Our lab is looking for any information you may have to offer regarding an ELISA competitive antibody test that we have developed and are in the process of validating. Briefly, the basis of our test is as follows:
coat plate with antigen block
add unknown serum (potentially with specific antibody)
add specific/known rabbit antiserum to the antigen on the plates
add anti rabbit HRP develop color with TMB substrate (if antibody is present in the unknown sample, it will reduce the absorbance that we expect with the known rabbit/anti rabbit combination and we can then calculate % inhibition).
Something that we have noticed repeatedly is that the higher the unknown serum concentration of some of the samples that we add to the test, the higher the absorbance, compared to a no serum control. Since we are looking for a reduction in the absorbance, this is a very big problem and it may be masking any antibodies that are present.Should we be substituting our conjugated secondary antibody with alkaline phosphatase?
Thanks so much in advance for any bit of information you may have and while I have your attention, I would also like to know if your lab has any way of getting any known SARS-CoV2 naturally infected serum (any species) that would help us in validating our ELISA. Our Biosafety Permit allows it.
If I understand correctly you have a three-layer system: (i) antigen, (ii) rabbit anti-antigen + serum (which may have unlabelled antibodies to the antigen) , (iii) anti-rabbit HRP detector.
If you increase the concentration of serum in the middle layer, you are increasing the concentration of total rabbit IgG (mostly not recognising your antigen), whose concentration will be very high (could easily be 10mg/ml, undiluted). Thus, I think you are seeing a wash problem here, with the HRP secondary is simply detecting non-specifically bound rabbit IgG originating from the serum.
There are two ways round this (i) change your wash procedure and verify that normal serum vs no serum gives the same (or very similar) signals. A better option may be to (ii) change the assay and label your rabbit anti-antigen antibody with HRP to give a direct assay format. You then have two layers (i) antigen (ii) rabbit anti-antigen HRP conjugate competing with specific antibodies (if present) in the unknown serum.
(the assay works "better "than the control? It could well be used as a control instead. I wonder if the signal would be masked on the opposite way, masking the control?...)
Could be an issue with blocking or with the rabbit serum (you are introducing high amounts of unspecific IgG). Have you tried if using purified (Protein A/G or antigen affinity) IgG is improving the results? And, as Nick Gee has mentioned, optimize the washing procedure. For best results, do repeated manual washing and beat the liquid out of the plates.
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