【摘要】 目的:优化灵芝多糖GLPL1免疫原,用于制备GLPL1单克隆抗体,为GLPL1免疫分析法建立提供物质基础。方法:GLPL1经1-氰基-4-二甲胺吡啶四氟硼酸盐(CDAP)活化后与牛血清白蛋白(BSA)共价偶联,制备系列拟糖蛋白GLPL1-BSA并考察其免疫原性;选择其免疫性强者免疫BALB/c小鼠,通过杂交瘤技术制备GLPL1特异性单抗。结果:共获得取代度(GLPL1/BSA摩尔比)分别为≈1、≈3、≈6三种拟糖蛋白GLPL1-BSA,其免疫小鼠后诱导产生强烈的抗GLPL1的IgM初次应答和IgG回忆应答,且GLPL1诱导小鼠主要分泌IgG2a独特型抗体;经细胞融合、筛选和亚克隆,获得一株分泌GLPL1特异性抗体杂交瘤细胞6G7,其抗体独特型为IgM。结论:成功优化、制备了灵芝多糖GLPL1的免疫用抗原和GLPL1特异性单克隆抗体。更多还原

【Abstract】 AIM:Antigen was optimized to prepare monoclonal antibody of GLPL1,a polysaccharide from Ganoderma lucidum for establishing immune analytical method of GLPL1.METHODS:GLPL1 was coupled to bovine serum albumin(BSA)by 1-cyano-4-dimethylaminopyridinium tetrafluoroborate(CDAP)to construct a series of neoglycoproteins,whose immunogenicities were evaluated in mice.Based on the result,one neoglycoprotein which had the best immunogenicity was injected into mice and monoclonal antibodies of GLPL1 were prepared.RESULTS:Three neoglycoproteins with different degrees of substitution(DSs;≈1,≈3 or ≈6 mol of GLPL1 hapten/mol of BSA carrier,respectively)were acquired.Neoglycoproteins induced vigorous primary IgM and boostered IgG responses to GLPL1.IgG2a was elicited as the dominant isotype antibody in mice by GLPL1,and IgM mab of GLPL1 named 6G7 was acquired.CONCLUSION:Mab of GLPL1 is acquired through antigen optimization.更多还原