隐孔雀石绿半抗原与全抗原设计、合成及鉴定

Design, Synthesis and Identification of Leucomalachite Green Haptens and Complete Antigens

从偶联位点、偶联活性基团的性质及半抗原的合成与偶联方法分析,设计了系列不同结构的半抗原和人工抗原及其合成方法。以N,N-二甲基苯胺、苯甲醛衍生物为原料,Amberlyst15树脂为催化剂,在温和条件下合成出3种隐孔雀石绿半抗原;进一步采用活泼酯和重氮化法分别将获得的半抗原与牛血清白蛋白(BSA)偶联,形成相应全抗原。半抗原和全抗原分别经减压硅胶柱色谱及透析纯化后,采用质谱、核磁共振及紫外扫描光谱等分析手段进行了鉴定,结果表明半抗原及免疫全抗原均合成成功,满足了进一步免疫、筛选高质量抗体和建立检测方法的需要。同时,采用分子模拟手段对抗原表面半抗原决定簇的前线分子轨道分布情况进行了仿真模拟分析,讨论了前线轨道的分布可能对抗体特性造成的影响,为进一步的半抗原构效关系研究提供理论支持。

Several complete antigens were prepared to produce the antibodies for leucomalachite green. Three haptens （4-MGC, 3-MGN and 4-MGN） with different active groups and linker substitution patterns were synthesized facilely with N, N-dimethylaniline and benzaldehyde derivatives as raw materials. The 4-MGC, 3-MGN and 4-MGN were conjugated to bovine serum albumin （BSA） with active ester and diazotization method respectively to form three complete antigens. The ratios of 4-MGC, 3-MGN, and 4-MGN to BSA are proved as 40：1, 15：1 and 13：1 respectively by UV spectra analysis and trinitrobenzenesulfonic acid （TNBS） method. Frontier molecular orbital （FMO） distribution of conjugate mimics was analyzed successfully by molecular modeling. The research lays a foundation for further discovering the structure-activity relations （SAR） of small molecule haptens and developing antibody and immunoassay method.