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.
Rait
Vladimir K
VK
Division of Biophysics, Department of Cellular Pathology and Genetics, Armed Forces Institute of Pathology, Rockville, MD 20850, USA.
Xu
Lixin
L
O'Leary
Timothy J
TJ
Mason
Jeffrey T
JT
D000818:Animals; D000941:Antigens / Q000737:chemistry / Q000378:metabolism; D002417:Cattle; D003432:Cross-Linking Reagents; D004797:Enzyme-Linked Immunosorbent Assay; D005404:Fixatives; D005557:Formaldehyde; D006358:Hot Temperature; D007150:Immunohistochemistry / Q000379:methods; D066298:In Vitro Techniques; D008954:Models, Biological; D015394:Molecular Structure; D011489:Protein Denaturation; D012259:Ribonuclease, Pancreatic / Q000737:chemistry* / Q000276:immunology / Q000378:metabolism*; D016707:Tissue Fixation
D000941:Antigens; D003432:Cross-Linking Reagents; D005404:Fixatives; D005557:Formaldehyde; D012259:Ribonuclease, Pancreatic