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immunochromatography, epitopes, and the sandwich principle

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Good Morning Everyone,

I have been studying various forensic test kits for body fluids, and a number of them from Abacus Diagnostics or Independent Forensics use lateral diffusion immunochromatography.  In a sandwich format the antigen forms a complex with both the soluble, labeled antibody and the antibody immobilized onto a membrane at the test (T) position.  I can see how this could easily work with a protein such as hemoglobin, which is a tetramer of two alpha and two beta subunits.  It is less clear how this assay works with human salivary alpha-amylase, which is a monomer from what I can gather.  In other words I can see how the sandwich forms when there are multiple epitopes, but it is less clear to me what is happening when there might be only one epitope.

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Epitopes are small structural elements in a good sized protein such as amylase you are likely to find a couple. Often epitopes are not larger than 5-15 AA in length so there is no need for elaborate quartenary structures. Even for insulin, which only has 50ish amino acids there are at least three different monoclonal antibodies. But crowding effects can be worse on smaller molecules. Partially it can be alleviated by using truncated antibodies (which is normally done).

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Thank you; that is very helpful.  I think that I had been assuming implicitly that the two antibodies (soluble versus immobilized) recognized the same epitope.  I now wonder whether the antibody at the Test position versus the gold-particle labeled antibody recognize the same or different epitopes.  Perhaps the answer is different, depending on which test is used.

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