Superheating Antigen Retrieval

Lee, Eung Seok; Yin, Hui; Kear, Megan; Haffajee, Zenobia; Pepperall, Debbie

doi:10.1097/00129039-200209000-00014

Cited by 21 publications

(14 citation statements)

References 17 publications

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“…However, the initial temperature at the start of the AR protocol may need to be even higher than the constant temperatures currently used in some 'superheating' protocols. 12 How does high temperature promote the reversal of formaldehyde-induced cross-links? In Figure 9, we show the structural diagrams of the amino methylol adduct formed by an Asn residue (a) and an Asn cross-linked with a Lys residue (b).…”

Section: Resultsmentioning

confidence: 99%

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

Rait

O'Leary

et al. 2004

Laboratory Investigation

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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. Laboratory Investigation (2004) 84, 300-306, advance online publication, 26 January 2004; doi:10.1038/labinvest.3700041Keywords: antigen retrieval; immunohistochemistry; immunoreactivity; formalin fixation; ribonuclease A; enzymelinked immunosorbent assayIn 1991, Shi et al 1 published their seminal observation that high-temperature incubation of formalinfixed paraffin-embedded tissue sections in buffers for short periods leads to improved immunohistochemical staining. However, more than a decade later, high-temperature antigen retrieval (AR) remains an empirical procedure with several critical parameters that require trial and error optimization. 2,3 Further improvements in AR will require an in-depth understanding of the chemistry of formaldehyde fixation and the molecular mechanism(s) underlying the AR method.It is commonly assumed that decreased immunoreactivity in fixed tissues results from formaldehyde-induced cross-linking. However, cross-linking can affect protein immunoreactivity on many levels. The extremely high concentration of proteins and other solutes within tissues 4,5 leads to the formation of a dense irregular network of cross-links (gelation) that can prevent antibody penetration to the location of its antigen within the tissue. 6 Even if this primary barrier is partially destroyed by enzymatic etching or thermal reversal of cross-linking, additional effects of formaldehyde can impede antigen-antibody interactions. Antibody binding can be inhibited by steric hindrance (excluded volume effect) arising from the shielding of epitopes on the target antigen due to the close proximity of adjacent molecules to which the antigen is cross-linked. Immunoreactivity can be further compromised by formalin-induced chemical mod...

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Section: Resultsmentioning

confidence: 99%

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

Rait

O'Leary

et al. 2004

Laboratory Investigation

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“…Rather, it is likely that ingestion of Cry1Ac by H. virescens and M. sexta caused some structural alteration that resulted in exposure of antigen-recognizing motif of this cadherin-like protein, or enrichment/relocation of cadherin-like proteins to membrane subdomains. It is fairly common that proteins may not be accessible to the antibody in immunohistochemical analysis, in which case antigen retrieval is necessary for signal detection (Leong et al, 2002). Potentially, the binding of Cry1Ac disrupts protein-protein/lipid interactions between this cadherin-like protein and its cell adhesion partners (or other molecules that bind the cadherin-like protein for its function), thereby exposing the cadherin-like protein for greater immunoreactivity.…”

Section: Discussionmentioning

confidence: 99%

Expression of Cry1Ac cadherin receptors in insect midgut and cell lines

Aimanova

Zhuang

Gill

2006

Journal of Invertebrate Pathology

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“…Such β -sheets would require substantial energy to induce suffi cient rehydration to reverse the formaldehyde cross -links within these sheets and regenerate protein monomers free of formaldehyde modifi cations. The required energy can be introduced using extremely high temperatures ≥ 100 ° C 79,80 or by the application of elevated hydrostatic pressure at moderate temperatures. 81 …”

Section: The Role Of Ethanol Dehydration In Armentioning

confidence: 99%

“…94,95 For example, a buffer that is pH 6.0 at room temperature will, at 120 ° C, be reduced to pH 3.06 (Tris), 94 pH 5.24 (phosphate), 96 or pH 5.69 (citrate). 95 Temperatures of 120 ° C, referred to as superheating AR, 80 are frequently attained during heat -induced AR using microwave, 97 pressure cooker, 98 or steamer 99 methods. The second complication is temperature -induced protein degradation reactions that can occur at temperatures ≥ 100 ° C. 100 -102 At acidic pH, these degradation reactions are deamination of glutamine or asparagine residues, and peptide bond hydrolysis on the carboxylate side of aspartic acid residues that results from the formation of an internal anhydride.…”

Section: General Comments On the Mechanism Of Armentioning

confidence: 99%