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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 a monoclonal antibody against O-specific polysaccharide (O-SP) and establish a competitive ELISA for quantitative detection of whole bacteria ELISA and O-SP for the identification of Salmonella paratyphi A serotype. Methods Mice were immunized with Paragonimmunopathogen A (Paratyphoid A) and fused by conventional methods. O-SP conjugated with tetanus toxoid (TT) as carrier was used to screen for the detection antigen. The indirect ELISA was used to screen anti-A Paratyphoid O-SP specific antibody hybridoma cell lines; respectively Parasitic A and Paramyxovirus O-SP-TT and Typhoid Vi-TT as coating antigen, the detection of the prepared monoclonal antibodies and different serotypes of bacteria Polysaccharide cross-reaction; different Salmonella bacteria coated antigen, prepared with a Paraben Paratyphus O-SP-specific monoclonal antibody indirect bacterial conjugation, detection of bacterial serotypes; with the monoclonal antibody To compete for antibodies, a competitive ELISA method for the quantitative detection of O-SP in samples was established. Results Six hybridoma positive cell lines secreting anti-paratyphoid O-SP antibody were screened out, of which two showed specific reaction only with O-SP paratyphoid A and the other four were associated with paratyphoid B O-SP Was cross-reacted with one of the monoclonal antibodies in the indirect bacterial strain ELISA showed that the strain monoclonal antibody only Salmonella Paratyphi A reaction, but not with Salmonella typhi, Salmonella paratyphi Salmonella enteritidis and enteritis Bacterial reaction; competitive ELISA quantitative detection of O-SP detection range of 0.4 ~ 0.003 2μg / ml the most accurate. Conclusions The monoclonal antibody against specific anti-paratyphoid O-SP can be used to detect the serotype of Paratyphoid A strain and the competitive ELISA for quantitative determination of O-SP.