Preparation of monoclonal antibody against lignin

抗木质素单克隆抗体的制备

• 批准号: 17580143
• 负责人: YOSHINAGA Arata
• 金额: $ 2.34万
• 依托单位: Kyoto University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2005
• 资助国家: 日本
• 起止时间: 2005 至 2007
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-17580143/
• 关键词: Tree cell wall; lignin; lignification; Immunolabeling; monoclonal antibody; 8-O-4 linkage; 8-5 linkage; 8-8 linkage; ヒノキ; 分化中木部; イチョウ; 8-0-4結合

To understand complex chemical structure of lignin in wood cell walls, monoclonal antibodies that recognize specific structure of lignin will be very important. The purpose of the present study is to develop the monoclonal antibody against lignin model compounds-protein conjugates. Three kinds of model compounds including three kinds of linkage (8-O-4, 8-5and8-8) of lignin were selected and conjugated with bovine serum albumin via p-aminohippuric acid. Monoclonal antibodies were raised with mice againstthree kinds of conjugates. As a result, 4hybridoma clones from 8-O-4 oligomer, 8 clones from 8-5 dimer and 8 clones from 8-8 dimer were developed.Enzyme-linked immunosorbent assay showed that the monoclonal antibody against8-O-4 oligomer conjugates did not react with p-aminohippuric acid-BSA conjugates and that the antibody against 8-5 and 8-8 dimers did not react with both p-aminohippuric acid-BSA conjugates and 8-O-4 dimer (guaiacyl glycerol-beta-guaiacyl ether)-p-aminohippuric acid-BSA conjugates. These results suggested that these monoclonal antibodies could recognize different linkage structure of lignin.Immunogold labeling of differentiating xylems of Japanese cypress, oak and poplar showed that the monoclonal antibody from at least one hybridoma clone from the three different antigens reacted with lignifying and lignified cell walls. Since the labeling sites were different among the three kinds of antigens, these monoclonal antibodies could recognize different chemical structure of lignin in wood cell walls. Although the specificity of these antibodies must be analyzed in detail, these monoclonal antibodies will be very effective probes to clarify complex structure of lignin and its formation process in wood cell walls.

为了了解木材细胞壁中木质素的复杂化学结构，识别木质素特异结构的单克隆抗体将是非常重要的。本研究的目的是研制抗木质素模型化合物-蛋白质偶联物的单克隆抗体。选择了三种模型化合物，包括木质素的三种键（8-O-4、8- 5和8 -8），通过对氨基马尿酸与牛血清白蛋白偶联。用三种偶联物制备了小鼠单克隆抗体。结果，从8-O-4寡聚体，酶联免疫吸附试验表明，抗8-O-4寡聚体的单克隆抗体不与对氨基马尿酸-BSA偶联物反应，抗8-5和8- 8二聚体的单克隆抗体不与对氨基马尿酸-BSA偶联物和8-O-4偶联物反应。4二聚体（愈创木基甘油-β-愈创木基醚）-对氨基马尿酸-BSA缀合物。免疫金标记的结果表明，来自这三种不同抗原的杂交瘤细胞株中至少有一株能与木质化和木质化细胞壁发生反应。由于三种抗原的标记位点不同，这些单克隆抗体可以识别木材细胞壁中不同化学结构的木质素。尽管这些抗体的特异性还有待于进一步的研究，但这些单克隆抗体将成为阐明木质素复杂结构及其在木材细胞壁中形成过程的有效探针。