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抗将是非常重要的。本研究的目的是研制木素模型化合物-蛋白质结合物的单抗。选择木质素的3种键(8-O-4、8-5和8-8)作为模型化合物，通过对氨基马尿酸与牛血清白蛋白偶联。用三种结合物与小鼠免疫，制备了单抗。酶联免疫吸附试验表明，抗8-O-4寡聚体的单抗不与对氨基马尿酸-牛血清白蛋白偶联物反应，8-O-4寡聚体的单抗与对氨基马尿酸-牛血清白蛋白偶联物不反应，8-O-4二聚体的抗体不与对氨基马尿酸-牛血清白蛋白偶联物和8-O-4-二聚体(愈创木甘油-β-愈创木酚醚)-对氨基马尿酸-牛血清白蛋白偶联物反应。用免疫金标记法对柏木、栎木和杨木的木质部进行免疫胶体金标记，结果表明至少有一个杂交瘤克隆的单抗与木质化和木质化的细胞壁发生了反应。由于三种抗原的标记部位不同，这些单抗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要详细分析，但这些单抗将是阐明木质素的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.