Sandwich ELISA giving too many positive results

[fishgirl]

Hi everyone,
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!

[NATHANALANPATRICKROGERS]

Originally Posted by fishgirl

Hi everyone,
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!

In my experience, overly-strong positives are usually the result of sample contamination/residue in supposedly clean plates, under-washing, or unsufficient blocking [of non-specific binding].

[fishgirl]

Thanks Nathanalan . . . - I agree that something must be contaminating the samples, since they are the only ones that are turning out high positive. I'm trying to eliminate anything and everything as a source -

is there a chance I leave my sample whirlpaks open for too long when I'm adding the HBSS and they're exposed to environmental factors? Or could a low pH in the HBSS result in a false positive sample? Could it be a simple case of cross contamination from sample to sample (I feel like this is not as likely, just because it would take one very high positive sample to contaminate the other 59 samples)? Once my samples are homogenized, they go into tubes with PBS-T20 w/5% goat serum and citrisolv - are there any known common problems with PBS-T20, goat serum, or citrisolv that can result in high positive samples?

Sorry to go on and on, this has just been plaguing me for a bit and I'm trying to pick people's brains as to what the problem is. Thanks!