Multiplexed method to calibrate and quantitate fluorescence signal for allergen-specific IgE.
Multiplexed method to calibrate and quantitate fluorescence signal for allergen-specific IgE.
复制标题
DOI：
10.1021/ac202212k
复制
发表时间：
2011-12-15
影响因子：
7.4
通讯作者：
Uenlue, M. Selim
中科院分区：
文献类型：
--
作者：
Monroe, Margo R.;Reddington, Alexander P.;Collins, Austin D.;LaBoda, Craig;Cretich, Marina;Chiari, Marcella;Little, Frederic F.;Uenlue, M. Selim
使用微阵列平台进行过敏诊断允许测试对多种过敏原的特异性IgE敏感性，同时仅需要少量血清。然而，探针固定在微阵列上的变化阻碍了在免疫诊断中做出定量、断言和统计学相关结论的能力。为了解决这个问题，我们已经开发了一种校准的，廉价的，多路复用的，和快速的蛋白质微阵列方法，直接相关的表面探针密度捕获标记的二抗在临床样品。我们已经确定了我们的校准荧光增强（CaFE）技术的三个主要技术优势：（i）在针对荧光特别优化的分层基底上的宽范围的荧光团上荧光发射的显著增加;（ii）在每个斑点中执行探针的无标记定量同时保持特定荧光团的荧光增强的方法;和（iii）校准的定量技术，其结合荧光和无标记模式以精确测量探针密度和各种抗体-抗原对的结合靶。在本文中，我们建立了CaFE方法的有效性，提出了强的线性依赖性的结合蛋白质的量产生的IgG，β-乳球蛋白，和过敏原特异性IgE的二抗的荧光信号的Ara h 1（花生主要过敏原）和Phl p 1（梯牧草主要过敏原）在人血清中。
Using a microarray platform for allergy diagnosis allows for testing of specific IgE sensitivity to a multitude of allergens, while requiring only small volumes of serum. However, variation of probe immobilization on microarrays hinders the ability to make quantitative, assertive, and statistically relevant conclusions necessary in immunodiagnostics. To address this problem, we have developed a calibrated, inexpensive, multiplexed, and rapid protein microarray method that directly correlates surface probe density to captured labeled secondary antibody in clinical samples. We have identified three major technological advantages of our calibrated fluorescence enhancement (CaFE) technique: (i) a significant increase in fluorescence emission over a broad range of fluorophores on a layered substrate optimized specifically for fluorescence; (ii) a method to perform label-free quantification of the probes in each spot while maintaining fluorescence enhancement for a particular fluorophore; and (iii) a calibrated, quantitative technique that combines fluorescence and label-free modalities to accurately measure probe density and bound target for a variety of antibody–antigen pairs. In this paper, we establish the effectiveness of the CaFE method by presenting the strong linear dependence of the amount of bound protein to the resulting fluorescence signal of secondary antibody for IgG, β-lactoglobulin, and allergen-specific IgEs to Ara h 1 (peanut major allergen) and Phl p 1 (timothy grass major allergen) in human serum.
登录
查看更多内容
影响因子：
4.4
作者：
Hessner MJ;Singh VK;Wang X;Khan S;Tschannen MR;Zahrt TC
通讯作者：
Zahrt TC
影响因子：
3.4
作者：
Balboni, Imelda;Limb, Cindy;Tenenbaum, Jessica D.;Utz, Paul J.
通讯作者：
Utz, Paul J.
DOI：
10.1364/josab.14.001149
发表时间：
1997-05-01
影响因子：
1.9
作者：
Sullivan, KG;Hall, DG
通讯作者：
Hall, DG
影响因子：
3
作者：
Wang X;Jia S;Meyer L;Xiang B;Chen LY;Jiang N;Moreno C;Jacob HJ;Ghosh S;Hessner MJ
通讯作者：
Hessner MJ
影响因子：
2.9
作者：
Schabacker, Daniel S.;Stefanovska, Ivana;Chandler, Darrell P.
通讯作者：
Chandler, Darrell P.