I had similar problems .

*I used 3 times wash in between each steps.

*Dried the well plate tapping on a paper towel ( make sure there is no liquid left from the previous step).

*TMB time can be reduced.

* Be careful about contamination /Change tips if you think it touched the plate while adding solution.

How did you select the dilution of your conjugate? Is it the recommended one? I would suggest to try more diluted conjugate, even to optimize conjugate dilution in a small experiment. Too concentrated conjugate can give rise to huge a background. Are you diluting the conjugate in the same blocking solution?, this is quite important.

As previously mentioned by others, I think the background might be from the Blocking buffer and can you try a different blocking buffer (like BSA) as your negative ctrl also has positive signal.

The problem as I can see it is that even your negative control is giving high background so I am excluding the washing steps factor

I do agree with Dr. Rojas answer, however, in my opinion, even though if you put enough antigen (in your case 1ug/ml), probably it will bind but not too well, therfore, it is important that we have proper coating buffer which is 1XPBS which is pH 7.4

These points are highly important to have a successful binding between the antigen and the antibody phage

Thanks for all of the advice. Unfortunately I am still getting high background readings on the negative control (as high as the positives).

Just to clarify:

- I am washing at least 3 times between steps with PBST and PBS

- I always ensure the plate is dry by tapping hard onto a paper towel

- If I reduce the TMB time the negative reading is still as high as the positive reading, albeit with a lower OD

- When adding the test antibodies I am using unique pipette tips for all, so no risk of cross contamination

- I have tried blocking with 4% milk, with 4% casein, and with 1% BSA

- The secondary antibody is diluted 1/1 000 in accordance with protocol, although I have tried a 1/10 000 dilution with the same results

- I am coating with 1 µg/mL antigen in PBS

- I have tried an alternative secondary antibody and I get the same results.

My next attempt will involve using 3% FCS as a blocking agent, and diluting the secondary by 1/100 000. Any additional advice and input will be warmly welcomed.

Many thanks.

Two additional questions

Are you diluting the secondary antibody in blocking solution as well? This can be crucial.

Is there any possibility that the anti-myc binds the coating antigen? Could it be myc-tagged?

As Gertrudis says, maybe the problem is about some specificity lack of your primary antibody, and if so, there is nothing you can do but generate a new one, because casein and BSA would be good enough for blocking, they work very well as blockers usually.

Anyway there is still some tricks to perform, check your neg control is not contaminated with labelled Ab; also try to rise the Tween concentration to 0.05 or 0.1%, and the washes number starting with 5 for coating, reducing to 4 for primary Antibody and 3 for the conjugate.

Thank you all for the advice. I managed to finally bring the background down by blocking with 3% BSA, diluting the secondary in blocking solution, and by only adding 50µL of secondary instead of 100µL. These three things in combination seemed to have done the trick. I'm not sure if it was any one of them specifically or a combination of all three.

Thanks again.

Good. Usually, mostly of ELISA works fine with 1% BSA blocking, but sometimes it is not concentration enough. In my experience Nunc Maxisorp plates are more powerful for protein adsorption than the same characteristic for Costar or Greiner ones., that's why troubleshooting with background could be seen.