人血清胰岛素原单克隆抗体夹心酶联免疫法的建立

A monoclonal antibody-based sandwich enzymeimmunoassay for human serum proinsulin

目的建立灵敏、特异的酶联免疫分析法测定人血清胰岛素原，用以评价胰岛β细胞功能。方法选用可同时与胰岛素原形成夹心结合的抗人C肽单抗和抗人胰岛素单抗，分别包被塑料微孔条作团相抗体和生物素化后作液相抗体，再以亲和素连接的辣根过氧化物酶放大检测信号，通过酶底物显色，判定胰岛素原浓度。结果方法的灵敏度为0．9pmol／L，曲线工作范围0．9～200pmol／L，批内批间变异系数分别小于8．9％和11．2％，平均回收率94％（82％～114％），稀释曲线和标准曲线平行，方法与高浓度的人胰岛素和C肽（5000pmol／L）均未见交叉反应。结论本方法具有灵敏度高、特异性强、操作简便和无同位素污染等优点，适于大批量标本测定胰岛素原及其临床意义的深人探讨。

Objective To establish a sensitive and specific ELISA for human serum proinsulin (PI) to evaluate theislet 5-cell function. Methods Based on two monoclonal antibodies (MCA) and biotin--avidin (BA) system, the anti-Cpeptide MCA is absorbed to plastic microtitre stript wells as solid phase, the biotinylated anti--insulin MCA as the liquidphase and avidin-horseradish peroxidase as the amplifier for the detecting signals during the two--step BA--ELISA.Results With a sensitivity of 0. gpmol/L, the assay provides a working range up to 200 pmol/L and does not crossreact with human insulin or C-peptide at a high dosage of 5000 pmol/L. The interassay and intraassay CVs are less than8. 9 % and 11. 2 % respectively and the mean recovery of the assay is 94 % (82 % ~ 114 % ). The dilution curve of a samplewith high PI level is parallel well to the standard curve. Conclusion Our BA--ELISA for human PI is thus verysensitive, specific and easy to perform on a large scale, and can provide a new method to evaluate the 6-cell function forclinical research and diagnosis.