Preparation of monoclonal antibody against lignin

为了了解木材细胞壁中木质素的复杂化学结构，识别木质素特定结构的单克隆抗体将是非常重要的。本研究的目的是制备针对木质素模型化合物-蛋白偶联物的单克隆抗体。选择木质素的3种连锁（8-O-4、8-5和8-8）3种模式化合物，通过对氨基马嘌呤与牛血清白蛋白偶联。用小鼠培养了针对三种缀合物的单克隆抗体。结果：共获得4个8- o -4低聚体杂交瘤克隆、8-5二聚体杂交瘤克隆和8-8二聚体杂交瘤克隆。酶联免疫吸附实验表明，抗8-O-4寡聚物的单克隆抗体与对氨基河马酸- bsa偶联物不发生反应，抗8-5和8-8二聚体的单克隆抗体与对氨基河马酸- bsa偶联物和8-O-4二聚体（愈创木酰甘油- -愈创木酰醚）-对氨基河马酸- bsa偶联物均不发生反应。结果表明，这些单克隆抗体能够识别不同的木质素连锁结构。对日本柏树、橡树和杨树分化木质部的免疫金标记表明，至少有一个由这三种不同抗原组成的杂交瘤克隆的单克隆抗体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的木质素化学结构。虽然这些抗体的特异性还需详细分析，但这些单克隆抗体将是研究木质素复杂结构及其在木材细胞壁中形成过程的非常有效的探针。

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.