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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3.91~62.5 ng/ml,最低检测限为3.0 ng/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F1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 a sandwich ELISA test kit for detecting Y. pestis F1 antigen. Methods Rabbit was immunized with Yersinia pestis F1 antigen to prepare antiserum. The antiserum was purified by salting out with 50% saturated ammonium sulfate and purified by Sephacryl S-300 gel column chromatography. Antibody ascites was purified and purified. HRP-labeled monoclonal antibody was used as enzyme-labeled antibody. Sandwich ELISA method was established for the detection of Yersinia pestis F1 antigen. Three sets of diagnostic kits were continuously prepared and internal quality control was established. The kit for intraplate precision, sensitivity, linear range, specificity, repeatability, accuracy and stability verification. Results The coefficient of variation (CV) was 5.2% and the coefficient of variation (CV) was 8.23%. The linear range of the kit was 3.91 ~ 62.5 ng / ml, the lowest detection limit was 3.0 ng / ml. The results showed that the test kit was positive for Yersinia pestis and did not cross-react with Yersinia pseudotuberculosis. The coefficient of variation (CV) of the F1 antigen in high, medium and low test kits ranged from 4.54% to 8.4% The recovery rate is between 104% and 108%. The kit has good stability and the validity period is at least 1 year. Conclusion The sandwich ELISA test kit of Yersinia pestis F1 antigen was successfully prepared and provided a rapid detection method for the clinical diagnosis and epidemiological surveillance of plague. It also provided a method for the detection of the content of F1 antigen in the novel plague component vaccine.