鼠疫菌F1抗原双抗体夹心ELISA诊断试剂盒的研制

Preparation of double antibody sandwich ELISA diagnostic kit for F1 antigen of Yersinia pestis

目的研制鼠疫菌F1抗原双抗体夹心ELISA诊断试剂盒，并进行验证。方法用鼠疫菌F1抗原免疫家兔制备抗血清，经50％饱和硫酸铵盐析2次后，再经Sephacryl S-300凝胶柱层析纯化，作为包被抗体；制备鼠疫菌F1抗原单克隆抗体腹水，并进行纯化，用HRP标记单抗，作为酶标抗体；建立鼠疫菌F1抗原双抗体夹心ELISA检测方法，连续制备3批诊断试剂盒，并建立内部质控品。对试剂盒进行板内板间精密性、灵敏度、线性范围、特异性、重复性、准确性和稳定性验证。结果制备的试剂盒检测精密性参比品的板内变异系数为5．2％，板间变异系数为8．23％；试剂盒的线性范围为3．91～62．5ng／ml，最低检测限为3．0ng／ml；试剂盒检测鼠疫菌菌液的结果为阳性，而与近缘假结核耶尔森菌无交叉反应；试剂盒检测高、中、低3种浓度Fl抗原含量的变异系数为4．54％～8．4％，回收率在104％-108％之间；试剂盒稳定性良好，有效期至少为1年。结论成功制备了鼠疫菌F1抗原双抗体夹心ELISA诊断试剂盒，为鼠疫的临床诊断及流行病学监测提供了一种快速检测手段，也为新型鼠疫组分疫苗F1抗原含量的检测提供了方法。

Objective To develop and verify a double antibody sandwich ELISA diagnostic kit for F1 antigen of Yersinia pestis. Methods Antisera were prepared by immunizing rabbits with F1 antigen of Y. pestis, subjected to salting out with 50% ammonium sulfate, the purified by Sephacryl S-300 gle column chromatography and used as the coating antibody. The murine ascites containing monoclonal antibody （McAb） against F1 antigen was prepared, purified, labled with HRP and used as the enzyme-labeled antibody. Three batches of double antibody sandwich ELISA kits were prepared, and veri-fied for precsion, sensitivity, linear range, specificity, reproducibility, accuracy and stability by intra-and inter-assays us-ing internal reference. Results The CVs of results of intra-and inter-assays on reference for precision by the prepared kit were 5. 2% and 8. 23% respectively. The linear range and minimum detection limit of the kit were 3. 91 - 62. 5 and 3. 0 ng/ml respectively. The detection result of Y. pestis was positive, and no cross reaction with Y. pseudotuberculosis was observed. The CVs of F1 antigen at high, moderate and low contents determined by the kit were 4. 54% - 8. 4%, while the recovery rates were 104% - 108%. The kit showed high stability, of which the validity period was not less than 12 months. Conclusion A double antibody sandwich ELISA diagnostic kit for F1 antigen of Y. pestis was successfully prepared, which provided a rapid method for clinical diagnosis and epidemiological surveillance of plaque, and a tool for determination of F1 antigen content in novel plaque component vaccine.