在吲哚乙酸不同位点偶联载体蛋白对其抗体特异性的影响

The Effect of Linking Carrier Protein to Different Position of Indole-3-acetic Acid on the Specificity of Its Antibodies

分别选择吲哚乙醇分子上的Ｃ１位羧基和吲哚环上的Ｎ位作为偶联载体蛋白的位点，用混合酸酐法和甲醛搭桥法分别合成了两种免疫原ＩＡＡ—ＣＯ—ＮＨ一ＨＳＡ和ＩＡＡ－Ｎ－ＢＳＡ，并进而制得了对吲哚乙酸侧链识别能力不同的两种多克隆抗体，分别可特异识别甲酯化ＩＡＡ和游离态ＩＡＡ；用碳化二亚胺法和甲醛搭桥法分别合成ＩＡＡ—ＣＯ—ＮＨ－ＢＳＡｂＩＡＡ—Ｎ—ＯＶＡ两种复合物，以之为包被物，建立了两种ＩＡＡＥＬＩＳＡ。其灵敏度分别为０．３５ｐｍｏｌ和１．８０ｐｐｍｏｌ；检测范围分别为０．７８～８００ｐｍｏｌ和１．９５～２０００ｐｍｏｌ；批内变异系数分别为４．４５％和４．７９％；批间变异系数分别为１．１５％和１．５０％。笔者用这两种ＥＬＩＳＡ检测了兰花气生根和桑树苗样品中ＩＡＡ的含量，发现两种检测结果相当一致。

Indole-3-acetic acid (IAA)-protein conjugates were prepared by linking humanserum albumin (HSA) to C1-COOH of IAA via mixed anhydride, or linking bovine serumsalbumin (BSA) to the indolic ring of IAA via formadehyde, respectively. Subsequently, twokinds of polyclonal antibodies with differet specificity, which could recognize methylated andfree IAA respectively, were obtained by immunizing rabbits with IAA-CO-NH-HSA orIAA-N-BSA. Further, IAA-CO - NH-BSA and IAA-N-OVA were synthesized with1-ethyl-3-(3-dimethylaminopropyl )-carbodiimide or via forrnadehyde respectively.Therefore, two kinds of indirect enzymelinked immunosorbent assays (ELISAI andELISA2) were developed, utilizing IAA-CO-NH-BSA or IAA-N-OVA for coatingwells. The sensitivities of ELISA1 and ELISA2 were 0. 35 pmol and 1. 80 pmol, and the detection ranges were 0. 78-800pmol and 1. 95-2000pmol respectively. The intra and interassay coefficience of variation of ELISAl were 4. 45% and 1. 15%, while that of ELISA2were 4. 79% and 1. 50% respectively. On the other hand, endogenous levels of IAA werequantitatively determined in the aerial roots of Vanda and the seedlings of mulberry by thesetwo kinds of ELISAs. It was found the data were approximately equal.