Possible Mechanisms of Antigen Retrieval

On the other hand, available evidence supports the occurrence of the breakage of protein crosslinks, which allows the antibody access to the antigen. Conventional heat, dry or steam, breaks down reversible protein-protein, protein-nucleic acid, and protein-carbohydrate crosslinks introduced by formaldehyde and thereby unmasks the epitopes, as well as allowing the antibodies access to the epitopes. It is well known that most, if not all, crosslinks formed during formaldehyde fixation are destroyed upon heating even at 37°C 

That heat is not the only method to unmask epitopes is exemplified by enzyme digestion or detergent treatment. The exact mechanism responsible for epitope retrieval with ultrasound is not clear, although intense heat is produced for an exceedingly short duration. It is known, however, that ultrasound and/or heat decreases the amount of negative charges on the cell surface (Joshi et al., 1983 Adler et al., 1988). Mechanical vibrations of molecules caused by ultrasound and heat are thought to unfold the protein molecule and to expose the epitopes. 

Some information is available on the possible mechanism underlying masking of epitopes in the presence of calcium ions. Such a masking of epitopes and their unmasking are discussed in detail on page 120. 

Evidence indicates that microwaves affect the kinetics of conformation changes of proteins such as P-lactoglobulin (Bohr and Bohr, 2000). It is thought that even approximately a few GHz can excite protein molecules. Consequently, the kinetics of conformational changes of the protein molecule are enhanced, and this denaturing effect is 

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