C-肽化学发光免疫分析方法学研究

The methodological study of C-peptide chemiluminescent immunoassay

目的：建立人血清C-肽化学发光免疫分析法。方法：采用两株高特异的抗C-肽单克隆抗体，以一株包被微孔板制成固相抗体，另一株标记碱性磷酸酶，以金刚烷衍生物为底物，双抗体夹心法测定人血清C-肽浓度。结果：以5μg/ml单克隆抗体包被微孔板，酶结合物以1：5000稀释，在C-肽浓度0．1～15ng／ml范围内线性良好，线性方程为y=0．9575x-0．0869，相关系数0．99；灵敏度为0．01ng／ml，批内、批间变异系数分别为5．8％、8．4％；平均回收率98．4％，范围91．5％～105．0％。结论：文中建立的人血清C-肽化学发光免疫分析法，首次建立小分子人血清C-肽双抗体夹心化学发光免疫法测定。线性好，灵敏度较常规高出一个数量级，准确度和精密性高，能够满足一般临床诊断和科研的应用。

Objective：To develop chemiluminescent immunoassay for serum C-peptide. Methods：Two specific anti-C-peptide monoclonal antibodies were used in this assay, one coated in microplate, and the other labeled with alkaline phosphatase, adamantine derivative as substrate, determine the serum C-peptide with sandwich principle. Results： A monoclonal antibody was coated at 5 μg/ ml, enzyme-monoclonal antibody conjugate was diluted at 1：5 000. There was a good linearity at 0. 1-15 ng/ml of C-peptide concentration, linearity equation was y = 0. 957 5x -0. 086 9, correlation rate was 0. 99. The sensitivity was 0. 01 ng/ml, CV of intraassay and interassay were 5.8% ,8.4% respectively. The mean recovery rate was 98.4%, and range was 91.5%-105.0%. Conclusion：It is first time to report the small molecule polypeptide determined by two monoclonal antibodies sandwich CLIA. This assay had a good linearity, low detection limit and a excellent accuracy and precision. It can be used in clinical diagnosis and research.