抗玉米素核苷的单克隆抗体制备及内源玉米素核苷的酶联免疫吸附测定

THE PREPARATION OF MONOCLONAL ANTIBODIES AGAINST ZEATIN RIBOSIDE AND THE DETECTION OF ENDOGENOUS CYTOKININS BY ENZYME-LINKED IMMUNOABSORBENT ASSAY (ELISA)

通过二次成功的细胞融合,共获得三株分泌抗玉米素核苷(ZR)单克隆抗体的稳定杂交瘤系F_2E_6、F_3G_6和F_1B_9。其中F_2E_6分泌的单抗属IgG_1、F_3G_6、F_1B_9分泌的单抗均属IgM。以F_2E_6、F_3G_6单抗为基础的ELISA对ZR的检测灵敏度为0.05pmol(—18pg),线性检测范围为0.05—50pmol。除玉米素(Z)外,F_2E_6、F_3G_6单抗与IPA、6-BA、KT和腺苷几种ZR类似物的交叉反应值都小于0.18％。用ELISA测定F_2E_6单抗与ZR反应的亲和常数为2.36±0.99×10^(-8)M。本文还报道了包埋抗原的ELISA在植物内源细胞分裂素测定中的应用,并用此方法测定了黄化玉米(Zea mays L.)幼苗中ZR的含量。

Three hybridomas, F2E6, F1B9, F3G6, which secreted monoclonal anti-zcatin ribosidc antibodies, were produced irorn two successive fusions of BALB/e spleen cells with Sp2/0 myeloma cells. The mice had been hyparimmuni-zed with zeatin riboside-bovinc scrum albumin conjugate lor about 6 months ami the positive hybridomas were screened by indirect enzyme-linked immunoabsorbent. assay. Among these hybridomas, F2E6 secretes antibody of the IgG1, both F3G9 and F1B9, secrete the IgM. Two of the monoclonal antibodies, F2E6, F3G6, were characterized by EEISA and found that the detection limits were 0.05 pmol (-18pg) of zeatin riboside and the measuring ranges extended from 0.05-50 pmol. Competitive binding trials againat trans-Zeatin, cis-Zeatin, Isopentenyladenoine, 6-Benzylaminopurine, 6 -Furfurylaminopurinc, and Adenosine showed that except Zcatin, the cross-reactivities towards these structurally related cylokinins were negligible (less than 0.18%). So lliesc monoclonal antibodies have high spelfieity. The, affinity constnt of F2E6 monoclonal antibody, measured by EL1SA, was 2.36 ×10^(-8)M, which indicates that this monoclonal antibody has high affinity for zeatin riboside. The use of immobilized antigen ELISA in plant cytokinin detection was discussed and the content of cylokinins (calculated as zeatin riboside) in etiolated Zca mays L. seedling extracts was analysed with a little handlement. The method reported here not only has high sensitivity and speeilicity, but also has the advantages of being simpler safer, time-saving (3 hours), requiring less expensive equipment and handling a lot of samples simultaneously, so which is worth being spread widely.