Hey Lejla,

I have had a hybridoma culture with two different monoclonals against different epitopes of the same target protein. So, in theory, it could as well be a mixed culture with mAbs against two different receptors.

Have you tried a limiting dilution to get them apart?

Christiane

hi Lejla

 I think you should do a limiting dilution on mixture of two monoclonal antibodies. after  supernatant of each clone should be tested by indirect ELISA using specific antigen or you can test  supernatant of each clone using western blotting to identify the specific antigen that recognized.

Hey Christiane and Samaneh thank you so much for your reply! This is greatly appreciated! 

I have been working on characterizing my antibody and I’ve been doing a lot of SDS-gels as well which are showing an extra band around 25-30 kDa. We think that it is a glycosylation variant. When we also cloned and sequenced the cDNA to find the sequences thus coding for the glycosylation variant, we found nothing. This has become very puzzling and was very curious if you and others have experienced the same phenomenon.

So right now I have started all over again where I have made dilutions and currently doing ELISA and WB to identify the monoclone producing the one antibody that will recognize the receptor protein I’m working with.

Best

Lejla