颗粒型环瓜氨酸肽抗原的制备及条件优化  

Preparation of particulate cyclic citrullinated peptide antigens and optimization of experimental conditions

作　　者：付琳[1] 裘宇容[1] 姜云飞 夏佳音 王海芳[1] 

机构地区：[1]南方医科大学南方医院临床检验中心,广东广州510515 [2]广州市哈毕特实验仪器有限公司,广东广州510730

出　　处：《中国实验诊断学》2013年第5期798-801,共4页Chinese Journal of Laboratory Diagnosis

基　　金：广东省教育厅产学研基金(编号:1012220700033);广州市科技计划项目(编号:12A62121523)

摘　　要：目的探讨颗粒型环瓜氨酸肽抗原的制备方法,并对实验条件进行优化。方法通过碳二亚胺交联法将人工合成的CCP与BSA共价偶联,使偶联产物蛋白大小在70-170KD之间,经紫外吸收光谱鉴定偶联成功。采用碳二亚胺两步法将偶联成功的CCP与BSA复合物又与聚苯乙烯微球偶联,优化偶联条件,根据蛋白偶联量和偶联效率判断偶联效果。结果 (1)BSA与CCP偶联质量比为1∶1,EDC与NHS用量比为2∶3,偶联pH为6.0,BSA与CCP偶联可以达到较好的效果。BSA与CCP偶联产物在70KD-170KD之间,紫外吸收峰有明显左移。(2)微球与偶联蛋白的吸附效率随着EDC用量的增大而增大,当EDC用量达到7.5 mg/ml时,偶联蛋白最大效率达到63.66%。(3)随着偶联时间的延长,蛋白偶联微球的效率不断增加,在偶联时间为0.5-2h内,偶联效率增加最快,当反应时间2h后,反应趋于平衡。(4)对不同偶联反应pH值条件进行筛选,pH为7.5时偶联效果最好,pH7.0-8.0为适宜偶联的pH范围。结论 CCP-BSA偶联产物蛋白与胶乳颗粒偶联,当EDC浓度为7.5mg/ml,偶联时间为2h,反应PH值为7.5,蛋白偶联效率可达到63.66%。Objective To investigate the preparation method of particulate cyclic citrullinated peptide antigens,and optimize the experimental conditions. Methods The artificially synthesized CCP was covalent coupled with BSA by the method of carbodiimide cross-linking,and the protein sizes on the coupled products were between 70 to 170 KD. The covalent coupling was tested by ultraviolet absorption spectrum. Then the successful coupling complex of BSA and CCP were covalently coupled to the microspheres,and the coupling conditions were optimized. The covalent coupling effect was determined according to the protein coupling quantity and the coupling efficiency. Results （1） When the mass ra- tio of BSA and CCP was 1 ~ 1,the ratio of EDC and NHS was 2 ： 3,and the pH of coupling was 6.0,the coupling result of BSA and CCP could be accepted. The size of the coupled protein was between 70 KD-170 KD,and had left shift in ul- traviolet absorption spectrum. （2） The adsorption efficiency of coupled protein on the particles was increased in pace with the increased amount of EDC. When the amount of EDC was 7.5mg/mL,the maximum＇efficiency of coupling pro- tein reached 63.66%. （3） The efficiency of the protein-coupled particles was increased with the extension of the cou- pling time. And the fastest increase was between 0.5 hours and 2 hours. Then the reaction tended to balance after 2 hours. （4） When optimized the pH of coupling,the coupling effect of pH 7.5 was the best,and the suitable coupling pH range was 7.0-8.0. Conclusion When the EDc concentration was 7.5 mg/mL,the coupling time was 2 h, and the pH of reaction was 7. 5, the coupling efficiency of CCP-BSA cross-linking complex to the latex particles could be Up to 63.66%,

关 键 词：环瓜氨酸肽 胶乳颗粒 化学偶联 

分 类 号：R392[医药卫生—免疫学]