Monoclonal antibody-based serological assays for detection of Potato virus S in potato plants

基于单克隆抗体的检测马铃薯植物中马铃薯S病毒的血清学方法（英文）

Potato virus S （PVS） often causes significant losses in potato production in potato-growing countries. In this study, the ordinary strain of PVS （PVS 0） was purified from PVS-infected potato plants and used as the immunogen to produce hybridomas secreting monoclonal antibodies （MAbs）. Five highly specific and sensitive murine MAbs （1A3, 16C10, 18A9, 20B12, and 22H4） against PVS were prepared using conventional hybridoma technology. Using these MAbs, tissue print-enzyme-linked immunosorbent assay （ELISA）, dot-ELISA, and double-antibody sandwich （DAS）- ELISA were developed for sensitive and specific detection of PVS infection in potato plants. The results of sensitivity assays revealed that PVS could be reliably detected in PVS-infected leaf crude extracts diluted at 1：10240 and 1：163840 （w/v, g/ml）in phosphate buffer saline （PBS） by dot-ELISA and DAS-ELISA, respectively. Twenty-two samples collected from potato fields in Yunnan Province, China were tested for PVS infection using the serological assays we had developed, and 14 of them were found to be positive. This indicates that PVS is now prevalent in potato fields in Yunnan Province.

目的:建立基于单克隆抗体的检测马铃薯植物中马铃薯S病毒(PVS)的血清学方法,为我国PVS的田间调查和检测及无毒种薯的生产提供快速、实用的检测技术。创新点:首次制备了抗PVS的高度特异和灵敏的单克隆抗体,并以制备单抗为核心建立三种能快速、准确、灵敏、特异地检测PVS的血清学检测方法。方法:以差速离心方法提纯的PVS病毒粒子作为免疫原免疫BALB/c小鼠,通过杂交瘤技术获得了能稳定传代并分泌PVS单克隆抗体的杂交瘤细胞株;扩繁的杂交瘤细胞注射到小鼠腹腔获得单抗腹水,并以纯化的制备单抗为核心,根据血清学原理建立检测马铃薯植物中PVS的双抗夹心酶联免疫吸附试验(DAS-ELISA)、斑点酶联免疫吸附试验(dot-ELISA)和组织印迹酶联免疫吸附试验(tissue print-ELISA)血清学检测方法;并对田间马铃薯样品进行PVS检测,分析建立的血清学方法检测PVS的有效性。结论:利用杂交瘤技术获得了五株能稳定传代并分泌PVS单克隆抗体的杂交瘤细胞株,以制备杂交瘤细胞分泌的单抗为核心建立了检测马铃薯植株中PVS的DAS-ELISA、dot-ELISA和tissue print-ELISA三种血清学方法。三种血清学方法检测感染PVS的马铃薯植株呈阳性反应,而检测健康的马铃薯及感染其他三种马铃薯病毒的植株呈阴性反应,且建立的DAS-ELISA和dot-ELISA方法检测马铃薯病叶的灵敏度分别达到1:163 840和1:10 240倍稀释(w/v,g/ml)。田间样品检测结果发现,建立的血清学方法的检测结果与逆转录聚合酶链反应(RT-PCR)的检测结果一致,表明建立的血清学方法可有效地用于马铃薯植物中PVS的检测,从而为我国PVS的田间调查和检测及无毒种薯的生产提供快速、实用的检测技术。