应用单克隆抗体不依赖 1 抗原检测和分离食品和水中的鼠疫耶尔森菌。

Detection and isolation of Yersinia pestis without fraction 1 antigen by monoclonal antibody in foods and water.

机构信息

Center for Food Safety, University of Georgia, Griffin, Georgia 30223-1797, USA.

出版信息

J Food Prot. 2012 Sep;75(9):1555-61. doi: 10.4315/0362-028X.JFP-11-514.

Abstract

Most available immunoassays for Yersinia pestis are based on the detection of fraction 1 antigen (F1) when yersiniae are grown at 37°C. A monoclonal antibody (MAb) was developed based on the detection of surface antigens that are not F1. F1-deficient Y. pestis cells were induced and used to immunize BALB/c mice from which MAb (immunoglobulin G1), which specifically recognizes Y. pestis, with or without F1, was obtained. This MAb (6B5) did not cross-react with enteric bacteria, including Yersinia enterocolitica. Enzyme-linked immunosorbent assay results revealed that MAb 6B5 is specific for Y. pestis, with the exception of a minor cross-reaction with Yersinia pseudotuberculosis. Western immunoblot analysis revealed that MAb 6B5 recognizes a Y. pestis outer membrane protein of ca. 30 kDa. Magnetic beads that were coated with MAb 6B5 were compared with beads coated with polyclonal antibody (PAb; rabbit) against Y. pestis for the isolation of Y. pestis in food and water samples by using a PATHATRIX cell concentration apparatus. Enrichment cultures of Y. pestis in different foods by using two different times (6 and 24 h) in brain heart infusion broth at 37°C were evaluated. Results revealed MAb 6B5-coated magnetic beads were equivalent to magnetic beads coated with PAb against Y. pestis A1122 whole cells in concentrating Y. pestis for isolation, especially when samples were enriched for 6 h. However, the selectivity for Y. pestis of the magnetic beads coated with MAb 6B5 was greater than that coated with PAb.

摘要

大多数现有的鼠疫耶尔森菌免疫检测方法都是基于在 37°C 下培养鼠疫耶尔森菌时检测 F1 抗原（F1）。本研究基于检测非 F1 表面抗原，开发了一种单克隆抗体（MAb）。诱导产生 F1 缺陷型鼠疫耶尔森菌细胞，并用其免疫 BALB/c 小鼠，从而获得特异性识别鼠疫耶尔森菌的 MAb（免疫球蛋白 G1），无论是否存在 F1。该 MAb（6B5）与肠细菌（包括肠炎耶尔森菌）不发生交叉反应。酶联免疫吸附试验结果表明，除了与假结核耶尔森菌有轻微的交叉反应外，MAb 6B5 特异性识别鼠疫耶尔森菌。Western 免疫印迹分析表明，MAb 6B5 识别鼠疫耶尔森菌的外膜蛋白约 30 kDa。用 MAb 6B5 包被的磁珠与针对鼠疫耶尔森菌的多克隆抗体（兔）包被的磁珠进行比较，使用 PATHATRIX 细胞浓缩装置从食品和水样中分离鼠疫耶尔森菌。评估了在脑心浸液肉汤中 37°C 下不同时间（6 和 24 h）培养不同食品中的鼠疫耶尔森菌的富集培养物。结果表明，在浓缩鼠疫耶尔森菌进行分离时，用 MAb 6B5 包被的磁珠与用 PAb 包被的鼠疫耶尔森菌 A1122 全细胞的磁珠等效，特别是当样品富集 6 h 时。然而，用 MAb 6B5 包被的磁珠对鼠疫耶尔森菌的选择性大于用 PAb 包被的磁珠。