I think the use of pre-immune serum as a control for affinity purified antibodies is not a good idea, in part because it is so hard to compare similar dilutions but also because affinity purification changes the reagent so much. I have never found a rabbit serum (from the >300 I have looked at) that does not yield non-specific bands in Western blots, and often lots of such bands. Antigen competition is my favorite way to assess specificity. This entails incubating the primary antibody with purified antigen prior to adding to the blocked filter. This should clearly diminish, possibly (but not always, depending upon antigen concentration in the competition) eliminate the specific signal. This is particularly important in those cases when the antigen in question may not be migrating to the expected location in SDS gels. The same issues are relevant for immunofluorescence. A case in point is that I have been told by people in the field that ~50% of all rabbit sera stain centrosomes and it is common to pre-screen rabbits for such staining before use in raising antibodies to antigens that may localize to that organelle.

Hi Robert,

I would block for 1 hr at room temperature, even 30 mins is fine to block your membrane, anymore is a waste of time. I would put your primary antibody on overnight at 4 degree and increase concentration to 1:1000 or 1:500 (i have had to go to 1:100 with some antibodies), i know it is a lot of antibody. 

You mention you see a faint band with primary antibody, if it is a the correct MW then i would certainly increase antibody concentration. 

Hi Robert,

Your antibody looks promising, the faint band is ~52kDa, proteins not always migrate in SDS-PAGE as their calculated mw.

I use PBS with 0.2% Tween-20 (PBST) in all the solutions (you can use TBST if you prefer). The Tween will increase specificity. 

I use 5% skim milk in PBST with 0.02% sodium azide and block for 1hr (as Shane suggests).

Then I add the 1st AB to the blocking solution and let it shake at room temp over night (the sodium azide prevents the milk from getting sour). If you don't want to use sodium azide, incubate the 1st AB in 4°C over night.

Between the 1st and 2nd AB I wash with PBST (for 10 min X3). Then I add the 2nd AB (1:10,000) in 5% skim milk (without sodium azid) for 1hr.

If your band is still faint you can lower the 1st AB titer (1:1000/1:500) as suggested by Shane.

If to take a look at this problem from a different angle:

1) Is that possible that your tropical nematode worm (or structurally similar worms) might be a parasite in the rabbits you used for immunization ? In that case, the host (rabbit) might generate it's own specific antibodies to the protein you are interested in.

2) Another thing that you might want to consider is that some worm parasites might stimulate the immune system of the host causing non-specific activation of the total immune response. Try to change the animal host used for immunization. 

Robert, are you comparing an affinity purified antibody against the (unpurified) pre-immune serum?

You should use an antibody that is not biotinylated, probably the peptide  would be have  biotine fragments 

Thanks everyone for your answers:

Thanks Shane and Ziv for your recommendations: I will try to do the modifications you propose.

Hello Viktor: the information we have now indicates that this parasite is specific to humans only. But as you propose, we may try changing the animal used in immunisation.

Hello Wolfgang; I am actually comparing an affinity purified antibody against the (unpurified) pre-immune serum.

Juan Carlos, thanks for your point: the antibodies, I use are not biotinylated.

I think the use of pre-immune serum as a control for affinity purified antibodies is not a good idea, in part because it is so hard to compare similar dilutions but also because affinity purification changes the reagent so much. I have never found a rabbit serum (from the >300 I have looked at) that does not yield non-specific bands in Western blots, and often lots of such bands. Antigen competition is my favorite way to assess specificity. This entails incubating the primary antibody with purified antigen prior to adding to the blocked filter. This should clearly diminish, possibly (but not always, depending upon antigen concentration in the competition) eliminate the specific signal. This is particularly important in those cases when the antigen in question may not be migrating to the expected location in SDS gels. The same issues are relevant for immunofluorescence. A case in point is that I have been told by people in the field that ~50% of all rabbit sera stain centrosomes and it is common to pre-screen rabbits for such staining before use in raising antibodies to antigens that may localize to that organelle.