Kobayashi K, Hirano A, Ohta A, Yoshida T, Takahashi K, Hattori M
Department of Applied Biological Science, Faculty of Agriculture, Tokyo University of Agriculture and Technology, Tokyo 183-8509, Japan.
J Agric Food Chem. 2001 Feb;49(2):823-31. doi: 10.1021/jf000926q.
To reduce the immunogenicity of beta-lactoglobulin (beta-LG), two beta-LG-carboxymethyl dextran (CMD) conjugates (Conj. 40 and Conj. 162) were prepared by using water-soluble carbodiimide (EDC). The molar ratios of beta-LG to CMD in Conj. 40 and Conj. 162 were 8:1 and 7:1, respectively. Each conjugate maintained approximately 50% of the retinol binding activity of beta-LG. Structural analyses by intrinsic fluorescence, CD spectra, and ELISA with monoclonal antibodies indicated that the surface of beta-LG in each conjugate was covered by CMD without great disruption of native conformation. By conjugation with CMD, the antibody response to beta-LG was reduced in BALB/c, C3H/He, and C57BL/6 mice, which was eminent in Conj. 162. The results of B cell epitope scanning using overlapping synthesized peptides showed that the linear epitope profiles of the conjugates were similar to those of beta-LG, whereas the antibody response to each epitope was reduced, which was eminent in Conj. 162. It was concluded that conjugation with CMD of higher molecular weight is effective in reducing the immunogenicity of beta-LG and that masking of epitopes by CMD is responsible for the reduced immunogenicity.
为降低β-乳球蛋白（β-LG）的免疫原性，使用水溶性碳二亚胺（EDC）制备了两种β-LG-羧甲基葡聚糖（CMD）缀合物（缀合物40和缀合物162）。缀合物40和缀合物162中β-LG与CMD的摩尔比分别为8:1和7:1。每种缀合物均保留了约50%的β-LG视黄醇结合活性。通过内源荧光、圆二色光谱以及使用单克隆抗体的酶联免疫吸附测定进行的结构分析表明，每种缀合物中β-LG的表面被CMD覆盖，而天然构象未受到重大破坏。通过与CMD缀合，BALB/c、C3H/He和C57BL/6小鼠对β-LG的抗体反应降低，这在缀合物162中尤为明显。使用重叠合成肽进行B细胞表位扫描的结果表明，缀合物的线性表位谱与β-LG相似，而对每个表位的抗体反应降低，这在缀合物162中尤为明显。得出的结论是，与较高分子量的CMD缀合可有效降低β-LG的免疫原性，且CMD对表位的掩盖是免疫原性降低的原因。
