摘要
为建立ELISA测定中HRP/TMB显色体系的优化方法,开发一种灵敏度高、性能稳定的HRP/TMB显色液,实验采用单因素试验,考察了pH、TMB质量浓度、过氧化脲质量浓度、DMSO体积分数、甘油体积分数和显色时间对显色效果的影响;采用L9(34)正交实验,进行了工作条件优化;最后,对自制的显色液进行了贮藏稳定性研究,并与3种商品化同类产品进行了质量对比.结果显示:HRP/TMB显色体系的最佳工作条件为pH 5.0,TMB质量浓度0.4 g/L,过氧化脲质量浓度0.4 g/L,DMSO体积分数2%,甘油体积分数5%,显色时间10 min;4℃储藏90 d,稳定性良好;与商品化产品对比,自制显色液的灵敏度较高,显色时间较短,但稳定性较差.实验提供了一种HRP/TMB显色体系的优化方法及显色液配方,能满足大部分实验室ELISA研究需求,但其稳定性尚不能达到商品化产品需求,需进一步研究.
For establishing an optimization method for substrate chromogenic system of ELISA and providing a TMB/THR color liquid formula with high sensitivity and stable performance,single factor experiments were applied to analyze the effects of pH,concentration of TMB,urea hydrogen peroxide,DMSO and glycerol,reaction time on chromogenic reaction.Based on the single factor tests,a L9(34)orthogonal experiment was adopted to further optimize reaction parameters.The storage stability of the self-made HRP/TMB chromogenic solution,and its quality was compared with that of three commercial products of the same kind.The results showed that the optimal reaction parameters were pH 5.0,TMB 0.4 g/L,urea hydrogen peroxide 0.4 g/L,DMSO 2%,glycerol 5%and 10min of reaction;a good storage stability was showed within 90d at 4℃.Compared with commercial products,self-made chromogenic solution has higher sensitivity,shorter chromogenic time,but poor stability.The experiment provided an optimization method and formulation of HRP/TMB chromogenic system,which could meet the needs of most laboratory ELISA research,but the stability can not meet the needs of commercial products,so further research is needed.
作者
刘凤银
卢嘉辉
巫嘉琦
黄燕璇
邹红丽
梁岳
穆洪涛
LIU Fengyin;LU Jiahui;WU Jiaqi;HUANG Yanxuan;ZHOU Hongli;LIANG Yue;MU Hongtao(College of Biology and Food Engineering, Guangdong University of Education, Guangzhou, Guangdong, 510303, P.R.China)
出处
《广东第二师范学院学报》
2020年第3期75-82,共8页
Journal of Guangdong University of Education
基金
国家自然科学基金资助项目“基于人工免疫原构想模拟的半抗原设计与高亲和力抗氟乐灵抗体的诱导”(31601540)
广东省自然科学基金资助项目“基于半抗原表位预测的抗氟乐灵抗体亲和力提高研究”(2016A030310304)
广东省青年创新人才类基金资助项目“β-内酰胺酶抑制剂免疫检测方法研究与应用”(2017KQNCX142)
广东第二师范学院大学生创新创业计划训练项目“联苯四氮唑沙坦类药物广谱性抗体的制备及其多残留免疫检测试剂盒的研制”(201914278132)。
