新型针对乳腺癌组织样本中人类 Ki67 和 P53 肿瘤标志物的单克隆抗体

Novel Monoclonal Antibodies Specific for Human Ki67 and P53 Tumor Markers in Breast Cancer Tissue Samples.

机构信息

Department of Immunology, School of Public Health, Tehran University of Medical Sciences, Tehran, Iran.

Department of Pathology, Tehran University of Medical Sciences, Tehran, Iran.

出版信息

Iran J Immunol. 2023 May 31;20(2):177-189. doi: 10.22034/iji.2023.96926.2469. Epub 2023 May 7.

DOI:10.22034/iji.2023.96926.2469

PMID:37149778

Abstract

BACKGROUND

Ki67 and P53 are important diagnostic and prognostic biomarkers expressed in several cancers. The current standard method for evaluating Ki67 and P53 in cancer tissues is immunohistochemistry (IHC), and having highly sensitive monoclonal antibodies against these biomarkers is necessary for an accurate diagnosis in the IHC test.

OBJECTIVE

To generate and characterize novel monoclonal antibodies (mAbs) against human Ki67 and P53 antigens for IHC purposes.

METHODS

Ki67 and P53-specific mAbs were produced by the hybridoma method and screened by enzyme-linked immunosorbent assay (ELISA) and IHC techniques. Selected mAbs were characterized using Western blot and flow cytometry, and their affinities and isotypes were determined by ELISA. Moreover, using the IHC technique in 200 breast cancer tissue samples, we assessed the specificity, sensitivity, and accuracy of the produced mAbs.

RESULTS

Two anti-Ki67 (2C2 and 2H1) and three anti-P53 mAbs (2A6, 2G4, and 1G10) showed strong reactivity to their target antigens in IHC. The selected mAbs were also able to recognize their targets by flow cytometry as well as Western blotting using human tumor cell lines expressing these antigens. The specificity, sensitivity, and accuracy calculated for clone 2H1 were 94.2%, 99.0%, and 96.6%, and for clone 2A6 were 97.3%, 98.1%, and 97.5%, respectively. Using these two monoclonal antibodies, we found a significant correlation between Ki67 and P53 overexpression and lymph node metastasis in patients with breast cancer.

CONCLUSION

The present study showed that the novel anti-Ki67 and anti-P53 mAbs could recognize their respective antigens with high specificity and sensitivity and therefore can be used in prognostic studies.

摘要

背景

Ki67 和 P53 是几种癌症中表达的重要诊断和预后生物标志物。目前评估癌症组织中 Ki67 和 P53 的标准方法是免疫组织化学（IHC），因此需要针对这些生物标志物的高灵敏度单克隆抗体才能在 IHC 测试中进行准确诊断。

目的

生成并鉴定针对人 Ki67 和 P53 抗原的新型单克隆抗体（mAb），用于 IHC 目的。

方法

采用杂交瘤方法制备 Ki67 和 P53 特异性 mAb，并通过酶联免疫吸附测定（ELISA）和 IHC 技术进行筛选。使用 Western blot 和流式细胞术对选定的 mAb 进行鉴定，并通过 ELISA 测定其亲和力和同种型。此外，我们使用 IHC 技术在 200 例乳腺癌组织样本中评估了所产生 mAb 的特异性、敏感性和准确性。

结果

两种抗 Ki67（2C2 和 2H1）和三种抗 P53 mAb（2A6、2G4 和 1G10）在 IHC 中对其靶抗原表现出强烈的反应性。选定的 mAb 还能够通过流式细胞术以及使用表达这些抗原的人肿瘤细胞系的 Western blot 识别其靶标。针对克隆 2H1 的特异性、敏感性和准确性分别为 94.2%、99.0%和 96.6%，针对克隆 2A6 的分别为 97.3%、98.1%和 97.5%。使用这两种单克隆抗体，我们发现乳腺癌患者中 Ki67 和 P53 过表达与淋巴结转移之间存在显著相关性。