| Free ? human chorionic gonadotrophin(f-?-hCG)and Alpha fetoprotein(AFP)are commonly used in the detection of pregnancy-related diseases,as well as tumor markers in clinical practice.At present,chemiluminescence immunoassay and time-resolved immunofluorescence detection are the main quantitative methods for those markers.Chemiluminescence is one of the most important branches,but the domestic market is almost monopolized by foreign large companies.Most of the instruments and reagents used in clinic are imported products,and very expensive.Based on the previous work on the Chemiluminescence Immunoassay Platform by our research team,this study is intend to develop a Chemiluminescence Immunoassay method for the determination of free beta-human chorionic gonadotropin and alpha-fetoprotein.The laboratory performance of the two self-made reagents were comprehensively evaluated.Objective:To develop Chemiluminescent Immunoassay for the free ? human chorionic gonadotrophin(f-?-HCG)and alpha-fetoprotein(AFP)which meet the clinic usage.Methods:1.The two-step,double antibody sandwich method was choiced to establish a chemiluminescence immunoassay for the quantitative determination of free beta-human chorionic gonadotropin and alpha-fetoprotein.2.To evaluate the performance of two self-made chemiluminescent immunoassay reagents,the parameters of limitation,accuracy,repeatability,precision,specificity,reference range and equivalence within clinic samples were included.Results:After screening,the two-step reaction procedure was selected.The methodological evaluation results of two self-made chemiluminescent immunoassay reagents for free human chorionic gonadotropin and alpha-fetoproteinwere as follows.f-?-hCG reagent:The limit of detection was 0.006 ng/mL and the accuracy of relative deviation was between 2.56%and 4.43%.The linear was between 0-200 ng/m L with R2 value of 0.99999.The repeatability was less than 8%and the batch variation between groups were less than 15%.Compared to Roche's kit in the quantitative analysis,the consistency was 0.965,the linear regression equation of the reagent was y=1.131x+0.826 with the correlation coefficient of 0.992.AFP reagent:The limit of detection was 0.0083 ng/mL and the accuracy of relative deviation is 0.39%.The linear was between 0-1000 ng/mL with R2 =1.The repeatability was less than 8%(2.36%?3.60%)and the batch variation between groups were less than 15%.Compared to Roche's kit in the quantitative analysis,the consistency was 0.989,the linear regression equation of the reagent was y=0.980x+0.498 with the correlation coefficientof 0.959.Conclusions:The chemiluminescent immunoassay for free beta-human chorionic gonadotropin and alpha-fetoprotein have been successfully established.All the indices(detective limitation,accuracy,repeatability,inter-batch precision,specificity and so on)met the requirements of clinical detection.It is expected to have a promised future to share the filed as those similar reagents imported done. |
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