一种用于检测癌症组织标本中 ETV1 表达的 ETV1 兔单克隆抗体的开发和鉴定。

Development and characterization of an ETV1 rabbit monoclonal antibody for the immunohistochemical detection of ETV1 expression in cancer tissue specimens.

机构信息

Center for Prostate Disease Research, Murtha Cancer Center Research Program, Department of Surgery, Uniformed Services University of the Health Sciences, Bethesda, MD 20817, USA; Henry M. Jackson Foundation for the Advancement of Military Medicine, Inc., Bethesda, MD 20817, USA.

Uniformed Services University of the Health Sciences, Bethesda, MD 20817, USA.

出版信息

J Immunol Methods. 2023 Jul;518:113493. doi: 10.1016/j.jim.2023.113493. Epub 2023 May 16.

DOI:10.1016/j.jim.2023.113493

PMID:37196930

原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC10802095/

Abstract

BACKGROUND

Aberrant ETV1 overexpression arising from gene rearrangements or mutations occur frequently in prostate cancer, round cell sarcomas, gastrointestinal stromal tumors, gliomas, and other malignancies. The absence of specific monoclonal antibodies (mAb) has limited its detection and our understanding of its oncogenic function.

METHODS

An ETV1 specific rabbit mAb (29E4) was raised using an immunogenic peptide. Key residues essential for its binding were probed by ELISA and its binding kinetics were measured by surface plasmon resonance imaging (SPRi). Its selective binding to ETV1 was assessed by immunoblots and immunofluorescence assays (IFA), and by both single and double-immuno-histochemistry (IHC) assays on prostate cancer tissue specimens.

RESULTS

Immunoblot results showed that the mAb is highly specific and lacked cross-reactivity with other ETS factors. A minimal epitope with two phenylalanine residues at its core was found to be required for effective mAb binding. SPRi measurements revealed an equilibrium dissociation constant in the picomolar range, confirming its high affinity. ETV1 (+) tumors were detected in prostate cancer tissue microarray cases evaluated. IHC staining of whole-mounted sections revealed glands with a mosaic staining pattern of cells that are partly ETV1 (+) and interspersed with ETV1 (-) cells. Duplex IHC, using ETV1 and ERG mAbs, detected collision tumors containing glands with distinct ETV1 (+) and ERG (+) cells.

CONCLUSIONS

The selective detection of ETV1 by the 29E4 mAb in immunoblots, IFA, and IHC assays using human prostate tissue specimens reveals a potential utility for the diagnosis, the prognosis of prostate adenocarcinoma and other cancers, and the stratification of patients for treatment by ETV1 inhibitors.

摘要

背景

基因重排或突变导致 ETV1 异常过表达，常见于前列腺癌、圆形细胞肉瘤、胃肠道间质瘤、神经胶质瘤和其他恶性肿瘤。由于缺乏特异性单克隆抗体（mAb），限制了其检测和对其致癌功能的理解。

方法

使用免疫原性肽段制备了一种 ETV1 特异性兔 mAb（29E4）。通过 ELISA 探测其结合的关键残基，并通过表面等离子体共振成像（SPRi）测量其结合动力学。通过免疫印迹和免疫荧光分析（IFA）评估其对 ETV1 的选择性结合，并通过前列腺癌组织标本的单重和双重免疫组织化学（IHC）检测评估其对 ETV1 的选择性结合。

结果

免疫印迹结果表明，该 mAb 高度特异性，与其他 ETS 因子无交叉反应。发现其核心含有两个苯丙氨酸残基的最小表位是有效 mAb 结合所必需的。SPRi 测量结果显示，平衡解离常数在皮摩尔范围内，证实其具有高亲和力。在评估的前列腺癌组织微阵列病例中检测到 ETV1（+）肿瘤。免疫组化染色的全载玻片显示，细胞呈现马赛克染色模式，部分为 ETV1（+），并散布有 ETV1（-）细胞。使用 ETV1 和 ERG mAbs 进行的双重免疫组化检测到含有具有明显 ETV1（+）和 ERG（+）细胞的碰撞肿瘤。