Hi Namdev.

Both sample and conjugate dilution, should be evaluated. If you have clear background in the blank position, the conjugate dilution is ok. Frequentily, high antibody titer requires high dilution (over 1: 10 5) and still give strong signal.

Please describe more or give more information for us to help you. Information about the blank and so on.

What is your current protocol? How do you prepare and store your sera?

Dear Namgrev, a rather simple and convenient method for estimating quality and quantity of your antibody titer by ELISA is the plate trapped antigen ELISA, This means: coat an ELISA plate with the respective antigen (dilutions) 1:100, 1:500, 1:1000, 1:2000 as well as a negative control (other nonhomologuous antigen in the same way) in 2 rows for  each dilution in carbonate buffer pH 8,6, Let it incubate overnight at 4°C, rinse, block with BSA or not fat dry milk 2%  in TBS or PBS (20 min) empty the plate by simply turning it and throwing the blocker into a containment . Add prepared dilutions of the antiserum 1: 100, 1:500, 1: 1000, 1: 2000, 1: 5000 and 1: 10.000 (in PBS or TBS-+ 25.000 Polyvinyl Pyrrolidone (PVP) + 0,05% Tween) in 2 columns for each dilution, let it incubate for 2- 4 h at RT. Rinse 3 times with PBS- or TBS-Tween add a species specific conjugate diluted 1:1000 in conjugate buffer. Let it incubate for 2 h at RT. Rinse with PBS-Tween, tap it dry on paper towels. Add the respective substrate in the respective buffer, incubate and read or estimate the best combinations. That method works for estimating the quality of raw antisera against plant pathogenic viruses rather well.

you just have to do a 2 dimensional titration for detecting the optimum of your test based on an antigen coated plate as described above. By varying the conjugate (enzyme) versus the antibody concentration this will show you the optimum for your test and also show if you get background signal (forced by unspecific binding) or just too high signal.... which then may be downsized by shorter substrate incubation  times.

If your ELISA works in principle (you know the antigen concentration at the solid phase, you know the concentration of the labelled anti-species antibody, reaction times, potential sample dilutions, potential detection limit, presence of standards and/or controls, ...) (see above)

the titer detection is simple, you prepare a serial dilution (you can use a 1:3 ... 1:10 .. serial dilution) of your sample. The dilution with the last detectable dilution is the titer of your antobody. it''s always semiquantitative. Therefore you need to have a control/standard which have to be used in each assay. This has to give at each experiment the same "titer"

Titres are usually measured as the dilution by a factor 2 from the lowest dilution giving background. This requires serial dilutions of the antibody by a factor two and having the antigen coated in the microwells in saturating amount. Please be aware that the secondary antibody concentration needs to be optimized as well. You also need a cutoff value as your border line of signal versus background. Multiple wells as negative control (omitting the primary antibody) will give you an average of that value. So when you reach that value, say at 1:20,000 dilution, then your titre is 1:10,000.

Hello Namdev,

By over range, do you mean:

No matter how high the dilution, there is no break away from maximum absorption?  The blank is blank, but everything else is all the same, with maximum absorption?

If that is the case, I would recommend verifying the blocking agent/blocking step/blocking protocol

thank you all........

Make dilutions