联合UV及SDS-PAGE监测并优化碳二亚胺偶联法制备多肽免疫原

Combine UV and SDS-PAGE to Monitor and Optimize of Carbodiimide Coupling Method for Preparing Peptide Immunogen

建立了一种多肽免疫抗原偶联的双监测法,获得一种操作简便、成功率高、适用范围广的碳二亚胺偶联法,并应用于多种肿瘤相关多肽标志物的免疫抗原制备。以合成多肽SP0104为标准品,牛血清白蛋白(BSA)为载体蛋白,碳二亚胺(EDC)为偶联剂,对加样顺序、投料比、偶联时间等关键条件进行考察,采用UV和SDS-PAGE对反应产物进行联合监测,以偶联比和偶联率联合评价反应条件。加样顺序为多肽和载体蛋白先混合再加到碳二亚胺中,三者质量比为1∶1∶1,偶联时间为12 h,偶联比在4∶1～12∶1之间,偶联率在9%～20%之间,所得SP0104多抗效价达到1∶80 000,MALDI-TOF质谱分析证实该抗体准确捕获目标抗原。用优化的偶联方法制备多肽SPC09、SPC15、SPG01、SPG03、SPG04的免疫抗原,所得免疫血清效价均在1∶1万以上,最高可达1∶51.2万。所建碳二亚胺多肽偶联联合监测法简便实用,可提高多肽抗体制备的成功率。

A combined method for monitoring peptide coupling carrier protein prepared immunogen was established to get a carbodiimide coupling method which has the advantages of simple operation,high success rate and wide range of application.The optimized coupling method was applied in the preparation of immunogens for a variety of tumor-associated peptide biomarkers.Using synthesized SP0104 as the peptide,BSA（bovine serum albumin） as the carrier protein and EDC（carbodiimide） as the coupling reagent,the key conditions such as the activation method of reactants,the coupling time and the ratio of three reactants were investigated.The coupling ratio of UV and coupling rate of SDS-PAGE were used to monitor the compounds derived from the coupling reaction.The addition sequence of reagents was optimized as follows ∶ The peptide was mixed with BSA firstly,and then added into carbodiimide solution.The mass ratio of SP0104 ∶ BSA ∶ EDC was 1 ∶ 1 ∶ 1,and coupling time was 12 h.The coupling ratio was between 4 ∶ 1 and 12 ∶ 1 and the coupling rate was between 9% and 20%.The antibody titer of purified SP0104 was 1 ∶ 80 000,and the polyclonal antibody successfully captured the peptide antigen SP0104 by the analysis of MALDI-TOF mass spectrometry.The optimized coupling method was applied in the peptide immunogen preparation of SPC09,SPC15,SPG01,SPG03 and SPG04.All the antibody titers of five immune-serum were over 1 ∶ 10 000,and the maximum reached up to 1 ∶ 512 000.The established combined monitoring method for carbodiimide coupling peptide and carrier protein was simple and effective,and could improve the success rate of peptide antibody preparation.