抗人胰岛素原单抗的制备及其IRMA的建立

THE PREPARATION OF MONOCLONAL ANTIBODIES AGAINST HUMAN PROINSULIN AND THE DEVELOPMENT OF IMMUNORADIOMETRIC ASSAY

建立胰岛素原（ＰＩ）免疫放射测定法（ＩＲＭＡ）并初步用于临床．方法：通过杂交瘤技术制备抗ＰＩ单克隆抗体，建立了ＰＩＩＲＭＡ，测定了正常人、非胰岛素依赖型糖尿病（ＮＩＤＤＭ）患者及胰岛素瘤患者空腹和餐后１小时血清ＰＩ含量．结果：共获得４株抗人ＰＩ单抗（２Ａ６，４Ａ２，４Ｅ１０，４Ｈ９）．其中４Ｈ９单抗所针对的抗原决定簇位于胰岛素的Ａ链或Ｂ链与Ｃ肽的连接部位；另外３株单抗则针对ＰＩ的胰岛素部位．血清ＰＩＩＲＭＡ灵敏度为１．９ｐｍｏｌ／Ｌ，与胰岛素及Ｃ肽均无交叉反应．测定正常人空腹及餐后１，小时血清ＰＩ分别为５．５±３．３ｐｍｏｌ／Ｌ、１７．３±６．４ｐｍｏｌ／Ｌ，ＮＩＤＤＭ患者分别为９．４±４．５ｐｍｏｌ／Ｌ、３１．８±９．４ｎｍｏｌ／Ｌ，１例胰岛素瘤患者术前及术后１周空腹血清ＰＩ分别为１０６ｐｍｏｌ／Ｌ、５７ｐｍｏｌ／Ｌ．结论：血清ＰＩＩＲＭＡ具有特异性强、灵敏度高的特点，对于胰岛素瘤的诊断和研究ＰＩ在生理及病理情况下对人体的作用具有重要意义．

PURPOSE To prepare monoclonal antibodies against human proinsulin (hPI) and to develop a sensitive and specific two-site immunoradiometric assay for human proinsulin. MEHTODS Monoclonal antibodies (McAbs) against human hPi were prepared by the routine method. Two of these McAbs were selected to develop a sensitive and specific two-site IRMA for serum hPI. We used this assay to measure the fasting and postprandial serum concentrations of PI in healthy subjects, patients with NIDDM and those with insulinoma. RESULTS We obtained 4 mouse McAbs (2A6, 4A2, 4E10,4H9) against hPI. McAb 4H9 probably binds to an epitope that is located at the junction of A chain or B chain of insulin with c-peptide and the three others bind to epitopes that are located on the A chain or B chain of insulin. The detection limit of the IRMA for hPI was 1.9pmol / L and the assay showed no cross-reaction with insulin and C-peptide.The mean fasting concentrations of PI was 5.5 ± 3.3pmol / L and increased to 17.3 ± 6.4pmol/ L one hour after a glucose loading in healthy subjects. They were 9.4 ± 1 .5pmol / L and 31.8 ± 9.4pmol / L in patients with NIDDM. In a patient with insulinoma, the fasting concentration of PI was 106 pmol / L and 57pmol / L before and one week after operation respectively. CONCLUSIONS Based on the hPI specific McAbs produced in our laboratoty, we developed a sensitive and specific two-site IRMA for serum hPI. It will be useful in the diagnosis of insulinoma and in the elucidation of the physiological and pathophysiological significance of PI in human.