BD ProEx™ C检测中MCM2和TOP2A单克隆抗体的表征及临床验证：一种检测宫颈组织中异常S期诱导的免疫检测方法。

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.

作者信息

Dixon Eric P, King Lorraine M, Nelson Ramona, Simkins Stephen G, Knapp Steven L, Brough George H, Lenz Karen L, Henderson Dorian T, Whitehead Clark M, Hessling Janice, Brown Charlotte A, Malinowski Douglas P

机构信息

BD Diagnostics - Women's Health and Cancer, Durham, NC, USA.

出版信息

J Immunol Methods. 2017 Mar;442:35-41. doi: 10.1016/j.jim.2017.01.002. Epub 2017 Jan 16.

DOI:10.1016/j.jim.2017.01.002

PMID:28093271

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.

摘要

背景

巴氏涂片检查在降低宫颈癌发病率方面取得了成功；但在检测宫颈发育异常时敏感性较低。在巴氏试验中使用分子生物标志物可能会提高诊断准确性。

设计

基于其在组织微阵列中区分正常与病变宫颈组织的能力，选择了针对微小染色体维持蛋白2（MCM2）和DNA拓扑异构酶IIα（TOP2A）的单克隆抗体用于免疫组织化学。使用增强型绿色荧光蛋白免疫印迹分析来帮助鉴定MCM2和TOP2A抗体克隆特有的结合表位。通过溶液相亲和力测量确定抗体亲和力，并使用高亲和力的MCM2或TOP2A抗体在连续组织切片上进行免疫组织化学。

结果

基于其检测宫颈异常上皮中过表达的能力，选择了MCM2和TOP2A克隆的抗体。在免疫组织化学中，与MCM2 - CRCT2.1抗体相比，MCM2 - 27C5.6和MCM2 - 26H6.19在异常宫颈组织中表现出更好的染色效果。与在连续组织切片上单独使用任何一种抗体染色相比，MCM2和TOP2A抗体联合使用时染色效果更佳。阐明了每个MCM2和TOP2A克隆的独特线性表位。MCM2或TOP2A抗体的亲和力值（Kd）范围相似。在一项研究中，MCM2和TOP2A（BD ProEx™ C）抗体混合物显示随着发育异常程度增加，上皮染色增强。将BD ProEx™ C与苏木精和伊红染色联合使用可增强对发育异常和非发育异常的福尔马林固定石蜡包埋宫颈组织标本的免疫组织化学鉴别。