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Reversed FluoroSpot


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With your reversed FluoroSpot, have you tried using two different SA-biotin complexes? For example, could you separately conjugate two different biotinylated proteins with different SA-conjugated fluorophores and then mix them together, or would you expect competition or other issues to occur?

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We have not tried this I believe. 

In general this goes against my "gut-feeling" of successful immune assay development but reversed B-cell FluoroSpot is a special case. 

The fear is ofcourse that some SA-fluorophore from one antigen dislodges (or does not bind in the premix) during incubation and binds to the other antigen. No binding is ever 100%. 

The problem is that it becomes rather difficult to validate such a setup. Sure you can take one nonsense antigen plus one real antigen and see how much binding the nonsense antigen cause using this setup. However, another antigen might behave differently depending on biotinylation level, confirmation and so on. 

We at Mabtech have these published anti-TAG antibodies (anti-WASP and anti-BAM) where you attach a small known aminoacid sequence to your antigen. This can be done chemically but even better recombinantly. It will give you peace of mind. 


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