Hello,
End of last year I have developped an indirect ELISA method to detect a couple of caseins in fermentation supernatants. It worked great until a couple of weeks ago.
Since then, for each plate we are running (6 plates as of today), their are black aggregates developping after the addition of H2SO4 (stop solution). They are increasing with time starting from the addition. They are not present after incubation with TMB.
Does anybody experienced the same thing? And/or does somebody knows what it could be and how to fix it?
Thanks in advance,
Julie
Dear Julie
Since I don't know about the ingredients used in developing your ELISA kit, I cannot guide you precisely. Nevertheless, I recommend you to use HCl instead of H2SO4 as the stop solution. Appearance of black aggregates may be due to a kind of oxidation process caused by H2SO4 which is a known oxidant (unlike the HCl).
Dear Alireza,
thanks for your answer. I'll try that, it migth help.
We are using the same TMB reference for the last 6 months and the aggregates have been appearing since 3 weeks without changes in the protocol. The changes are different lot of plates and TMB. Secondary antibody seems to participate to these aggregates formation (tests of last week), even if we are using the same bottle since the begining.
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