PDF(Pubmed)
Abstract：
Campylobacter jejuni is a leading cause of foodborne illness worldwide, mainly due to consumption and handling of contaminated raw chicken. Rapid detection methods for C. jejuni are vital for monitoring contamination levels in chicken products and reducing human Campylobacteriosis cases. The \'gold standard\' culture-based method of Campylobacter detection takes 3-5 days and is too slow to permit effective intervention. Immuno-based methods are faster, but usually necessitate use of animals or hybridoma technology to produce antibodies; making them difficult and expensive to produce. Here, we report the generation and characterization of recombinant single-chain variable fragment (scFv) antibodies specific for C. jejuni cells, and evaluation of one scFv antibody for an immunomagnetic separation-quantitative PCR (IMS-qPCR) method to rapidly, sensitively, and specifically detect low numbers of C. jejuni. An scFv antibody phage-display library was constructed using spleen mRNA derived from a rabbit immunized with gamma-irradiated C. jejuni cells. This library was screened by surface biopanning against C. jejuni whole cells. Enriched clones were analyzed by enzyme-linked immunosorbent assay (ELISA). Two scFv antibodies that strongly and specifically recognized C. jejuni cell were expressed in Escherichia coli. Western blot analysis showed that one antibody, scFv80, was expressed as a soluble protein and retained its specific and strong binding to C. jejuni cells. This recombinant monoclonal scFv antibody was purified and used to covalently coat paramagnetic beads to be used for IMS-qPCR. The IMS-qPCR method was able to specifically and sensitively detect C. jejuni in mixed cultures within 3 h.
摘要：
空肠弯曲杆菌是全球食源性疾病的主要原因，主要是由于食用和处理受污染的生鸡肉。空肠弯曲杆菌的快速检测方法对于监测鸡肉产品中的污染水平和减少人类弯曲杆菌病的病例至关重要。基于“金标准”的弯曲杆菌检测方法需要3-5天，并且太慢，无法进行有效的干预。基于免疫的方法更快，但通常需要使用动物或杂交瘤技术来生产抗体；使它们难以生产且昂贵。这里,我们报道了特异性针对空肠弯曲杆菌细胞的重组单链可变片段（scFv）抗体的产生和表征，并评价一种scFv抗体的免疫磁珠分离-定量PCR(IMS-qPCR)方法,敏感,并特别检测到低数量的空肠弯曲杆菌。使用来源于用γ辐射的空肠弯曲杆菌细胞免疫的兔的脾mRNA构建scFv抗体噬菌体展示文库。通过针对空肠弯曲杆菌全细胞的表面生物淘选来筛选该文库。通过酶联免疫吸附测定(ELISA)分析富集的克隆。在大肠杆菌中表达强烈且特异性识别空肠弯曲杆菌细胞的两种scFv抗体。蛋白质印迹分析显示一种抗体，scFv80表达为可溶性蛋白并保留其与空肠弯曲杆菌细胞的特异性和强结合。纯化该重组单克隆scFv抗体并用于共价包被顺磁珠以用于IMS-qPCR。IMS-qPCR方法能够在3小时内特异性和灵敏地检测混合培养物中的空肠弯曲杆菌。
