Dear all,

In the lab I currently work at we have rabbit sera, immunized against antigens of interest. I have isolated IgGs from these with a column (Protein A IgG purification kit from Thermo Scientific), and from the obtained IgGs I labeled 500 ug with the Ez-Link Plus activate Peroxidase kit (thermo scientific).

So far so good - SDS-page with Coomassie blue tells me the IgG is obtained very pure, and the HRP is functional as well.

But then the problem:

In ELISA, there is a high background when using these antibodies. I cannot seem to get rid of it. I've tried a number of scenarios:

Coat with IgG, block, add antigen, add IgG-HRP -> Signal

Coat with IgG, block, no antigen, add IgG-HRP -> Signal

Don't coat, block, add IgG-HRP -> No signal

Don't coat, don't block, add IgG-HRP -> No signal

Coat with IgG, block, antigen, no IgG-HRP -> No signal (obviously, but still).

Any scenario where both the unlabeled IgG and a labeled IgG are present results in high background. This should be impossible, as the antibodies come from the exact same animal and serum.

Our protocol for an ELISA is this:

- Coat with IgG in bicarbonate, 50 ul/well, for 2 hours at 37c

- wash 3x (washing buffer: 0,1% Tween-20 in PBS). Usually around 10ug/ml or 5 ug/ml, much higher doesn't seem to have an effect in getting higher signals

- Block overnight at 4c, 200 ul/well

- wash 3x

-Add antigen of interest, 50 ul/well, and incubate 2 hours at 37c

- Wash 6x

-Add IgG-HRP, 50ul/well, and incubate 1 hour at 37c

-Wash 6x

-Add TMB, and stop with 0,5M H2SO4

We're trying/tried the following:

- Lowering detection antibody concentration didn't work, as the signal:noise ratio didn't get much better and the signal from the positive samples got lower as well

- Other blocking buffers/diluent buffers. We're trying a combination of things in this regard: Gelatin, Triton X-100, Tween, BSA, FCS etc. So far not much improvement here. Our go-to blocking buffer is 1% non-fat-dry-milk in PBS. This is also used as our diluent.

- We've washed away as much unbound HRP as possible after conjugation using an Amicon column, just in case that makes a difference.

- We've put the coat IgGs on a Western-Blot, and used the IgG-HRPs as detection. Nothing visible (thankfully). But then what is causing the reaction in the ELISA?

As I'm typing this one of our brave interns is testing different TMB mixtures and a different HRP-conjugation kit, but I'm not sure if the answer will be found there. We're also trying a direct ELISA, using the antigen as coat and then simply putting the IgG-HRP on top to see how that goes.

After that, I don't really have that many ideas left.

I was hoping anyone here could have an idea.

Looking forward to any replies,