产单核细胞李氏杆菌内化素G单克隆抗体的制备及双抗体夹心ELISA方法的建立

doi:10.11843/j.issn.0366-6964.2024.09.031

Abstract

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

CLC Number:

Jinrui MA, Wenjing SHI, Changqing TIAN, Zhijie DONG, Xuehui ZHAO, Ji ZHI, Qing CAO, Yanquan WEI, Weili SONG, Huiwen XUE, Huitian GOU. Preparation of Monoclonal Antibodies against Internalin G of Listeria monocytogenes and Their Preliminary Application[J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(9): 4069-4076.

Figures/Tables 5

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References 28

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试验菌株Experimental strain	ELISA反应结果ELISA Result	试验菌株Experimental strain	ELISA反应结果ELISA Result
产单核细胞李氏杆菌Listeria monocytogenes	+	沙门菌Salmonella	-
伊氏李斯特菌Listeria ivanovii	+	大肠杆菌Escherichia coli	-
韦氏李斯特菌Listeria welshimeri	+	枯草杆菌Bacillus subtilis	-
无害李斯特菌Listeria innocua	+	白色葡萄球菌Staphylococcus albus	-
		柠檬色葡萄球菌Staphylococcus citreus	-

样品增菌液Enrichment solution of samples	接菌浓度/(CFU·mL^-1) Concentration of inoculum	增菌时间/h Enrichment of time
样品增菌液Enrichment solution of samples	接菌浓度/(CFU·mL^-1) Concentration of inoculum	12	15	18	21	24
生牛乳Raw milk	1.0×10⁰	0.572	0.796	0.932	0.942	1.582
	1.0×10¹	0.751	0.880	0.938	1.040	1.740
	1.0×10²	0.827	0.899	1.097	2.096	3.267
	1.0×10³	0.831	1.354	1.834	2.385	3.669
	1.0×10⁴	0.932	1.688	2.583	2.523	4.636
	1.0×10⁵	1.366	2.350	2.946	2.846	5.210
	1.0×10⁶	2.767	3.065	2.980	4.546	5.504
生牛肉Raw beef	1.0×10⁰	0.825	0.876	1.001	1.746	1.805
	1.0×10¹	0.896	0.997	1.048	1.984	1.997
	1.0×10²	1.010	1.107	1.944	2.256	2.262
	1.0×10³	1.081	1.509	1.903	2.365	2.591
	1.0×10⁴	1.441	1.747	2.126	2.380	2.631
	1.0×10⁵	2.062	3.677	2.477	2.589	2.777
	1.0×10⁶	2.169	2.381	2.902	3.006	3.939

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

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