Zheng Z, Zhou X, Chen J
Department of Agronomy, Nanjing Agricultural University, China.
Chin J Biotechnol. 1995;11(4):221-6.
According to the heterogeneity of the immunogen of GA3-3-O-HSA, which contained other components such as GA3-7-CONH-HSA, two kinds of monoclonal antibodies (MAbs) against different antigen determinants of GA3 were prepared in a single process using a two-step screening assay for hybridomas. The results from a cross-reactivity experiment showed that MAb BG2 was specific for GA7/4 methyl esters (GA7/4me) with high affinity. Its affinity for GA7me was over 100 and 200 times higher than for GA3me and GA1me, respectively. Methylation of the 7-oic acid significantly increased the binding of MAb BG2 with GAs. On the contrary, the absence of a double bond or 3beta-OH in ring A and the breakdown of 19,10-gamma-lactone as well as the presence of 13-OH in ring D greatly reduced the binding of MAb BG2 with GAs. This antibody with high specificity can be used effectively to quantify and localize the main active GA7 and GA4 from the early non-hydroxylation pathway of GAs metabolism. Using this antibody, enzyme immunoassays with high sensitivity were developed which displayed linear ranges from 1.0 x 10(-14) to 1.0 x 10(-12) mol for GA4me and from 2.0 x 10(-15) to 2.0 x 10(-13) mol for GA7me.
由于GA3-3-O-HSA免疫原具有异质性，其中包含GA3-7-CONH-HSA等其他成分，因此通过两步杂交瘤筛选试验在单一过程中制备了两种针对GA3不同抗原决定簇的单克隆抗体（MAb）。交叉反应实验结果表明，单克隆抗体BG2对GA7/4甲酯（GA7/4me）具有特异性且亲和力高。它对GA7me的亲和力分别比对GA3me和GA1me高100倍和200倍以上。7-羧酸的甲基化显著增加了单克隆抗体BG2与赤霉素的结合。相反，A环中没有双键或3β-OH以及19,10-γ-内酯的断裂以及D环中13-OH的存在大大降低了单克隆抗体BG2与赤霉素的结合。这种具有高特异性的抗体可有效地用于定量和定位来自赤霉素代谢早期非羟基化途径的主要活性GA7和GA4。使用该抗体，开发了高灵敏度的酶免疫测定法，其对GA4me的线性范围为1.0×10^(-14)至1.0×10^(-12) mol，对GA7me的线性范围为2.0×10^(-15)至2.0×10^(-13) mol。
