Hi everyone,
i am currently testing new antibodies for its compatibility for a sandwich ELISA and at the moment, their cross-reactivity should be determined. Normally, we have to extract allergens freshly from our cross-reactive materials and use them with our new antibodies (both capture and conjugate).
There was an idea of coating the microtiter plate with cross-reactive allergens. Then, we can just add our antibody of interest (direct ELISA format) and detect the signal. Compared all measured values with the blank, we can then see the antibody cross-reactivity. I did this a few times but the results are variable and did not match well with the freshly-extracted-allergen approach. I was told that we have to do the later anyway but i think it's also a good idea to gain knowledge early on about the cross-reactivity of antibody.
Have anyone already done this before and what are your advice on this? Thank you guys in advance :).
Hi my Dear, for allergen assays, our team routinely tested pAbs and mabs first by plating the key cross reactive allergens using bicarbonate buffer to the elisa plate and then indirectly and directly testing the antibody by elisa. Any antibody that passed this initial cross reactivity screen was then considered as a candidate for sandwich and/ or competitive elisa assay testing using direct conjugates. Usually, antibodies that passed the first screen passed the second format, but there exceptions. Let me know if you have any more questions
There are many ways to perform such screenings, e.g.
Article Microplate-based screening methods for the efficient develop...
The coating of antigen/allergen on a plate has several issues, you should consider:
1. The antigen might be partially or completely denatured on the surface.
2. If you use an extract, the antigen of interest might not coat on the plate properly or might be displaced.
3. The denatured fraction of your antigen might lead to wrong results.
4. In sandwich assays, the total cross-reactivity is dependent on both antibodies. Sometimes cross-reacting antibodies might be useful in an optimized "matching pair".
5. The coating density of your antigen highly influences the results. Cross-reactivity might be overestimated due to multivalency effects.
6. The general rule for the characterization is to stay as close as possible to your final assay format.
I had a recent request from some else on the protocol we used, here it is reproduced:
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