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

Abstract

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

CLC Number:

S852.72

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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Establishment of Double-antibody Sandwich ELISA to Diagnose Acute Toxoplasmosis

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