ELISA help needed

rick schuman virion at radix.net
Mon Oct 21 11:20:33 EST 1996


I have been watching the responses to Martin Oaks concerning high "background" 
in his ELISA.  I suspect that the problem is not background, but cross 
reactivity.  As described, the assay procedure requires addition of human serum 
at one stage, and later the addition of HPR-labelled anti-human.  Unless the 
human serum has been stripped of Ig's, the immunoglobulins are probably 
sticking and are there for the reporter antibody to detect.  This is likely to 
happen even with blocking.  Even detergent will not eliminate this problem if 
the percentage of human serum is too high.

If detergent is not included in the wash buffer and serum diluent, its addition 
might help the problem.  A better solution would be to use the humanized 
antibody as the capture antibody and the mouse monoclonal to detect the 
analyte, followed by a rabbit or goat anti-mouse - HRP that has been human 
serum absorbed.  Another alternative is the use of an antibody from a different 
species on the top half of the sandwich.  It is also possible that the human 
serum contains sufficient amount of the analyte so that the human serum could 
be used as a coating antigen.  The analyte could then be detected with the 
mouse monoclonal.

Good luck, Rick Schuman



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