基于抗原包被的微孔板式化学发光酶免疫分析法测定人尿中的雌三醇

将雌三醇-6-(O-羧甲基)肟(E3-6-CMO)与牛血清白蛋白(BSA)形成的偶联物E3-6-CMO-BSA物理吸附于聚苯乙烯微孔板孔内作为固相抗原,与雌三醇(E3)标准溶液或者水解尿样中待测E3通过竞争法进行免疫反应.以对碘苯酚增强的辣根过氧化物酶(HRP)催化鲁米诺-过氧化氢化学发光体系作为信号检测系统,建立了一种高通量、简便快速、灵敏稳定的化学发光酶免疫分析方法用于测定人尿中E3的含量.考察和优化了包被液的酸碱性、抗原包被浓度、酶标抗体稀释比例及用量、温育时间、化学发光底物用量及化学发光反应时间的影响.在最优实验条件下,方法的灵敏度为0.20ng/mL,批内和批间变异系数均在15%之内,低、中、高浓度加标水解尿样的平均回收率分别为107.9%、100.9%和91.2%.使用抗原包被法和抗体包被法同时对10份水解尿样进行测定,结果显示相关性良好,相关系数为0.9984,表明本方法可以满足临床检测的要求.

Micro-plate chemiluminescence enzyme immunoassay with antigen coated method for determination of estriol in human urine

The conjugant of E3-6-CMO-BSA by estriol-6-（O-carboxymethyl） oxime to combine with bovine serum albumin（BSA） was physically adsorpted on polystyrene micro-plate holes as a solid-phase antigen which competitively immunoreacts with Estriol（E3） in standard solution or in the urine samples.A highly throughput,simple and rapid,sensitive and steady chemiluminescence enzyme immunoassay（CLEIA） was developed for determination of estriol in human urine.Luminol-hydrogen peroxide chemiluminescence system catalyzed by horseradish peroxidase（HRP） and enhanced by p-iodophenol was used as signal detecting system.The effect of several factors,such as acid-base properties of coated solution,coated E3-6-CMO-BSA solution concentration,E3-Ab-HRP dilution ratio and usage amount,incubation time,volume of chemiluminescence substrate mixed solution and chemiluminescence reaction time were optimized.The assay was evaluated with sensitivity as low as 0.20 ng/mL,the RSD was less than 15% for both intra-and inter-assay precision,the recoveries for low,middle and high concentration samples with a hydrolyzed urine sample added standard E3 solution were 107.9%,100.9% and 91.2%,respectively.This method has been synchronously applied to the evaluation of estriol in ten hydrolyzed urine samples.The correlative coefficient is 0.9984,which indicated that this method can meet the requirements of clinical testing.