Monoclonal Ab Western and ELISA issue - (Nov/07/2008 )

Hello all. I work in a lab that produces monoclonal antibodies for different labs around campus. For a multitude of reasons up until now we have been met with little success. However now we are producing some beautifully performing antibodies for people.

My question relates to one issue we seem to be having. Until recently we have always tested sera by ELISA as a check to verify we have antibodies in the mice and we give the majority of the sera to the investigators to see if it works in whatever assay they are using. When they tell us things look good enough to move to fusion we then test the fusion wells by ELISA. If the ELISA score is positive then we pull that sup and give it to the investigator to test. If the score is negative we trash the well. A few weeks ago we began having people test some of the negative wells and in more than once instance the investigators have come back and said one of the wells we thought was negative actually work rather well in their western blots.

Is there a rapid, efficient, reliable test we could perform in conjunction with the ELISA or perhaps replace the ELISA with that would catch these wells that we detect as negative in an ELISA? Most of the people we work with do not have the time or resources to commit to testing 900 or so wells at a time to see if something is there nor are we equipped to do so as we run maybe half a dozen fusions at one time.

Sometimes, the mAb was negative in ELISA, but positive in WB.

ELISA test the ability of mAb binding to the linear peptide; however, in WB the protein is denatured.

Therefore, some antibody companies will specify the use of mAb whether it is suitable for both ELISA and WB, or just WB.

Hope this may help.