trying for last several months.
hence, i'm earnestly requesting you to consider my case ...pls give me some suggestion.
i would like to know, whether my detection antibody choice is right or wrong.
is there any possibility that the detection antibody can bind with antigen?
pls help....give some suggestion...