1. Characterization and clinical validation of MCM2 and TOP2A monoclonal antibodies in the BD ProEx™ C assay: An immunoassay which detects aberrant S-phase induction in cervical tissue.
- Author
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Dixon EP, King LM, Nelson R, Simkins SG, Knapp SL, Brough GH, Lenz KL, Henderson DT, Whitehead CM, Hessling J, Brown CA, and Malinowski DP
- Subjects
- Biopsy, Blotting, Western, Cell Nucleus enzymology, Cell Nucleus immunology, Cell Nucleus pathology, Cervix Uteri enzymology, Cervix Uteri pathology, Epitope Mapping methods, Epitopes, Female, Humans, Poly-ADP-Ribose Binding Proteins, Predictive Value of Tests, Severity of Illness Index, Uterine Cervical Dysplasia enzymology, Uterine Cervical Dysplasia immunology, Uterine Cervical Dysplasia pathology, Uterine Cervical Neoplasms enzymology, Uterine Cervical Neoplasms immunology, Uterine Cervical Neoplasms pathology, Antibodies, Monoclonal immunology, Antigens, Neoplasm immunology, Cervix Uteri immunology, DNA Topoisomerases, Type II immunology, DNA-Binding Proteins immunology, Immunohistochemistry, Minichromosome Maintenance Complex Component 2 immunology, S Phase, Tissue Array Analysis methods, Uterine Cervical Dysplasia diagnosis, Uterine Cervical Neoplasms diagnosis
- Abstract
Background: The Papanicolaou (Pap) screen has been successful in reducing cervical cancer; but exhibits low sensitivity when detecting cervical dysplasia. Use of molecular biomarkers in Pap tests may improve diagnostic accuracy., Design: Monoclonal antibodies to Minichromosome Maintenance Protein 2 (MCM2) and DNA Topoisomerase II α (TOP2A) were selected for use in IHC based on their ability to differentiate normal from diseased cervical tissues in tissue microarrays. Enhanced Green Fluorescent Protein Western blot analysis was used to help identify binding epitopes specific to MCM2 and TOP2A antibody clones. Antibody affinity was determined by solution phase affinity measurement and immunohistochemistry was performed using high affinity MCM2 or TOP2A antibodies on serial histological sections., Results: Antibody clones to MCM2 and TOP2A clones were selected based on their ability to detect over expression in abnormal cervical epithelia. In IHC, MCM2-27C5.6 and MCM2-26H6.19 demonstrated superior staining in abnormal cervical tissue over the MCM2-CRCT2.1 antibody. A combination of MCM2 and TOP2A antibodies showed greater staining when compared to staining with any of the antibodies alone on serial histological sections. Distinct linear epitopes were elucidated for each of the MCM2 and TOP2A clones. Affinity values (Kd) for MCM2 or TOP2A antibodies had a similar range. In a research study, the MCM2 and TOP2A (BD ProEx™ C) antibody cocktail showed increased epithelia staining with increasing dysplasia. The use of BD ProEx™ C in combination with H&E staining enhanced immunohistochemical discrimination of dysplastic and non-dysplastic FFPE cervical tissue specimens., Conclusions: BD ProEx™ C containing MCM2 and TOP2A antibodies showed strong specific nuclear staining that correlated with increased dysplasia and lesion severity. Enhanced performance of the antibodies was linked to their unique topography recognition. BD ProEx™ C incorporates antibodies that enhance detection of CIN2+ cervical disease., (Copyright © 2017 Elsevier B.V. All rights reserved.)
- Published
- 2017
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