嗜肺军团菌快速检测胶体金试纸的研制

doi:10.3969/j.issn.1673-8640.2020.07.022

摘要/Abstract

摘要：

目的研制特异性检测嗜肺军团菌（Lp）的胶体金免疫层析试纸条。方法通过基因工程制备Lp-肽聚糖关联脂蛋白（PAL）重组蛋白并进行纯化,筛选分泌抗Lp-PAL重组蛋白单克隆抗体（简称单抗）的杂交瘤细胞株,采用间接酶联免疫吸附试验（ELISA）和免疫印迹法鉴定筛选到的Lp-1和Lp-2单抗的特异性,采用生物膜层干涉（BLI）技术鉴定抗原抗体反应的特异性。依据双抗体夹心原理制备胶体金免疫层析试纸条,并初步评价其检测性能（特异性、灵敏度及稳定性）。结果筛选到2株高效分泌抗Lp-PAL蛋白抗体的杂交瘤细胞株Lp-1和Lp-2,纯化得到2种单抗Lp-1和Lp-2。间接ELISA检测结果表明,Lp-1及Lp-2单抗只与Lp发生阳性反应,与其他10种呼吸道常见病原菌（肺炎链球菌、卡他莫拉菌、流感嗜血杆菌、肺炎支原体、金黄色葡萄球菌、肺炎克雷伯菌、产酸克雷伯菌、鲍曼不动杆菌、铜绿假单胞菌、奇异变形杆菌）无交叉反应。免疫印迹法结果显示,Lp-1和Lp-2单抗与Lp-PAL天然蛋白和重组蛋白有强反应,与肺炎链球菌、流感嗜血杆菌和卡他莫拉菌无目的反应条带。BLI技术的验证结果显示,抗原抗体的相互作用力均较强,解离速率慢,Lp-1和Lp-2单抗识别的是同一抗原的不同抗原表位,且只与Lp有强反应,与其他10种细菌均无特异性结合反应。制备的胶体金免疫层析试纸条最低检出限为1×10⁷ CFU/mL,与Lp常见4种血清型（Lp14、Lp12、Lp9和Lp6）均有特异性反应,与肺炎链球菌、卡他莫拉菌等10种常见呼吸道病原菌均无交叉反应,可在25 ℃环境下稳定6个月。结论 PAL蛋白具有高度的表面暴露性和较强的抗原性,可作为Lp的检测标志物,以此为基础制备的胶体金免疫层析试纸条具有快速、简便、灵敏的特点,适用于Lp感染的快速检测。

关键词: 嗜肺军团菌, 单克隆抗体, 肽聚糖关联脂蛋白, 生物膜层干涉技术, 胶体金免疫层析法

Abstract:

Objective To establish a novel colloidal gold immunochromatography assay for the specific detection of Legionella pneumophila（Lp）. Methods Lp-PAL recombinant protein was prepared and purified by genetic engineering,and hybridoma cell lines secreting anti-Lp-peptidoglycan-associated lipoprotein（PAL） recombinant protein monoclonal antibody were screened. The indirect enzyme-linked immunosorbent assay（ELISA） and immunoblotting were used to screen and identify the specificities of Lp-1 and Lp-2 monoclonal antibodies. The specificity of antigen-antibody response was identified using bio-layer interferometry（BLI） technology. A colloidal gold immunochromatography test strip was prepared based on double antibody sandwich principle,and its detection performance（specificity,sensitivity and stability） was initially evaluated. Results Totally,2 hybridoma cell lines that secreted anti-Lp-PAL protein antibodies were screened out,and 2 monoclonal antibodies,Lp-1 and Lp-2,were purified. Indirect ELISA showed that Lp-1 and Lp-2 monoclonal antibodies only positively reacted with Lp,and reacted with 10 other common respiratory pathogens（Streptococcus pneumoniae,Moraxella catarrhalis,Haemophilus influenzae,Mycoplasma pneumoniae,Staphylococus aureus,Klebsiella pneumoniae,Klebsiella oxytoca,Acinetobacter baumannii,Pseudomonas aeruginosa and Proteus mirabilis） had no cross-reactivity. The results of western blotting showed that Lp-1 and Lp-2 monoclonal antibodies had strong reactions with natural PAL protein and Lp-PAL recombinant protein,and had no purposeful reaction bands with Streptococcus pneumoniae,Haemophilus influenzae and Moraxella catarrhalis. The verification results of BLI technology showed that the antigen-antibody interactions were strong,and the dissociation rate was slow. Lp-1 and Lp-2 monoclonal antibodies recognized different epitopes of the same antigen,and only had a strong reaction with Lp. They had no specific binding reaction with 10 other pathogens. The minimum detection limit of the prepared colloidal gold immunochromatography test strip was 1×10⁷ CFU/mL,which specifically reacted with the 4 common serotypes of Lp（Lp14,Lp12,Lp9 and Lp6） and did not react with 10 other pathogens such as Streptococcus pneumoniae and Moraxella catarrhalis. The strips could be stored at 25 ℃ for at least 6 months. Conclusions Lp outer membrane protein PAL,which possesses a high degree of surface antigen accessibility and antigenicity,could be used as a molecular marker for Lp infection. The colloidal gold immunochromatography test strip with the features of rapidity,convenience and sensitivity provides a unique tool for the on-site surveillance and diagnosis of Lp infection.

