摘要
目的探索酶联免疫吸附测定(enzyme linked immunosorbent assay,ELISA)反应中反应条件对抗原(antigen,Ag)与抗体(antibody,Ab)结合的影响。方法以黄曲霉毒素B_(1)(aflatoxin B_(1),AFB_(1))抗原抗体为主要研究对象,通过分析ELISA中包被方法、包被液、离子浓度、pH、时间、温度、终止液等不同反应条件下的吸光度值(optical density,OD)和抑制率等指标,了解反应过程中抗原与抗体结合的差异情况,讨论反应条件对抗原-抗体结合的影响。结果两步包被抗体法效价高、抑制率好,包被液用pH 7.0、0.10 mol/L的磷酸氢二钠磷酸二氢钾抑制率高,封闭时间影响较小,25℃反应更适合操作,辣根过氧化物酶(horseradish peroxidase,HRP)标记抗原稀释液离子浓度0.020 mol/L pH=7.0时抗原-抗体结合最好,用盐酸比硫酸作为终止液更稳定。结论包被方法、包被液、离子浓度、pH、时间、温度、终止液等条件,均是影响抗原-抗体结合的因素。
Objective To explore the effect of reaction conditions on the binding of antigen(Ag)to antibody(Ab)in enzyme linked immunosorbent assay(ELISA).Methods Aflatoxin B_(1) antigen-antibody was taken as the main research object,the indicators such as optical density(OD)and inhibition rate in ELISA under different reaction conditions such as coating method,coating solution,ion concentration,pH,time,temperature,and termination solution were analyzed to understand the difference in antigen-antibody binding during the reaction,and the effect of reaction conditions on antigen-antibody binding was discussed.Results The two-step coating antibody method had high titer and good inhibition rate,the coating solution with pH 7.0 and 0.10 mol/L disodium hydrogen phosphate-potassium dihydrogen phosphate had high inhibition rate and the blocking time affects less,the reaction at 25℃was more suitable for operation,the antigen-antibody binding was the best when the ion concentration of horseradish peroxidase(HRP)-conjugated antigen dilution was 0.020 mol/L with pH=7.0,and hydrochloric acid was more stable than sulfuric acid as the termination solution.Conclusion Coating method,coating solution,ion concentration,pH,time,temperature,termination solution and other conditions are all factors that affect antigen-antibody binding.
作者
余厚美
王琴飞
林立铭
韦卓文
徐缓
张振文
YU Hou-Mei;WANG Qin-Fei;LIN Li-Ming;WEI Zhuo-Wen;XU Huan;ZHANG Zhen-Wen(Tropical Crops of Genetic Resources Institute,Chinese Academy of Tropical Agricultural Sciences,National Potato Processing Research&Developing Centre,Haikou 571101,China)
出处
《食品安全质量检测学报》
CAS
北大核心
2022年第19期6399-6408,共10页
Journal of Food Safety and Quality
基金
海南省自然科学基金项目(320MS101)
科技部科技伙伴计划项目(KY202002005)
国家现代农业产业技术体系建设专项(CARS-11-HNZZW)。
关键词
酶联免疫吸附测定
抗原-抗体
反应条件
enzyme linked immunosorbent assay
antigen-antibody
reaction condition
