What you have suggested shall work, and an indirect ELISA for detection of Ag-x shall be a better option. Coat the ELISA plate with 1:100 dilution of bovine anti-x serum prepared in bicarbonate buffer of pH 9.3, followed by blocking with BLOTTO or BSA. Put diluted Ag-x preferably 10-fold serial dilutions (1 mg/ml starting conc) followed by addition of Ant-Man mouse serum. Last step will involve use of anti-mouse-HRP or AP followed by addition of appropriate substrate-chromogen solution. This method shall work.

1. For coating capture antibody use a range of 5 - 25 ug/mL in bicarbonate buffer as suggested by Shamsher. Incubate for an hour in humid environment, wash and block. Try different concentration until you find the best signal/ratio.

If these are polyclonal antibodies and Ag-X is a large molecule (i.e. will possess multiple epitopes) I suggest using antibody from the same animal for both capture and cap to prevent high background from anti-species antibodies.

Capture and probe antibodies have to be different in indirect ELISA. You need to use anti-species enzyme (HRP or AP) conjugate. If you use both antibodies of same species, false color shall develop in all well irespective of Wether the Ag has been captured or not.

That is correct if a layer of reporter conjugated antibody is added and not if the reporter conjugated probe is anti-Ag-X

As Pamela shall be using whole serum, I believe anti-species Ab- enzyme conjugate shall be the best option.

Right!

Thanks you all!!. Just one question more. In the individual indirect ELISA, after coating the plates, I use the first antibody diluted in PBS, while the detection Ab was diluted in blocking buffer... it worked well.

So, for the indirect sandwich ELISA, I will coate the plates as mentioned by Shamsher Singh Kanwar, then I will block the remaining binding sites with BSA blocking buffer. So, I think I have to dilute the Ag_x in PBS (dilution buffer), but I am not pretty sure about the buffers to be used for the second Ab (mice serum) and for the detection antibody (Anti_mice-). I will use ABTS as substrate.

Somebody have an advice?

Yes, second antibody and anti-mouse-HRP you can use PBS pH 7.2, washing need to be performed using PBS containing Tween 20 (just a drop in 500 ml buffer, it's a detergent). The substrate (0.15%) is hydrogen peroxide and chromogen may be OPD or ABTS (0.5 mg/ml) prepared in phosphate citrate buffer pH 5.2. this step needs incubation for 15-20 at 37 C in a humid chamber and further reaction is blocked with addition of 1 M sulfuric acid. Read at 492 nm.

You can coat with purified antibodies at 1 µg/ml in PBS, pH 7.2 at room temperature overnight. You can use mouse serum with antibodies to your antigen as secondary antibodies and then use goat or (any other species) anti-mouse IgG conjugated to alkaline phosphate or peroxidase. It should work. You can also label your purified bovine antibodies with an enzyme, then use the same species antibodies for a sandwich ELISA - purified antibodies for coating or capture of antigen at 1 µg/ml and enzyme labelled purified antibodies for detection or secondary antibodies.

PBS, pH 7.2 is a better buffer for coating for most antigens. You can skip the blocking step, if you include BSA (0.5%) in your dilution buffer and tween 20 used for diluting your antigen, secondary antibodies and conjugate. Make sure your coating buffer does not have any detergent (tween 20) or any other protein.

Thanks you all!