BSA is highly immunogenic and is a relatively large polypeptide, so contains multiple epitopes that can be recognized by antibodies in the antiserum.  If you immunize with BSA, conjugated or not, you will always get a high titer of anti-BSA antibodies. Steroids are relatively small molecules, less clones can produce antibodies recognizing them after immunization.

Do these anti-BSA antibodies interfere in the final application of your anti-steroid antiserum or not? If not, I dont see any problem in having them. If you expect an interference, you could try other carriers (like KLH) or even the conjugation of your hapten to small T cell epitopes, that could provide the carrier effect without  generating high antibody responses against themselves.

Dear Gertrudis Rojas,

Thanks for the suggestion. Yes, it seems that anti-BSA antibodies are interfering in ELISA  and not giving any gradient in serially diluted standards or samples. I am getting the constant OD in whole plate. Can I remove the anti-BSA antibodies from anti-steroid antiserum?

With regards

Vinod Kumar

Do your standards and samples contain free steroid or BSA-conjugated steroid. If the steroid is free, I would not expect any inteterference, but I dont know how is your assay designed. Perhaps the problem is a different one.

You can remove the anti-BSA antibodies with a BSA column or with BSA-coupled beads, but if the titers are really high, it will be difficult (although not impossible) to achieve 100% depletion.

Alternatively you can use BSA in your ELISA diluent to block anti-BSA antibodies without removing them.

Could you give more details about the assay to understand what could the anti-BSA be doing?

Thanks for the answer. I did direct ELISA in which I coated some wells BSA only and some with Steroid: CMO: BSA (same immunogen used for immunization) then I added the serially diluted antiserum followed the incubation and secondary antibody with HRP. Eventually, wells containg only BSA have OD almost similar to the wells have steroid:CMO: BSA. At serially diluted antiserum , BSA containg wells should show less antibody titre than steroid :CMO BSA?

If we use high persentage of BSA for blocking the non-specific binding sites, will BSA antibodies bind to the BSA??

With regards

If you want to know the specific  titer against your hapten the best way is to present the hapten in a form that is different to the one used in immunizations. For instance, you could coat the hapten alone (if its possible) or conjugated to a totally unrelated carrier. In that way, the anti-BSA antibodies would not interfere with the titration.

The comparison of the absorbances against BSA and conjugated BSA (the experiment I think you did ) could give you a clue, but if anti-BSA titers are really high (as they are expected to be, by the way) detecting the differences can be very difficult. 

You could also add to the antibody diluent high BSA concentrations to make sure that anti-BSA antibodies cannot bind to the coating conjugate, while anti-steroid can still bind. But the most robust solution would be to use the hapten without VBSA in the ELISA.

Thanks for the suggestion!