Antigens, denaturation

Zwitterionic detergents (0.1-0.4%) in the electrotransfer buffer may restore the antibody-binding capacity of conformational epitopes of antigens denatured by SDS (Mandrell and Zollinger, 1984). 

The approximate location of the epitopes for more than 40 monoclonal anti-ATPase antibodies has been mapped to various regions within the cytoplasmic domain of the Ca " -ATPase [285,302-304]. All antibodies were found to bind with high affinity to denatured Ca -ATPase, but the binding to the native enzyme showed significant differences depending on the location of antigenic sites within the ATPase molecule. 

Munn, E.A., Smith, T.S., Smith, EL, Smith, F. and Andrews, S.J. (1997) Vaccination against Haemonchus contortus with denatured forms of the protective antigen Hll. Parasite Immunology 19, 243—248. 

In general, the mechanism of heat and alkaline solution for DNA extraction may be based upon a hypothesis, previously proposed for the AR technique.32 Strong alkaline solution may denature and hydrolyze proteins, resulting in breaking cell and nuclear membranes as well as disrupting cross-linkages due to formalin fixation. It is no surprise to observe the similarity between retrieval of nucleic acid and retrieval of protein (antigen) based on a similar chemical reaction of formaldehyde with these two kinds of macromolecules (Fig. 3.1).15"19... 

Figure 7.6 Peptides do not denature after baking (dry heat) or deparaffinization and antigen retrieval (wet heat). Peptide-coupled slides were treated as indicated on the Y-axis and then immunohistochemically stained. In this particular example, an ER peptide with an ER MAb was used. The resulting peptide spot intensity (mean pixel intensity on a 1-256 scale) was measured and is shown on the y-axis.The data represent the means and SD or triplicate measurements. The experiments on the left (solid bars) and the right (hatched bars) were conducted at different times and have no connection to one another. Adapted with permission from Sompuram et al.6...

Chemical treatment with formaldehyde may not necessarily result in significant denaturation. A recent study of RNase A indicated that treatment with formaldehyde does not significantly alter secondary structure.21,22 Although formalin treatment may induce subtle changes in secondary structure, alpha helices and beta-pleated sheets are left essentially intact after formalin treatment. It is reasonable to assume, however, that boiling (as per antigen retrieval protocols) will significantly alter secondary structure. 

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