专一识别甲酯化赤霉素7,4的单克隆抗体的制备

Preparation of Monoclonal Antibodies Specific for Methyl Esters of Gibberellins A_7 and A_4

合成GA_3-3-O-HSA免疫原的同时,往往产生GA_3-7-CONH-HSA等其它成分,从而有可能通过特定的ELISA分步筛选法,在一次单抗研制流程中兼得两类针对不同GA_3抗原决定簇的单克隆抗体(MAbs)。交叉反应结果表明,其中的MAb BG2对GA_(7/4)甲酯具有高亲和力和专一性,它与GA_7me的亲和力比GA_3me与GA_1me分别高出100与200倍。7位羧基的申酯化可显著增加GA与该抗体的结合,而A环上双键或3β羟基的缺失,19,10-r-内酯环的破坏及D环上13位羟基的存在却严重降低GA与它的结合。这种高专一性的单抗将可用于高等植物和真菌体内早期非羟化途径中的GA_7与GA_4的免疫定量与定位研究。用该抗体建立的GA_4me与GA_7meELISAs具有极高的灵敏度,两者的检测范围分别为1.0×10^(-14)～1.0×10^(12)mol与2.0×10^(-15)～2.0×10^(-13)mol。

According to heterogeneity of immunogen of GA3-3-O-HSA which contains other components,such as GA3-7-CONH-HSA,in combination with development of two-step screening assay for hybridomas,two kinds of monoclonal antibodies (MAbs)against different antigen determinants of GA3 were prepared in single process of preparing antibody. The results from the experiment of cross-reactivities show that MAb BG2 has high affinity for and specificity to GA7/4 methyl esters (GA7/4me). It exhibits over 100 and 200 times higher affinity for GA7 me than for GA3 me and GA1me, respectively. Methylation of the 7-oic acid significantly increases the binding of MAb BG2 with GAs. On the contrary, the absence of double bond or 3B-OH in A cycle and the breakdown of 19, 10-r-lactone as well as the presence of 13-OH in D cycle magnificantly reduce the binding of MAb BG2 with GAs. This antibody with high specificity can be effectively used to quantify and localize main active GA7 and GA4 from early non-hydroxylation pathway of GAs metabolism. Based on this antibody, enzyme-linked immunosorbent assay with high sensitivity were developed which display linearity ranges from 1. 0×10-14 to 1.0×10-12mol of GA4me and from 2.0×10-15to 2. 0×10-13 mol of GA7me.