Preparation and characterization of monoclonal antibodies against human cytomegalovirus PP65 antigen

Shuting ZHANG; Haixia TIAN; Qingqing LIU; Xing KANG; Weiping FAN

doi:10.16016/j.1000-5404.202002059

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Preparation and characterization of monoclonal antibodies against human cytomegalovirus PP65 antigen

Basic Medicine | 更新时间：2026-01-09

- Preparation and characterization of monoclonal antibodies against human cytomegalovirus PP65 antigen
- Journal of Third Military Medical University Vol. 42, Issue 12, Pages: 1182-1187(2020)
- 作者机构：
  
  030001 太原，山西医科大学微生物学与免疫学教研室
- 作者简介：
- 基金信息：
  
  the Special Project for Patent Exploitation and Promotion of Shanxi Province(20171009);the Science and Technology Development Foundation of Shanxi Province(033068)
- DOI：10.16016/j.1000-5404.202002059
  CLC： R373.9; R392-33; R392.11
- Received：22 January 2020，
  
  Revised：2020-03-08，
  
  Published：30 June 2020
- 稿件说明：
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Shuting ZHANG, Haixia TIAN, Qingqing LIU, et al. Preparation and characterization of monoclonal antibodies against human cytomegalovirus PP65 antigen[J]. Journal of Third Military Medical University, 2020, 42(12): 1182-1187.
DOI：

Shuting ZHANG, Haixia TIAN, Qingqing LIU, et al. Preparation and characterization of monoclonal antibodies against human cytomegalovirus PP65 antigen[J]. Journal of Third Military Medical University, 2020, 42(12): 1182-1187. DOI： 10.16016/j.1000-5404.202002059.

摘要

目的

制备人巨细胞病毒(human cytomegalic virus

HCMV)PP65抗原的单克隆抗体，并通过免疫组化鉴定单克隆抗体与HCMV PP65抗原的反应性。

方法

诱导PQE30-PP65重组质粒表达PP65蛋白并Ni柱亲和层析纯化。PP65抗原免疫BALB/c小鼠，将血清抗体阳性小鼠脾细胞与骨髓瘤细胞(SP2/0)融合，经HTS筛选，扩大培养获取单克隆抗体。经Protein A亲和层析纯化，并通过间接ELISA鉴定其特异性、效价及亚型。将制备的单克隆抗体应用于临床样本的玻片免疫组化检测。

结果

获得8株高特异性抗HCMV PP65抗原的单克隆抗体，其亚型均为IgG1型，轻链均为κ链，效价均在1 :2 048 000以上。其中6株单克隆抗体能够通过玻片免疫组化法准确鉴定HCMV激活性感染。

结论

成功制备了HCMV PP65单克隆抗体。单抗与白细胞中HCMV PP65抗原反应良好。

Abstract

Objective

To prepare monoclonal antibodies (mAbs) against human cytomegalovirus (HCMV) PP65 antigen and characterize their reactivity to HCMV PP65 antigen by immunohistochemistry.

Methods

The recombinant plasmid PQE30-PP65 was transformed into competent

E.coli

M15 cells to obtain the recombinant PP65 antigen

which was purified with Ni column affinity chromatography. BALB/c mice were immunized with the purified an

tigen

and the spleen cells of the mice were isolated and fused with myeloma cells (SP2/0). The mAbs were obtained by HTS screening and purified by protein A affinity chromatography

and their specificity

titer and subtype were identified by indirect ELISA. The performance of the mAbs in immunohistochemical detection of HCMV PP65 antigen was tested on slides of clinical samples.

Results

Eight highly specific mAbs against HCMV PP65 antigen were obtained. All these mAbs were identified to be IgG1 subtype containing κ light chains

with titers all above 1 :2 048 000. Six of the mAbs were shown to accurately identify an active infection of HCMV by slide immunohistochemistry.

Conclusion

We successfully prepared HCMV PP65 mAbs

and 6 of these mAbs have good reactivity with HCMV PP65 antigen in leukocytes.

关键词

Keywords

references

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