I am running an ELISA on mouse serum that has worked beautifully in the past, and for some reason now it doesn't work for either the standard or the samples. I keep getting almost the same ODs for all the samples and I can't figure out why! I thought maybe the new HRP conjugated streptavidin was more concentrated than the old one, so I made sure to wash the plate very well the last time before adding substrate, but this did not work.
Has this happened to anyone else?
Thanks,
Alex
Among possible reasons, you might want to consider that your HRP conjugated streptavidin might interact with some components in your blocking / washing buffer. For example, we got some evidence showing that TBS(T) Blocking Buffer from Pierce (which is used for ELISA and western blot) somehow interferes with the biotin-avidin reaction both in ELISA and western blot. In ELISA we observed very high background eliminating any differences in absorbance (similar to your observations). Try to change your Blocking/Washing Buffer.
Do you have negative samples or other kind of negative control? If so, does the problem affect them as well? I would try to know whether you have non-specific background or specific ELISA signals that are too high and cannot be distinguished. The solutions for these problems can be different. If you changed the conjugate, probably you need to titrate the new one, testing several dilutions, using a smalll model experiment (for instance, only with the standards and the engative controls).
If negative controls are not showing the same OD as your test sample i.e., if negative control of serum and that of antigen are not giving false positive results, then it might be due to the use of a) more concentrated serum b) less amount of antigen, because of which gradient/difference was not obtained. Determine the n titre value of serum and use dilutions accordingly.
Please, please, when asking a question like this, give a brief but full description of the assay. Plate type X was coated with antigen Y in Buffer Z for so many hours at temperature C. Washed with buffer B, n times, Blocked with ?? for ? at ?. It take a lot of the guess work what fault may have developed. If you must be vague because of commercial reasons, say so. You state you have a new HRP conjugated strepavidin. Is this a new batch or a new supplier?
I agree with Richard: It seems a systematic error, but without the experimental details, it will remain every one's guess where the problem is.
All steps your ELISE necessary to control once more. Start from conjugates HRP-strept. and biotin-protein. Need to titrate, to detecte work-dilutions of conjugates. Titers lower sometimes.
So far as mouse serum is it mono-Ab?
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