1种检测弓形虫急性感染的夹心ELISA方法

doi:10.11843/j.issn.0366-6964.2020.12.019

畜牧兽医学报 ›› 2020, Vol. 51 ›› Issue (12): 3101-3110.doi: 10.11843/j.issn.0366-6964.2020.12.019

1种检测弓形虫急性感染的夹心ELISA方法

李祎¹, 王先梅¹, 杨旭¹, 邓均华³, 王飞¹, 刘群^1,2, 许建海¹, 刘晶^1,2*

1. 中国农业大学动物医学院, 国家动物寄生原虫实验室, 北京 100193;
2. 中国农业大学动物医学院, 农业部动物流行病学重点实验室, 北京 100193;
3. 洛阳普泰生物技术有限公司, 洛阳 471000

收稿日期:2020-06-01 出版日期:2020-12-25 发布日期:2020-12-23
通讯作者: 刘晶,主要从事寄生虫病研究,E-mail:liujingvet@cau.edu.cn
作者简介:李祎(1993-),女,山东烟台人,硕士,主要从事寄生虫病诊断研究,E-mail:liyi931119@126.com
基金资助:
国家重点研发计划（2017YFD0501304）

Establishment of Double-antibody Sandwich ELISA to Diagnose Acute Toxoplasmosis

LI Yi¹, WANG Xianmei¹, YANG Xu¹, DENG Junhua³, WANG Fei¹, LIU Qun^1,2, XU Jianhai¹, LIU Jing^1,2*

1. National Animal Protozoa Laboratory, College of Veterinary Medicine, China Agricultural University, Beijing 100193, China;
2. Key Laboratory of Animal Epidemiology of the Ministry of Agriculture, College of Veterinary Medicine, China Agricultural University, Beijing 100193, China;
3. Luoyang Taipu Biological Engineering, INC., Luoyang 471000, China

Received:2020-06-01 Online:2020-12-25 Published:2020-12-23

摘要/Abstract

摘要： 弓形虫（Toxoplasma gondii）是一种人畜共患机会性致病原虫，其急性感染可导致宿主产生明显的临床症状和严重的病理损伤。弓形虫致密颗粒蛋白1（dense granuleprotein 1，GRA1）是一种良好的诊断抗原，也是弓形虫急性感染的标志物循环抗原（circulating antigen，CAg）的重要组分。本研究利用TgGRA1单克隆抗体建立双抗体夹心ELISA方法，为急性弓形虫感染的检测提供依据。将免疫GRA1-His的小鼠脾细胞与SP2/0进行融合，筛选出能稳定分泌抗体的杂交瘤细胞。选择其中一种单抗与HRP标记后的鼠源GRA1多抗配对，建立1种双抗体夹心ELISA方法，检测人工感染弓形虫的猪和小鼠血清样品，并将检测效果与巢式PCR（nest PCR，nPCR）和商品化试剂盒进行比较。结果筛选到4株杂交瘤细胞，腹水效价为10⁶~10⁷，亚型均为IgG1；IFA和Western blot结果显示，4株单抗均具有良好的反应性和特异性。选择1G2单抗和HRP标记多抗配对，建立了循环抗原双抗体夹心ELISA方法，最低能够检测到血清中1.563 ng·mL^-1 GRA1抗原，或者100 ng·mL^-1 ESA。该方法与nPCR相比具有较高的一致性，较市售商品化试剂盒更为准确可靠。本研究第1次将GRA1抗原作为急性弓形虫感染的诊断指标，建立相应的检测方法，成功地在人工感染样品中检测到弓形虫急性感染，可为弓形虫急性感染的诊断提供参考，对临床上急性弓形虫病的治疗有指导意义。

关键词: 弓形虫急性感染, GRA1单克隆抗体, 循环抗原, 双抗体夹心ELISA

Abstract: The zoonotic protozoa Toxoplasma gondii is an opportunistic pathogen and distributes worldwide. Acute Toxoplasma infection causes serious pathological damages. Dense granule protein 1(GRA1) secreted by dense granule is an important component of Circulating antigen, which is an indication of acute toxoplasmosis. We aimed to use a monoclonal antibody against TgGRA1 to establish an enzyme-linked immunosorbent assay that targets antigen GRA1 in serum for acute toxoplasmosis diagnosis. First, the spleens of TgGRA1-His immunized mice were fused with SP2/0 cells,then we screened hybridomas that can constantly secret monoclonal antibody to the supernatant and injected them into mice to produce a large amount of mAbs. After the identification and purification of ascites, we choose one mAb as a capture antibody, HRP conjugated mouse anti-TgGRA1 polyclonal antibody as a detection antibody to develop sandwich ELISA. This method was used to detect samples from swine and mice artificially infected with Toxoplasma gondii. Besides, the results were compared with that of nPCR and two commercial kits to evaluate the efficiency of sandwich ELISA. We successfully got 4 mAbs with ascitic titers of 10⁶-10⁷, their subtypes are IgG1. Indirect fluorescent assay and Western blot showed that all of them can react specifically with TgGRA1.1G2 mAb and HRP conjugated mouse anti-TgGRA1 polyclonal antibody were used subsequently to establish sandwich ELISA for diagnosing acute infection. After optimization, sandwich ELISA can specifically detect 1.563 ng·mL^-1 GRA1 or 100 ng·mL^-1 ESA in serum. When detecting experimental animal samples, the sandwich ELISA exhibited the high consistency with the results of nPCR and showed higher efficiency than the commercial kits. In summary, we established a sandwich ELISA for acute toxoplasmosis diagnosis that captures one certain toxoplasma antigen GRA1, samples of artificially infected animals can be detected by this method, which makes acute toxoplasmosis diagnosis more reliable. It has guiding significance for clinical treatment of acute toxoplasmosis.

Key words: acute toxoplasmosis, GRA1 monoclonal antibody, circulating antigen, sandwich ELISA

中图分类号:

S852.72

李祎, 王先梅, 杨旭, 邓均华, 王飞, 刘群, 许建海, 刘晶. 1种检测弓形虫急性感染的夹心ELISA方法[J]. 畜牧兽医学报, 2020, 51(12): 3101-3110.

LI Yi, WANG Xianmei, YANG Xu, DENG Junhua, WANG Fei, LIU Qun, XU Jianhai, LIU Jing. Establishment of Double-antibody Sandwich ELISA to Diagnose Acute Toxoplasmosis[J]. Acta Veterinaria et Zootechnica Sinica, 2020, 51(12): 3101-3110.

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1种检测弓形虫急性感染的夹心ELISA方法

Establishment of Double-antibody Sandwich ELISA to Diagnose Acute Toxoplasmosis

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