A Molecular Model of Antigen Retrieval Using a Peptide Array

Sompuram, Seshi R.; Vani, Kodela; Bogen, Steven A.

doi:10.1309/dceq-d30v-5uej-a5gn

Cited by 25 publications

(28 citation statements)

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“…We examined the kinetics of formalin fixation and antigen retrieval in our peptide array model (Sompuram et al 2006a,b). We were particularly interested to determine whether the kinetics of antigen retrieval resembles the time course often associated with tissue sections.…”

Section: Similarity Of Reaction Kineticsmentioning

confidence: 99%

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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 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 bonding. 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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Section: Similarity Of Reaction Kineticsmentioning

confidence: 99%

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

Section: Figmentioning

confidence: 99%

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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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“…Our results are the first to demonstrate that heating could remove the masking of epitopes hidden due to the molecular configuration of native molecule (Kakimoto et al 2004). Sompuram et al (2004Sompuram et al ( , 2006 have reported on employing a model system with short peptide antigens covalently bound to glass slides to address the mechanism of formalin fixation and AR. Some short peptides including Ki-67 are sensitive to formalin and antigenicity is recovered by heating.…”

Section: Effects Of Heating Ar In Ihc and The Dot-blot Analyses With mentioning

confidence: 99%

Hypothesis for the mechanism for heat-induced antigen retrieval occurring on fresh frozen sections without formalin-fixation in immunohistochemistry

et al. 2008

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The mechanism involved in heat-induced antigen retrieval (AR) remains unproven but probably utilizes the breaking of formalin-induced cross-linkages. We investigated the effectiveness of heat-induced AR on immunohistochemistry and dot-blot analysis using rat uterus tissue sections and protein extracts without formalin-fixation. The unfixed frozen sections, which did not show immunostaining with nine antibodies, were clearly stained after heating the sections. In the dot-blot analysis, the immunoblot sensitivity of detection was greatly enhanced by heating the protein-blotted membrane. These results indicate that other mechanisms of breaking formalin-induced cross-linkages may be present. We propose that one of the other mechanisms for heat-induced AR is that accessibility to the target epitopes of antigenic proteins is limited by natural steric barriers even in the fresh state caused by the antigenic protein itself.

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“…Unlike cells and tissues, peptides can be printed on glass slides in a high throughput and reproducible fashion. The peptides can also be fixed in formalin, similar to formalin fixation of tissues, and behave immunochemically like the native antigen in tissue 15, 16. By comparing the staining of formalin-fixed peptides to unfixed peptides, IHC staining errors associated with antigen retrieval can be distinguished from errors in other analytic steps of the assay 12.…”

Section: Introductionmentioning

confidence: 99%

Experimental Validation of Peptide Immunohistochemistry Controls

Bogen¹,

Vani²,

McGraw³

et al. 2009

Applied Immunohistochemistry &Amp; Molecular Morphology

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Peptide immunohistochemistry (IHC) controls are a new quality control format for verifying proper IHC assay performance, offering advantages in high throughput automated manufacture and standardization. We previously demonstrated that formalin-fixed peptide epitopes, covalently attached to glass microscope slides, behaved (immunochemically) in a similar fashion to the native protein in tissue sections. To convert this promising idea into a practical clinical laboratory quality control tool, we tested the hypothesis that the quality assurance information provided by peptide IHC controls accurately reflects IHC staining performance amongst a diverse group of clinical laboratories. To test the hypothesis, we first designed and built an instrument for reproducibly printing the controls on microscope slides and a simple software program to measure the color intensity of stained controls. Automated printing of peptide spots was reproducible, with CVs of 4−8%. Moreover, the peptide controls were stable at ≤4° C for at least seven months, the longest time duration we tested. A national study of 109 participating clinical laboratories demonstrated a good correlation between a laboratory's ability to properly stain formalin-fixed peptide controls to their ability in properly staining a 3+ HER-2 formalin-fixed tissue section mounted on the same slide (r = 0.87). Therefore, peptide IHC controls accurately reflect the analytical component of an IHC stain, including antigen retrieval. Besides its use in proficiency survey testing, we also demonstrate the feasibility of applying peptide IHC controls for verifying intra-laboratory IHC staining consistency, using Levy-Jennings charting.

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A Molecular Model of Antigen Retrieval Using a Peptide Array

Cited by 25 publications

References 0 publications

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

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

Hypothesis for the mechanism for heat-induced antigen retrieval occurring on fresh frozen sections without formalin-fixation in immunohistochemistry

Experimental Validation of Peptide Immunohistochemistry Controls

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