R&D Center, NH Foods Ltd, 3-3 Midorigahara, Tsukuba, Ibaraki 300-2646, Japan.
R&D Center, NH Foods Ltd, 3-3 Midorigahara, Tsukuba, Ibaraki 300-2646, Japan.
J Microbiol Methods. 2020 Jun;173:105919. doi: 10.1016/j.mimet.2020.105919. Epub 2020 Apr 11.
Vibrio parahaemolyticus is a major foodborne pathogen worldwide. Contamination of V. parahaemolyticus in foods must be detected as quickly as possible because raw seafood, a major source of V. parahaemolyticus infection, is shipped immediately after production due to its short expiration date. In this study, we generated monoclonal antibodies (mAbs) against V. parahaemolyticus to develop a rapid and specific detection assay. Obtained mAbs were categorized into four groups according to their specificity. Of the groups, Group 1 (mAb VP7, VP11, and VP24) reacted to O1-O12 of V. parahaemolyticus without cross-reaction with human pathogenic Vibrio spp. (V. alginolyticus, V. cholerae, V. fluvialis, V. furnissii, V. mimicus, and V. vulnificus). We developed an immunochromatographic (IC) strip for the rapid detection of V. parahaemolyticus in the field using VP7 as a membrane-immobilized antibody and VP24 as a colloidal gold-conjugated antibody. The IC strip detected any and all serogroups (O1 to O12) or isolates (clinical, food, and environmental strains) of V. parahaemolyticus, regardless of the presence of virulence factors thermostable direct hemolysin (TDH) or TDH-related hemolysin (TRH). It did not cross-react with any other non-V. parahaemolyticus strains tested. To elucidate the target of the IC strip, we analyzed the antigen recognized by these mAbs. Group 1 mAbs showed two specific bands at molecular masses of approximately 11 and 16 kDa by western blotting analysis. Nano liquid chromatography mass spectrometry (LC-MS)/MS analysis revealed that the candidate antigen recognized by these mAbs was outer membrane (OM) lipoprotein Q87G48. We verified that mAb VP7 detected His-tagged OM lipoprotein synthesized by reconstituted cell-free protein synthesis reagent. Reactivity to an N-terminus deletion form and protease digestion form of the OM lipoprotein showed that the extent of epitope recognized by VP mAbs was 22nd-41st amino acids (AAs) from N-terminus of the OM lipoprotein, with the sequence "SDDAATANAAKLDEL." This region was also confirmed to be a V. parahaemolyticus-specific sequence by comparing putative orthologs of OM lipoprotein among Vibrio spp. The C-terminus deletion form (1st-39th AAs) including the sequence primarily recognized by VP mAbs (22nd-36th AAs) showed poor reactivity, indicating that the sequence after 40 residues of OM lipoprotein is also important for recognition by VP mAbs and VP mAbs recognize a conformational epitope. Bioinformatics research demonstrated that the OM lipoprotein is an ortholog of the lpp protein conserved throughout many bacteria. Lpp is an abundant and constitutively expressed protein and exists on the bacterial surface, suggesting it may be a good target for detection of V. parahaemolyticus.
副溶血性弧菌是一种主要的食源性病原体。由于生海鲜是副溶血性弧菌感染的主要来源，其保质期很短，因此必须尽快检测食品中的副溶血性弧菌污染情况。在本研究中，我们针对副溶血性弧菌生成了单克隆抗体（mAb），以开发一种快速且特异的检测方法。根据特异性，获得的 mAb 分为四组。其中，第 1 组（mAb VP7、VP11 和 VP24）与 O1-O12 群的副溶血性弧菌反应，而与人类致病性弧菌属（副溶血性弧菌、霍乱弧菌、弗氏弧菌、费氏弧菌、类志贺邻单胞菌和创伤弧菌）无交叉反应。我们使用 VP7 作为膜固定抗体和 VP24 作为胶体金偶联抗体，开发了一种用于现场快速检测副溶血性弧菌的免疫层析（IC）条带。该 IC 条带可检测到所有血清群（O1 至 O12）或分离株（临床、食品和环境分离株）的副溶血性弧菌，无论是否存在耐热直接溶血素（TDH）或 TDH 相关溶血素（TRH）等毒力因子。它与测试的任何其他非副溶血性弧菌菌株均无交叉反应。为了阐明 IC 条带的靶标，我们分析了这些 mAb 识别的抗原。通过 Western blot 分析，第 1 组 mAb 显示出两个约 11 和 16 kDa 的特异性条带。纳米液相色谱-质谱（LC-MS）/MS 分析表明，这些 mAb 识别的候选抗原是外膜（OM）脂蛋白 Q87G48。我们验证了 mAb VP7 可以检测到由重组无细胞蛋白合成试剂合成的 His 标记 OM 脂蛋白。针对 OM 脂蛋白的 N 端缺失形式和蛋白酶消化形式的反应性表明，VP mAb 识别的表位程度为 OM 脂蛋白 N 端第 22-41 个氨基酸（AA），序列为“SDDAATANAAKLDEL”。通过比较弧菌属 OM 脂蛋白的假定同源物，还证实了该区域是副溶血性弧菌的特异性序列。C 端缺失形式（第 1-39 个 AA），包括 VP mAb 主要识别的序列（第 22-36 个 AA），反应性较差，表明 OM 脂蛋白的 40 个残基之后的序列对 VP mAb 的识别也很重要，并且 VP mAb 识别构象表位。生物信息学研究表明，OM 脂蛋白是一种在许多细菌中保守的 lpp 蛋白的同源物。Lpp 是一种丰富且组成型表达的蛋白质，存在于细菌表面，表明它可能是检测副溶血性弧菌的良好靶标。
