Sandwhich ELISA using pig serum - (Sep/16/2008 )

Hello all,

I was wondering if you could provide me with some feedback. We are trying to use sandwhich ELISAs to look at cytokine levels in pig serum at certain time points following surgery.

The standards are working perfectly, and so are the positive controls, but the readings for the serum samples are all over the place and completely not what we expected. This is no minor surgery so we should certainly see significant cytokine concentrations, but that is not at all what we're seeing. The values are extremely low and we are even getting some negative values once we subtract background.

We tried to spike one of the serum samples with standard but when we do, we only get about 40-50% recovery.

Could it be that the cytokines in our samples are being bound by something in the serum (for instance, albumin)? We've tried everything we could think of, including treating the samples with acetonitrile and filtering the serum but we're getting NOTHING.

This is quickly becoming an expensive problem, so if anyone could provide us with some feedback I would greatly appreciate it!

Heterophile abs. Abs in the samples against abs in the assay. Only way to remove is review your data and locate high false positives. Add into your sample buffer non-specific ab (IgG) of the species used as capture and detector in your system and check with the false positive. <<use this buffer and pre-incubate the samples to bind up the heterophile ab>> Once the false positive is reduced or eliminated test a select few of your samples. Hopefully, the samples will line up according to the conentration you expect.

In addtion to this try diluting the samples by a factor of 10 and do a small screening test with FP, FN, TP and TN. If the dilution step corrects the problem you can avoid having to try to block the heterophile abs.

Everyone sees this problem. A more complicated time consuming approach is to use F(ab)'2 abs (remove Fc) which will also reduce heterophile.

Or change your capture/detector ab species.

Or in place of species non-specific IgG you can try neat sera (of same species as you system abs).

Lastly, there are commercial blocking reagents which may work for you: Scantibodies, Omega, Bioreclamation are the company sources.

I hope this helps!

hi,
i would assume the same, your antibody is binding to some other proteins in the serum.

just to figure it out, run the serum sample in the gel in different concentrations, and probe with your antibody, you can see the contaminating bands (size) then implement some minor purification protocols if possible.

if your cytokine exists in too low concentration, perform IP in pig serum, then perform western blot with same antibody. if you see multiple bands.. that would give you hint about contaminating protein profile. If this is the case then you should go for more specific antibody for your experiment.

gud luk