KLH-based immunization protocols: suitable for hybridoma production purposes? - (Jun/09/2008 )
Have any of you tried to immunize mice, for hybridoma production purposes, with the desired antigen conjugated to KLH? My concern is that the high immunogenicity of KLH will make that the vast majority of hybridoma clones will produce Abs against KLH, thus making it difficult to find clones producing antibodies against your desired antigen, is this true?
You can make two different antigen conjugations, one to KLH to immunize, and another to BSA or OVA to the ELISA screenings, thus the KLH specific antibodies will be discarded and your positive wells will be for a specific response against your peptide.
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Surely, KLH-conjugation is a pretty standard procedure and it has worked well in our lab. Also, it is not absolutely necessary to prepare an additional conjugate for screening, you can also screen against KLH alone and remove all clones that produce antibodies reacting with it and retain only those that react with the antigen but not KLH.
Smoky, thanks for your answer, and now a very direct question: in our hands, for each mAb against our antigen of interest, we have obtained 100 or more against KLH. Is this your experience as well?
It can happen. I guess it depends on the immunogenicity of the antigen bound to KLH.
I think that one time we actually got only clones reacting with KLH.
But usually a few good mAbs are sufficient, so even if you get many more against KLH, the quality of those against antigen is what matters.