芝麻是八大类食物过敏原之一,快速准确识别芝麻过敏原对预防其过敏有重要意义。核酸适配体可以高效识别靶标过敏原,在过敏原检测中有良好的应用前景。为了获得芝麻主要过敏原Ses i 2的特异性核酸适体,本研究以Ses i 2为靶标,通过磁珠筛...芝麻是八大类食物过敏原之一,快速准确识别芝麻过敏原对预防其过敏有重要意义。核酸适配体可以高效识别靶标过敏原,在过敏原检测中有良好的应用前景。为了获得芝麻主要过敏原Ses i 2的特异性核酸适体,本研究以Ses i 2为靶标,通过磁珠筛选法(磁珠-SELEX)开展10轮筛选,经由高通量测序获得6条候补序列(S1~S6),并进行家族性、同源性分析及二级结构预测。结果表明,6条候选核酸适体的重复率可达46.38%,其自由能在-9.02到-2.47 kcal·moL^(-1)之间,根据自由能能量稳定原则,S1和S5吉布斯自由能最低最稳定,分别为-6.70和-9.02 kcal·moL^(-1)。利用ELISA试验进行亲和力测试,结果表明核酸适体S1和S2的亲和能力较强,S1:KD=67.02 nmol·L^(-1),R2=0.925 8,S2:KD=97.65 nmol·L^(-1),R2=0.795 1。核酸适体S1与过敏原Ses i 2的结合力和其他过敏原蛋白相比有显著差异,可视为具有特异性。本研究最终获得一条兼具良好亲和力和特异性的核酸适体S1,为芝麻过敏原快速检测提供了技术支撑。展开更多
Objective:To investigate the hook effect in the detection of beta2-microglobulin(β2-MG)with different reagents as well as on two fully automated biochemical analyzers and their solutions.Methods:Using immunoturbidime...Objective:To investigate the hook effect in the detection of beta2-microglobulin(β2-MG)with different reagents as well as on two fully automated biochemical analyzers and their solutions.Methods:Using immunoturbidimetric assay forβ2-MG as the research object,β2-MG levels were detected by different concentration gradients ofβ2-microglobulin samples in Liedemann,Mack,and Myriad reagents of three manufacturers on two automatic biochemical analyzers,and the difference of the hook effect was comparatively analyzed.Results:Leadman reagent showed the hook effect on the Beckman AU5800 automated biochemistry analyzer,while both Maccura and Mindray reagents did not show the hook effect.After the experiments,we found the limit value of the pre-zone check of Leadman reagent,and changed the parameters of the instrument,when the limit value of the pre-zone check was reached,the instrument automatically diluted the specimen five times and then detected it again.After changing the parameters of the instrument,the correlation between the three methods of detecting samples of different concentrations was r>0.99.Conclusion:Before selecting the application of immunoturbidimetric reagents,we have to carry out the risk assessment of the hook effect and selectively set the parameters of the pre-zone check based on the highest concentration that may occur in the clinic;for the items that may have the hook effect,we have to selectively set the parameters of the pre-zone check when the ratio of average reaction rate and the immediate reaction rate is at the limit value,and the phenomenon of antigen excess may occur,the instrument will carry out automatic dilution before detection,so as to avoid the issuance of erroneous results of high-value samples due to the hook effect.展开更多
文摘芝麻是八大类食物过敏原之一,快速准确识别芝麻过敏原对预防其过敏有重要意义。核酸适配体可以高效识别靶标过敏原,在过敏原检测中有良好的应用前景。为了获得芝麻主要过敏原Ses i 2的特异性核酸适体,本研究以Ses i 2为靶标,通过磁珠筛选法(磁珠-SELEX)开展10轮筛选,经由高通量测序获得6条候补序列(S1~S6),并进行家族性、同源性分析及二级结构预测。结果表明,6条候选核酸适体的重复率可达46.38%,其自由能在-9.02到-2.47 kcal·moL^(-1)之间,根据自由能能量稳定原则,S1和S5吉布斯自由能最低最稳定,分别为-6.70和-9.02 kcal·moL^(-1)。利用ELISA试验进行亲和力测试,结果表明核酸适体S1和S2的亲和能力较强,S1:KD=67.02 nmol·L^(-1),R2=0.925 8,S2:KD=97.65 nmol·L^(-1),R2=0.795 1。核酸适体S1与过敏原Ses i 2的结合力和其他过敏原蛋白相比有显著差异,可视为具有特异性。本研究最终获得一条兼具良好亲和力和特异性的核酸适体S1,为芝麻过敏原快速检测提供了技术支撑。
基金Baoding Science and Technology Bureau(2141ZF313)。
文摘Objective:To investigate the hook effect in the detection of beta2-microglobulin(β2-MG)with different reagents as well as on two fully automated biochemical analyzers and their solutions.Methods:Using immunoturbidimetric assay forβ2-MG as the research object,β2-MG levels were detected by different concentration gradients ofβ2-microglobulin samples in Liedemann,Mack,and Myriad reagents of three manufacturers on two automatic biochemical analyzers,and the difference of the hook effect was comparatively analyzed.Results:Leadman reagent showed the hook effect on the Beckman AU5800 automated biochemistry analyzer,while both Maccura and Mindray reagents did not show the hook effect.After the experiments,we found the limit value of the pre-zone check of Leadman reagent,and changed the parameters of the instrument,when the limit value of the pre-zone check was reached,the instrument automatically diluted the specimen five times and then detected it again.After changing the parameters of the instrument,the correlation between the three methods of detecting samples of different concentrations was r>0.99.Conclusion:Before selecting the application of immunoturbidimetric reagents,we have to carry out the risk assessment of the hook effect and selectively set the parameters of the pre-zone check based on the highest concentration that may occur in the clinic;for the items that may have the hook effect,we have to selectively set the parameters of the pre-zone check when the ratio of average reaction rate and the immediate reaction rate is at the limit value,and the phenomenon of antigen excess may occur,the instrument will carry out automatic dilution before detection,so as to avoid the issuance of erroneous results of high-value samples due to the hook effect.
