甲型副伤寒细菌单克隆抗体的制备及其初步应用

Preparation and preliminary application of monoclonal antibodies against Salmonella paratyphi A

目的制备甲型副伤寒特异性多糖（O-specific polysaccharide,O-SP）单克隆抗体,建立鉴定甲型副伤寒沙门血清型细菌的全菌体ELISA和O-SP的竞争ELISA定量检测方法。方法用甲型副伤寒全菌体免疫小鼠,通过常规方法融合,以破伤风类毒素（tetanus toxoid,TT）为载体偶联的O-SP为筛选检测抗原,间接ELISA法筛选分泌抗甲型副伤寒O-SP的特异性抗体杂交瘤细胞株;分别以甲型和乙型副伤寒的O-SP-TT及伤寒Vi-TT为包被抗原,检测制备的单克隆抗体与不同血清型细菌多糖的交叉反应;以不同沙门菌菌体为包被抗原,用制备的1株甲型副伤寒O-SP特异性单克隆抗体进行菌体间接ELISA,检测细菌血清型;用该株单克隆抗体为竞争抗体,建立定量检测样品中O-SP的竞争ELISA方法。结果筛选出6株分泌抗甲型副伤寒O-SP抗体的杂交瘤阳性细胞株;其中2株仅与甲型副伤寒O-SP呈特异性反应,其余4株与乙型副伤寒O-SP呈交叉反应;用其中1株单克隆抗体进行的菌体间接ELISA显示,该株单克隆抗体仅与甲型副伤寒沙门菌反应,而不与伤寒沙门菌、乙型副伤寒沙门菌和肠炎沙门菌反应;竞争ELISA定量检测O-SP的检测范围在0.4~0.003 2μg/ml最准确。结论制备的特异性抗甲型副伤寒O-SP的单克隆抗体可用于甲型副伤寒细菌血清型快速鉴定和O-SP的竞争ELISA定量检测。

Objective To prepare monoclonal antibodies（Mc Abs）against O-specific polysaccharide（O-SP）of Salmonella paratyphi A and develop a whole cell enzyme-linked immunosorbent assay（ELISA）for identification of serotype of S.paratyphi A and a competitive ELISA for O-SP.Methods The hybridoma cell lines secreting specific antibodies to OSP of S.paratyphi A were established by conventional fusion method of myeloma SP2 / 0 cells with spleen cells of mice immunized with whole cell S.paratyphi A,and screened by indirect ELISA using tetanus toxoid（TT）-coupled O-SP as detection antigen.The cross reactions of prepared Mc Ab with bacterial polysaccharides of various serotypes were tested using O-SP-TT of S.paratyphi A and B as well as Vi-TT as coating antigens respectively.Serotype was identified by indirect ELISA with prepared Mc Ab against O-SP of a S.paratyphi A strain using various Salmonella bacteria as coating antigens.A quantitative competitive ELISA method for quantitative determination of O-SP in test samples was developed using the Mc Ab.Results Six hybridoma cell strains secreting Mc Abs against O-SP of S.paratyphi A were obtained,of which two showed only specific reactions with O-SP of S.paratyphi A while the other four showed cross reaction with O-SP of S.paratyphi B.Indirect ELISA proved that the specific Mc Ab secreted by a hybridoma cell strain showed only reaction with S.paratyphi A while no reactions with S.typhi,S.paratyphi B and S.enteritidis.The quantitative detection range of O-SP by competitive ELISA was 0.4 ~ 0.003 2 μg / ml.Conclusion The prepared Mc Abs against O-SP of S.paratyphi A may be used for the rapid identification of serotype of S.paratyphi A and quantitative detection of O-SP by competitive ELISA.