I am performing an antibody pairs expriment for constructing a ELISA assay. A good antibody pairs could theoretically differentiate positive serum and normal serum, but I found that the antibody pairs used in my experiment could not differentiate the positive and normal serum well， could you give me any advice?
Are them monoclonal or polyclonal? If they are polyclonal, the assay should work in principle after you optimize the concentration of the reagents and the conditions. If both are monoclonal antibodies, you should make sure that they does not compete for binding to the antigen. Otherwise, they would never bind to the same antigen molecule, so they will not work in a sandwich ELISA. If one of them is monoclonal and the other polyclonal, you should also check that the polyclonal does not inhibit the monoclonal binding.
Can you detect the pure antigen (not in sera) with your pair of antibodies. This would be a good control for the antibodies usefulness. Then you would have to deal with possible matrix effects in serum and with sensitivity if sera concentrations are low.
In any case, you need to optimize the conditions. And perhaps the specificity of the antibodies is ok, but the affinity is not enough to obtain a sensitive assay. An amplification system could help in the later case. Is one of the antibodies directly labelled with an enzyme?
@Gertrudis Rojas Thanks for your advice. some antibody pairs I choosed could detect serum well but have no response for pure antigen, why? different structures for pure recombinant antigen and target natural antigen in serum?
you are right, one of the antibodies was directly labelled with HRP, are there some adverse effects for do so?
Perhaps the recombinant antigen is not properly folded, this is always a possibility.
On the other hand, you say that the antibodies are not detecting the purified antigen, but they are able to detect the antigen in sera. At the same time, you have problems to detect the antigen in sera, I dont understand the situation. Are you able to detect the antigen in sera or not? If so, are you sure that the signal is specific and not serum background? Specificity problems could be an alternative explanation of why your ELISA doesnt work with the recombinant antigen and apparently works with sera.
Of course you can conjugate one of the antibodies, but if you need more sensitivity you could biotinylate one of them and use streptavidin-HRP to amplify the signals.
@Gertrudis Rojas Thanks for your response. Maybe I didn't express clearly. The antibody pairs used can detect serum well but have no response for pure recombnant antigen, this may be caused by different structures? Another, why the antibody pairs can't differentiate positive serum and normal serum well (signal from positive and normal serum is similar)?
Yes, I understood, but my interpretation is different.
What I think is that , if signals with positive and negative sera are the same, perhaps you dont have specific recognition, but non-specific background. In that case, your ELISA is simply not working and thats the reason why you cannot detect purified antigen. How do you know that you are not having just background with sera (positive and negative).
Of course, your recombinant antigen could be misfolded, but until now I dont see evidences that the antigen in serum is being recognized. I would suggest to focus on possible background issues with your sera ELISA.
Which is the dilution of your serum samples? Are you diluting sera in any blocking solution? Which one? If you try to dilute more the samples or to use a better blocking agent perhaps the signal for positive sera is maintained and it disappears for negative samples. On the other hand, is signal disappears for both kind of samples, you would have to conclude that everything was background. By the way, are you totally sure that your negative samples are negative?
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