Key words: Legionella pneumophila, Monoclonal antibody, Peptidoglycan-associated lipoprotein, Bio-layer interferometry technology, Colloidal gold immunochromatography assay

中图分类号:

R446.1

张秋, 李杰, 王猛, 王毅, 杨波, 胡征. 嗜肺军团菌快速检测胶体金试纸的研制[J]. 检验医学, 2020, 35(7): 726-733.

ZHANG Qiu, LI Jie, WANG Meng, WANG Yi, YANG Bo, HU Zheng. Development of colloidal gold immunochromatography assay for the rapid detection of Legionella pneumophila[J]. Laboratory Medicine, 2020, 35(7): 726-733.

图/表 11

图1 AMC传感器的检测流程图

图2 PCR扩增结果和重组质粒双酶切鉴定结果注：（a）PCR扩增结果,M为DNA Marker,1~4为不同样本的PCR扩增产物;（b）重组质粒双酶切结果,M为DNA Marker,1为重组质粒双酶切鉴定电泳结果

图3 Lp-PAL融合蛋白的诱导表达和Ni柱亲和纯化结果注：M为蛋白质Marker;1为pET-28a（+）诱导后全菌;2为融合蛋白诱导前全菌;3为融合蛋白诱导后全菌;4为融合蛋白诱导上清液;5为融合蛋白诱导沉淀;6为穿透液;7~11分别为5、20、40、100、150 mmol/L咪唑洗脱液

图4 抗Lp-PAL蛋白单抗的SDS-PAGE结果注：M为蛋白质Marker;1为Lp-1穿透液;2为Lp-1洗脱液;3为Lp-2穿透液;4为Lp-2洗脱液

图5 Lp-1和Lp-2单抗的免疫印迹法结果注：M为蛋白质Marker;1、6为肺炎链球菌;2、7为卡他莫拉菌;3、8为流感嗜血杆菌;4、9为Lp;5、10为Lp-PAL重组蛋白

表1 Lp-1和Lp-2单抗与抗原Lp-PAL重组蛋白的相互作用动力学常数

单抗	亲和力常数/M	结合常数/（1/Ms）	解离常数/（1/s）	误差值	拟合度
Lp-1	1.32×10^-9	8.23×10⁵	1.09×10^-3	0.057 7	0.911 1
Lp-2	9.53×10^-10	6.65×10⁵	6.33×10^-4	0.059 2	0.978 6

图6 Lp-1和Lp-2单抗与抗原Lp-PAL重组蛋白的结合、解离图

图7 Lp-1和Lp-2单抗相互竞争抗原Lp-PAL结合位点实验结果

图8 交叉反应实验结果

图9 胶体金免疫层析试纸条的特异性注：（a）胶体金免疫层析试纸条对Lp不同血清型的检测特异性;（b）胶体金免疫层析试纸条对11种常见呼吸道病原菌的检测特异性

图10 胶体金免疫层析试纸条的灵敏度

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