After the conjugation processes, what kind of tests should be used to find the best way of conjugation? How can we know the conjugation reaction efficiency? There is a lot of commercially available conjugation kits, but how can we know the yield for sure?
This is a very complex question. The chemical yield is not very relevant in this context and hence does not need to be determined. The conjugate is a mixture of products of different structures and labeling density (dye-to-protein ratio) in nearly all cases. You should determine the conjugation density, eg. by MALDI-TOF mass spectrometry or UV/VIS. You have to know that often lower conjugation densities (approx. not more than 5 dyes per IgG molecule) are better than higher ones. High conjugation densities may lead to a loss of antibody activity, precipitation, lower stability, non-specific binding, and self-quenching of the fluorophore (low fluorescence).
It depends on the method. If you are using antibody with a fluorophore, you may say It is "quantitative" where you need to separate the antibody from the substrate (heterogenous and homologous. It depends on the method, but it a factor to bare in mind.). Why do we stop reactions with different substances like in the use of enzymes? The substrate must separate to quantity. However, you may either indirect or direct ELISA.In flow cytometry you detect the light and you quantify. The light in a spectroscopy you measure by structure. It depends how you set the protocol and the method. If It glows you might expect to be qualitatitive assay. There must be a standard on your kit for you to do this. Maybe measure the intensity of light. Maybe it glows for a time and then fades?
The antibodies are All the same? You measure antigen?and then you set the antibody with the fluorophore.? Is the fluorophore proportional? I Am not sure if you can use quantum yield. (Measurement of intensity of light, the presence of light or the electrones that make light?)
Usually, unconjugated antibodies are no problem at all. It is a statistical question. If you have a mean conjugation density of 5, the amount of unconjugated antibodies is negligible. A separation of unconjugated antibodies from conjugated ones is practically impossible. And it would not improve the performance of your assay, anyway.
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