Modeling formalin fixation and antigen retrieval with bovine pancreatic RNase A II. Interrelationship of cross-linking, immunoreactivity, and heat treatment

In this study, gel electrophoresis and capture enzyme-linked immunosorbent assay were used to assess the effect of formaldehyde treatment on the structural and immunological properties of bovine pancreatic ribonuclease A (RNase A). Prolonged incubation of RNase A in a 10% formalin solution leads to the formation of extensive intra- and intermolecular cross-links. However, these formaldehyde cross-links do not completely eliminate the recognition of RNase A by a polyclonal antibody. Comparative immunotitration of monomers, dimers, and oligomers greater than pentamers isolated from formalin-treated RNase A demonstrated that reduction of immunoreactivity due to intramolecular modifications prevails over the excluded volume effect of intermolecular cross-links. The latter only becomes important for intermolecular cross-links involving four or more molecules. The restoration of RNase A immunoreactivity during heating correlates with the reversal of formaldehyde cross-links if the incubation temperature does not exceed the denaturation temperature of the formalin-treated RNase A preparation. We conclude that formaldehyde cross-links stabilize antigens against the denaturing effects of high temperature, but the reversal of these cross-links is necessary for the restoration of immunoreactivity.