How many washes do you use with the initial wash step? What is the dilution of your secondary antibody?

The plates are washed at least 3 times initially. The secondary antibody is diluted 1/5000. I have also tried fish gelatin as a blocking buffer and high background is still obtained.

Hi Catherine, streptavidin is notorious for giving high background because it is very positively charged.

You could try doing your incubation steps with the  1' and 2' antibodies in the presence of 5-10% FBS. If that doesn't work, use RIPA buffers to wash.

Alternatively, you could try using Neutravidin from Pierce, it is an engineered streptavidin that is less charged.

try removing BSA and use another blocker instead (i.e. casein). I think your secondary Ab (rabbit anti-bovine) is recognizing Bovine Serum Albumin, probably because it was present in trace amounts when the rabbit was immunized with bovine Ig. I presume your secondary Ab is directly conjugated to HRP. 

As was mentioned, dilute your antibodies with a protein containing buffer (i.e. 0.2% casein with tween). If your detection Ab is reacting with BSA, using FBS would not work well.

Also, why are you blocking plates that have been pre-blocked?What were they pre-blocked with?

Thanks for both your suggestions, I will try diluting both antibodies in blocking buffers.

Hi Evan - The company simply states that the plates have been pre-blocked with "super-block blocking buffer". I have read the manual and it doesnt specify the exact blocking buffer.

I have blocked after the addition of my biotinylated peptides to ensure that any streptavidin sites still free on the plate can be blocked. I have also performed a test with no blocking buffer at all and the background is even higher.

Hi Catherine, the biotin-avidin interaction is the strongest interactions of all biological molecules. Their Kd is around 10-15 M. This is about a log off from a covalent bond.

In short, there is no way that you can block the biotin-avidin binding by using blocking buffers containing  proteins, serum, etc . (Unless they have free biotin in them, and almost all of them don't).

Essentially, you can shovel proteins into your assay all day long, but this will not interfere with the high affinity binding of  avidin-biotin.. 

I think that what you are seeing is non-specific interactions of the charged streptavidin with your Abs in buffers that have very-or-little proteins in them. 

So load up a truck with (non-biotin) proteins and dump it in your assay.  

If all else fails, try a blocking with pre-immune rabbit antibody, before using the final rabbit-antibovine antibody, The idea is to suck up all the sites that the second antibody  would non-specifically stick to before using that final reporter antibody.

Rethinking my previous answer, try blocking BEFORE the bovine antibody with a pre-immune sera from any species your rabbit antibody doesn't recognize. Pre-immune rabbit sera should work well here, or goat, horse, donkey, etc. ; anything that your rabbit AB doesn't react to. You need to reduce the non-specific adsorption of both detection antibodies, not just the second.

Thanks for all the suggestions. I will give them a go...fingers crossed.

It is necessary to control the background that added Tween 20 in washing buffer and antibody dilution buffer.

Further dilute second antibody. Dilution buffer should contain high amount of inert protein (up to 5%). If you do this non-specific binding may reduce. If non-specific binding still persist then switch over to neutravidin.