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Aptamer-Based ELISA Assay for Highly Specific and Sensitive Detection of Zika NS1 Protein
Analytical Chemistry ( IF 6.7 ) Pub Date : 2017-11-21 00:00:00 , DOI: 10.1021/acs.analchem.7b02862 Kyung Hyun Lee 1 , Huaqiang Zeng 1
Analytical Chemistry ( IF 6.7 ) Pub Date : 2017-11-21 00:00:00 , DOI: 10.1021/acs.analchem.7b02862 Kyung Hyun Lee 1 , Huaqiang Zeng 1
Affiliation
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We report here a few Zika NS1-binding ssDNA aptamers selected using the conventional SELEX protocol, and their application in an ELISA assay for sensitive diagnosis of Zika NS1 protein. Among the aptamers identified, aptamers 2 and 10 could recognize different binding epitopes of Zika NS1 protein. This complementary in binding site, when coupled with an extraordinarily high binding affinity by 2 (41-nt, KD = 45 pM) and high specificity by 10, was used successfully to construct an ELISA-based assay where 2 and 10 serve as the capture and detection agents, respectively, giving rise to a highly specific detection of Zika NS1 with a detection limit of 100 ng/mL in buffer. Further testing of a few in-house anti-Zika NS1 antibodies show that 2 could also pair with an anti-Zika NS1 antibody. Such aptamer-antibody pairing not only lowers the detection sensitivity by 3 orders of magnitude to 0.1 ng/mL in buffer but also enable highly sensitive detection of as low as 1 and 10 ng/mL of Zika NS1 to be carried out in 10% and 100% human serum, respectively. These results suggest that the selected aptamers would be useful for medical diagnosis of Zika virus infection in various aptamer-based diagnostic devices including ELISA assay.
中文翻译：
基于适体的ELISA分析用于Zika NS1蛋白的高特异性和灵敏检测
我们在这里报告了一些使用常规SELEX协议选择的与Zika NS1结合的ssDNA适体，它们在ELISA检测中对Zika NS1蛋白的敏感诊断中的应用。在鉴定的适体中，适体2和10可以识别Zika NS1蛋白的不同结合表位。在结合位点该互补，当通过用一个非常高的结合亲和力耦合2（41-NT，ķ d = 45时），并通过高特异性10，被成功地用于构建基于ELISA的测定法，其中2和10分别用作捕获剂和检测剂，可对Zika NS1进行高度特异性的检测，在缓冲液中的检测限为100 ng / mL。对一些内部抗Zika NS1抗体的进一步测试表明2种抗体也可以与抗Zika NS1抗体配对。这种适体-抗体配对不仅可将缓冲液中的检测灵敏度降低3个数量级，降至0.1 ng / mL，而且还能够以1％和10 ng / mL的Zika NS1进行低灵敏度的高灵敏度检测，且分别为100％人血清。这些结果表明，所选的适体将在包括ELISA测定在内的各种基于适体的诊断装置中用于寨卡病毒感染的医学诊断。
更新日期：2017-11-22
中文翻译：
基于适体的ELISA分析用于Zika NS1蛋白的高特异性和灵敏检测
我们在这里报告了一些使用常规SELEX协议选择的与Zika NS1结合的ssDNA适体，它们在ELISA检测中对Zika NS1蛋白的敏感诊断中的应用。在鉴定的适体中，适体2和10可以识别Zika NS1蛋白的不同结合表位。在结合位点该互补，当通过用一个非常高的结合亲和力耦合2（41-NT，ķ d = 45时），并通过高特异性10，被成功地用于构建基于ELISA的测定法，其中2和10分别用作捕获剂和检测剂，可对Zika NS1进行高度特异性的检测，在缓冲液中的检测限为100 ng / mL。对一些内部抗Zika NS1抗体的进一步测试表明2种抗体也可以与抗Zika NS1抗体配对。这种适体-抗体配对不仅可将缓冲液中的检测灵敏度降低3个数量级，降至0.1 ng / mL，而且还能够以1％和10 ng / mL的Zika NS1进行低灵敏度的高灵敏度检测，且分别为100％人血清。这些结果表明，所选的适体将在包括ELISA测定在内的各种基于适体的诊断装置中用于寨卡病毒感染的医学诊断。
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