CYFRA21-1双抗体夹心酶联免疫检测方法的研制

摘要

Objective:To establish the detection method of double-antibody sandwich ELISA about CYFRA 21-1 in human ser-um.Methods:The paired antibody were screened among four strains mAbs of CYFRA 21-1, which was marked by sodium periodate method.The detecting method of double antibody sandwich ELISA was optimizted , and evaluated by specificity , stability and sensitivi-ty.Results:The results showed that a paired of antibody , which was 2F9 as the coated antibody and 6F11 as the labeled antibody, was selected from four mAbs .It was the optimum condition of double antibody sandwich ELISA that the coating antigen concentration of 2F9 was 0.50 μg/ml, while the labeled antibody of 6F11 was diluted 6 000 times.The linear range of standard curve was 0.7-25 ng/ml with r2 =0.990 8, while the limit of detection was 0.666 8 ng/ml, the recovery rate was 98.14%.The cross-reactions with the oth-er analogues in serum were less than 0.1%.The coefficient of variation in group (n=10) was 6.8%, whereas coefficient of variation among group(n=5) was 11.4%.The correlation compared with other foreign ELISA kit was 91.42%.Conclusion:In brief, we suc-cessfully established the method of double antibody sandwich ELISA detecting CYFRA 21-1 level in human serum , laying the foundation for the production of CYFRA21-1 ELISA kit.%目的：建立人血清CYFRA21-1双抗体夹心酶联免疫检测方法。方法：以高碘酸钠法标记4株自制的CY-FRA21-1单克隆抗体，从中筛选配对抗体，建立双抗体夹心ELISA检测方法，并对其特异性、稳定性和灵敏度进行评价。结果：在4株单克隆抗体中筛选出1对配对抗体：2 F9株做包被抗体、6 F11株做酶标抗体。以0．50μg/ml的包被浓度、1∶6000的酶标抗体稀释度建立双抗体夹心检测方法。标准曲线在0．7～25 ng/ml范围内成线性关系，相关性为99．08％，最低检测限为0．6668 ng/ml，回收率为98．14％；与血清类似物的交叉性反应低于0．1％；批内（n＝10）、批间（n＝5）精密度分别为6．8％和11．4％，与国外试剂盒检测对比的相关性为91．42％。结论：成功地建立了双抗体夹心ELISA检测人血清CYFRA21-1的方法，为后续的检测试剂盒的生产奠定基础。