I'm new to this site, but I'm desperately seeking some assistance. I've been running a sandwich ELISA in my lab for the past 2 years and never had any major problem until a few months ago. I did not make any drastic changes to my protocol or reagents used, but I suddenly started getting very high positive results, either for just the samples I was testing, or for the entire plate (standards and negative control and all).
I thought I solved the problem by sterilizing everything and ordering new supplies of everything, because my ELISA was working for about a month, until recently. I tested some new samples, and they all came out as high positives (which is very unlikely), but when I tested some older (tested previously) samples, the results came out exactly as I expected (i.e., not all high positives).
I have a few questions: is there some sort of generic problem that could be causing this? Like error in washing, too much conjugate, etc.? Also, I dilute my samples with HBSS (Hank's Balanced Salt Solution) - could there be some error in the HBSS that could be causing complications? Like a low/high pH or some such thing?
I appreciate any advice/assistance that I can get. Thanks!