Hi, I preformed a competitive ELISA (for SEMAGLUTIDE protein) and the blank STD which have the highest OD, turned out lower than the usual ELISA I do with these kits. Usually in Semaglutide ELISA I am waiting for OD at 620nm to reach 1 and than I stop the reaction to reach OD ~3., which take around 30 minutes. This time, using the same lot of the kit, I waited 1hour and still did not reach 1 OD, only 0.6 OD at 620nm (~2 at 450nm). What could be the problem? I want to perform another test but don't want this problem to repeat.
If the signal in your ELISA test turned out lower than usual even after extending the color development time, several factors could be contributing to this issue. Here are some potential reasons and troubleshooting steps:
Start from the basics: What did you differently from other times you ran the same ELISA? Maybe you don't remember but there has to be something different. Washing steps are particularly critical, as you probably know.
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