Acta Veterinaria et Zootechnica Sinica ›› 2024, Vol. 55 ›› Issue (9): 4069-4076.doi: 10.11843/j.issn.0366-6964.2024.09.031

• Preventive Veterinary Medicine • Previous Articles     Next Articles

Preparation of Monoclonal Antibodies against Internalin G of Listeria monocytogenes and Their Preliminary Application

Jinrui MA1(), Wenjing SHI1, Changqing TIAN1, Zhijie DONG1, Xuehui ZHAO1, Ji ZHI1, Qing CAO1, Yanquan WEI1, Weili SONG2, Huiwen XUE1, Huitian GOU1,*()   

  1. 1. College of Veterinary Medicine Gansu Agricultural University, Lanzhou 730070, China
    2. Animal Husbandry and Veterinary Station, Longmen Town, Lintao County, Lintao 730500, China
  • Received:2023-11-13 Online:2024-09-23 Published:2024-09-27
  • Contact: Huitian GOU E-mail:Mjr_2473@163.com;gouht@gsau.edu.cn

Abstract:

This study aimed to establish a rapid method for the detection of Listeria monocytogenes. Listeria monocytogenes (LM) internalin G (InlG) gene was amplified by PCR, pET-32a-InlG recombinant expression vector was constructed, and recombinant protein of InlG was obtained by induced expression, which was purified and immunized to mice (100 μg per mouse). Three positive hybridoma cell lines were obtained by cell fusion, cloning, and screening, and were designated as 1D2, 1D2-1 and 2H10. The subtypes of the antibodies were identified as IgG1. Using the prepared 1D2-1 as the capture antibody and the Listeria monocytogenes rabbit-derived polyclonal antibody as the detection antibody, a preliminary double-antibody sandwich ELISA method for detecting Listeria monocytogenes was established. WESTERN BLOT results showed that all three monoclonal antibodies (mAb) reacted specifically with InlG. The results of the specificity test of the method showed that there was not reaction to Salmonella, Escherichia coli, Bacillus subtilis, Staphylococcus albicans and Staphylococcus citreus. The results of the sensitivity test showed that the limit of the method for the detection of LM pure cultures was 1.0×106 CFU·mL-1. The results of the reproducibility test showed that the coefficients of variation for each group ranged from 5%-10% (< 10%). In conclusion, this study established a double antibody sandwich ELISA method using anti-internalin G protein mAb and rabbit polyclonal antibody. The method showed good specificity, sensitivity and repeatability, and can be used for rapid diagnosis and detection of Listeria monocytogenes infection.

Key words: Listeria monocytogenes, internalin G, monoclonal antibody, enzyme-linked immunosorbent assay

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