Antibodies Immunoreactive With Formalin-Fixed Tissue Antigens Recognize Linear Protein Epitopes

Sompuram, Seshi R.; Vani, Kodela; Hafer, Laurie J.; Bogen, Steven A.

doi:10.1309/6h0a-rqf7-k3y6-08eh

Cited by 38 publications

(41 citation statements)

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“…The reduction of cross-linking accompanies fixation. These conclusions are supported further by the work of Sompuram et al who demonstrated both that antibodies reacted with linear epitopes and that the presence of an irrelevant protein positioned to block access of the primary antibody to the reactive epitope reduced immunoreactivity (Sompuram et al 2004(Sompuram et al , 2006a(Sompuram et al , 2006b). …”

Section: Effects Of Fixation On Intraand Intermolecular Cross-linkingmentioning

confidence: 65%

Protein fixation and antigen retrieval: chemical studies

O'Leary

Fowler

Evers

et al. 2009

Biotechnic & Histochemistry

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Fixation with formaldehyde is the first process to which most biopsy and necropsy specimens are exposed prior to dehydration and embedding in paraffin wax. Tissue specimens that have been fixed in formaldehyde have architectural characteristics that are familiar to virtually every pathologist and these facilitate routine diagnosis. Nevertheless, formaldehyde fixation has some deleterious effects including reduction in immunoreactivity and degradation of nucleic acids. Development of methods to counteract these deleterious effects requires an understanding of the chemical events that occur during tissue fixation and subsequent tissue processing. This short review illustrates some of the chemical consequences of formaldehyde fixation and ethanol dehydration. It also provides some insight into the molecular events accompanying heat-induced antigen retrieval.

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Section: Effects Of Fixation On Intraand Intermolecular Cross-linkingmentioning

confidence: 65%

Protein fixation and antigen retrieval: chemical studies

O'Leary

Fowler

Evers

et al. 2009

Biotechnic & Histochemistry

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“…This cross -link is reversible upon heating at alkaline pH 21 . Lysine and cysteine can also form a cross -link at the ortho positions of the phenolic group of tyrosine through a Schiff -base intermediate.…”

Section: Reaction Of Formaldehyde With Proteinsmentioning

confidence: 97%

“…Instead, they support the model proposed by the Bogen laboratory, which suggests that antibodies chosen for their immunoreactivity for FFPE tissue sections recognize contiguous linear epitopes rather than the more common conformational epitopes. 21 Antibodies developed against denatured proteins are also likely to consist of paratopes that recognize short contiguous amino acid epitopes. 50 In this model, heat -induced AR is only required to regenerate and expose the primary amino acid sequence of the protein in order to restore immunoreactivity.…”

Section: Effect Of Formalin On the Secondary And Tertiary Structure Omentioning

confidence: 99%

Study of Formalin Fixation and Heat‐Induced Antigen Retrieval

Mason

Fowler

O'Leary

2010

Antigen Retrieval Immunohistochemistry Based Research and Diagnostics

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“…The grid lines were superimposed after scanning the image to enhance clarity. Reproduced from Sompuram et al (2006b).…”

Section: Antibodies Useful In Formalin Fixed Paraffin Embedded (Ffpementioning

confidence: 99%

Molecular mechanisms of antigen retrieval: antigen retrieval reverses steric interference caused by formalin-induced cross-links

Bogen

Vani

Sompuram

2009

Biotechnic & Histochemistry

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The overwhelming majority of antibodies useful for formalin fixed, paraffin embedded (FFPE) tissues require antigen retrieval to reverse the effect of formalin fixation and re-establish immunoreactivity. How this reversal happens is poorly understood. We developed a new experimental model for studying the mechanism of formalin fixation and antigen retrieval. Epitope mapping studies on nine antibodies useful for FFPE tissues revealed that each consisted of a contiguous stretch of amino acids in the native protein (linear epitope). Small peptides representing the epitopes of antibodies to human epidermal growth factor receptor type (HER2), estrogen, and progesterone receptors were attached covalently to glass microscope slides in a peptide array. Most peptides retained immunoreactivity after formalin fixation. Immunoreactivity was completely abrogated for all peptides, however, if an irrelevant large protein was present during formalin-induced cross-linking. We hypothesize that cross-linking the irrelevant protein to the peptide epitopes sterically blocked antibodies from binding. Antigen retrieval dissociates irrelevant proteins and restores immunoreactivity. Because the epitopes for clinical antibodies require only primary protein structure, the fact that antigen retrieval probably denatures the secondary and tertiary structure of the protein is irrelevant. The same mechanism may occur in tissue samples subjected to formalin fixation and antigen retrieval.

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Antibodies Immunoreactive With Formalin-Fixed Tissue Antigens Recognize Linear Protein Epitopes

Cited by 38 publications

References 0 publications

Protein fixation and antigen retrieval: chemical studies

Protein fixation and antigen retrieval: chemical studies

Study of Formalin Fixation and Heat‐Induced Antigen Retrieval

Molecular mechanisms of antigen retrieval: antigen retrieval reverses steric interference caused by formalin-induced cross-links

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