异戊烯基腺苷组细胞分裂素的快速酶联免疫吸附测定法的建立

Rapid Enzyme-Linked Immunosorbent Assay for Isopentenyladenosine Group Cytokinins

从样品制备，ＥＬＩＳＡ类型及免疫反应时间三方面着手建立了异戊烯基腺苷（ｉＰＡ）组细胞分裂素的快速酶联免疫吸附测定法。用ＮａＩＯ４氧化合成免疫原ｉＰＡ－Ｎ９－ＢＳＡ（ＢＳＡ：牛血清白蛋白）。由此获得的免抗血清效价达１：３４５００。交叉反应试验表明该抗血清只识别ｉＰＡ组细胞分裂素。因此本研究中采用直接型（即固相抗体型）ＥＬＩＳＡ，并将最佳免疫反应平衡时间缩短至３０ｍｉｎ（４℃）。所建立的ｉＰＡ组ＥＬＩＳＡ检测极限为４．７ｆｍｏｌｉＰＡ／孔，线性检测范围０．０１～５０ｐｍｏｌｉＰＡ／孔，板内、板间的变异系数分别为３．７％和５．３％。样品的稀释曲线与标准曲线平行，回收率为８７．１％，表明本研究中采用的８０％甲醇提取、Ｓｅｐ－ｐａｋＣ１８柱初纯化的快速制样程序适合于该ＥＬＩＳＡ。对大豆植株不同部位ｉＰＡｓ含量测定结果表明，根瘤与根尖内ｉＰＡｓ含量显著高于幼叶与顶芽内的ｉＰＡｓ含量。

The rapid ELISA used specifically for the determination of iPAs (ip,iPA,iPA-5'P) has been developed. The hapten iPA has been selected and conjugated,via its hydroxy groups of the ribose moiety,to free amino groups of bovine serum albumin(BSA) with NaIO4 oxidation and the immuogen iPA-N9-BSA has been synthesised. Anti-iPA antiserum has been raised in rabbit and its titer,determined by ELISA,reaches 1: 34500. This antiserum basically recognized iPA group cytokinins. Because of higher quality of this antiserum, direct ELISA (that is,poly antibodies as solid phase)has been set up and it has been observed that the immunoreaction almost reaches to equilibrium during 30 minutes at 4℃. The detection limit of this ELISA is 4. 7 fmol/ well and the linear detection range is 0. 01-50 pmol/well. The coefficient variation(CV) within plates is 3. 7 % and the CV between plates is 5. 3 %. The dilution curves of sample extracts parallel with standard curve in this ELISA. And the recovery is as high as 87. 1%. These results suggest that the simple sample preparation procedure(i. e. extraction with 80% methanol,then passing througn Sep-pak C18 column)used in this ELISA is effective. The iPA-group cytokinins contents in various parts of soybean plants have been determined with this ELISA. It is shown that iPAs levels in root nodules and root tips are greatly higher than those in young leaves and apical buds of soybean plants.