Question:

I am working on an ELISA antigen capture test and the detection antibody (monoclonal) is biotinylated. So far, I have evaluated 2 different lots of commercial reagent, but I think that both are over-biotinylated, one has a biotin/IgG ratio of 8 and the other is 11. I am thinking that I need something in the 2-5 range. If that is the case, can the process be controlled well enough to provide that type of reagent reproducibly? Assuming that I can supply purified monoclonal, how much would it cost and how long would it take to get reagent with the above referenced ratio?

Resolution:

This can certainly be done - I would suggest a titration of reactants within a standard protocol. To conserve antibody, we can biotinylate 100 - 200 µg in each of 4 - 5 reactant ratios, separate the unreacted biotin, quantitate the antibody and have you test each of them for specific activity. The concentration will not be high enough for biotin:antibody ratio determination but once you pick something that works, we can determine the ratio on a larger prep.

I will get quote to you on this next week.

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