Indirect IgM ELISA Background - (Dec/14/2007 )
Hi Everyone
This question has probably been asked a lot but I am new to the forum so please forgive me.
I have developed an indirect ELISA based on a recombinant protein for the specific virus I am working with (plates are coated with the antigen). The ELISA works beautifully for detection of IgG antibodies in humans against the virus, but when I want to detect IgM I have non-specific binding (coat with antigen, block, react with serum, react with anti-human IgM HRPO, substrate). I get a lot of false-positives. I have tried to solve this problem, but I am still unsuccessful. I have tried converting it to a capture format ELISA where I coat with chicken (IgY) against human IgM, then the test serum, then the recombinant antigen, then the detection system because I read that rheumatoid factor is very similar to human IgM and could thus bind non-specifically. Another theory I have, which I still have to test, is that the fusion tag added to the recombinant protein by the expression vector, which is the thioredoxin bacterial protein for improved sollubility, might cause some non-specific binding. But why then only when I want to detect IgM, and not with IgG? Could it be heterophilic Abs? Does anyone have any advice for me please?
Thanks
hi,
u coat Ag then look for either igG or igM?
can u try to be bit simple with respect to ur system...
sravan payeli
This question has probably been asked a lot but I am new to the forum so please forgive me.
I have developed an indirect ELISA based on a recombinant protein for the specific virus I am working with (plates are coated with the antigen). The ELISA works beautifully for detection of IgG antibodies in humans against the virus, but when I want to detect IgM I have non-specific binding (coat with antigen, block, react with serum, react with anti-human IgM HRPO, substrate). I get a lot of false-positives. I have tried to solve this problem, but I am still unsuccessful. I have tried converting it to a capture format ELISA where I coat with chicken (IgY) against human IgM, then the test serum, then the recombinant antigen, then the detection system because I read that rheumatoid factor is very similar to human IgM and could thus bind non-specifically. Another theory I have, which I still have to test, is that the fusion tag added to the recombinant protein by the expression vector, which is the thioredoxin bacterial protein for improved sollubility, might cause some non-specific binding. But why then only when I want to detect IgM, and not with IgG? Could it be heterophilic Abs? Does anyone have any advice for me please?
Thanks