Preparation and purification of anti-human epididymis protein-4 monoclonal antibody and its application in lateral flow immunoassay

Yang Zhan; Xue Menghan; Wu Jinbo; Wang Zhizeng; Chen Chunxia

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Preparation and purification of anti-human epididymis protein-4 monoclonal antibody and its application in lateral flow immunoassay

更新时间：2025-09-23

- Preparation and purification of anti-human epididymis protein-4 monoclonal antibody and its application in lateral flow immunoassay
- Journal of Chongqing Medical University Pages: 1-6(2025)
- 作者机构：
  
  1.河南大学抗体药物开发技术国家地方联合工程实验室，开封 475004
  2.重庆大学医学院医学公共实验中心，重庆 401147
- 作者简介：
- 基金信息：
- DOI：
  CLC： R392.11
- Received：27 June 2025，
  
  Revised：2025-08-03，
  
  Accepted：19 August 2025，
  
  Published Online：23 September 2025，
- 稿件说明：
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杨展,薛孟涵,武晋博等.抗人HE4单克隆抗体的制备、纯化及在侧向免疫层析中的应用[J].重庆医科大学学报.

Yang Zhan,Xue Menghan,Wu Jinbo,et al.Preparation and purification of anti-human epididymis protein-4 monoclonal antibody and its application in lateral flow immunoassay[J].Journal of Chongqing Medical University.
杨展,薛孟涵,武晋博等.抗人HE4单克隆抗体的制备、纯化及在侧向免疫层析中的应用[J].重庆医科大学学报. DOI：

Yang Zhan,Xue Menghan,Wu Jinbo,et al.Preparation and purification of anti-human epididymis protein-4 monoclonal antibody and its application in lateral flow immunoassay[J].Journal of Chongqing Medical University. DOI：

摘要

目的

制备抗人附睾蛋白4（human epididymis protein 4，HE4）单克隆抗体，并将其应用于侧向免疫层析（lateral flow immunoassay，LFIA）试剂盒的研制。

方法

利用HE4重组蛋白作为免疫原进行动物免疫，采用杂交瘤技术筛选HE4融合细胞株，并通过动物体内诱生法大量制备抗人HE4单克隆抗体，并对抗体的亚型、亲和力、特异性等进行评价。将制备出的抗体应用于胶体金LFIA试剂盒，通过检测灵敏度、特异性等指标评价其应用价值。

结果

成功筛选了3株杂交瘤细胞（3F7、3E5、5F10）并制备出抗人HE4单克隆抗体，与HE4的亲和力依次为252 pM、89 pM、104 pM；抗体与CA125、AFP、CEA、CYFRA21-1、CA72-4、BSA、HCG均没有交叉反应。3F7和3E5抗体的亚型是IgG2b，5F10抗体的亚型IgG1型。成功应用至LFIA试剂盒的检测限为100 pmol/L，特异性良好。

结论

以人HE4重组蛋白为免疫原制备出三种具有高亲和力和良好特异性的抗人HE4单克隆抗体，并成功建立了胶体金LFIA试剂盒用于人血清HE4检测，为卵巢癌早期快速检测奠定基础。

Abstract

Objective

To prepare anti-human epididymis protein-4（HE4） monoclonal antibody，and to apply it in the development of lateral flow immunoassay（LFIA） kit.

Methods

Animal immunization was conducted using the recombinant protein of HE4 as the immunogen，and the hybridoma technique was used to obtain hybridoma cell lines secreting anti-HE4 antibodies.

In vivo

animal induction was performed to prepare anti-human HE4 monoclonal antibodies in large quantities，and the antibodies were evaluated in terms of subtypes，affinity，and specificity. The antibodies prepared were used to develop colloidal gold LFIA kit，and the application value of the kit was assessed based on the indicators such as sensitivity and specificity.

Results

Three hybridoma cell lines（3F7，3E5，5F10） were successfully screened，and anti-human HE4 monoclonal antibodies were prepared. The affinities between the three cell lines and HE4 were 252 pM，89 pM，and 104 pM，respectively. The antibodies showed no cross-reactivities with CA125，AFP，CEA，CYFRA21-1，CA72-4，BSA，and HCG. The subtypes of 3F7 and 3E5 antibodies were IgG2b，and the subtype of 5F10 antibody was IgG1. The LFIA kit using these antibodies had a limit of detection of 100 pmol/L，with good specificity.

Conclusion

Three anti-HE4 monoclonal antibodies with high affinity and good specificity are prepared using human HE4 recombinant protein as the immunogen，and a colloidal gold LFIA kit is successfully developed for the detection of human serum HE4，which lays a foundation for early and rapid identification of ovarian cancer.

关键词

Keywords

references

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