Antibody microarrays for native toxin detection
Antibody microarrays for native toxin detection
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DOI：
10.1016/j.ab.2005.01.030
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发表时间：
2005-04-15
影响因子：
2.9
通讯作者：
Herr, AE
中科院分区：
文献类型：
--
作者：
Rucker, VC;Havenstrite, KL;Herr, AE
我们已经开发了基于抗体的微阵列技术，用于多重检测霍乱毒素β亚基，白喉毒素，炭疽致死因子和保护性抗原，金黄色葡萄球菌肠毒素B，破伤风毒素C片段的加标样品。研究了两种检测方案：（i）直接测定法，其中荧光标记的毒素直接被抗体阵列捕获，和（ii）竞争测定法，其采用未标记的毒素作为报告物用于定量溶液中的天然毒素。在直接测定中，将在每个阵列元件处测量的荧光与标记的毒素浓度相关联以产生基线结合信息（朗缪尔等温线和亲和常数）。延伸到直接测定，竞争测定产生关于毒素的存在、身份和浓度的信息。竞争测定法优于所报道的谱分析测定法的显著优点是在分析之前所需的最少样品制备，因为竞争测定法消除了对荧光标记感兴趣样品中的天然蛋白质的需要。建立了两种测定形式的S形校准曲线和检测限。虽然直接测定的灵敏度上级于竞争测定，但竞争测定中未修饰毒素的检测限与先前报道的平面基底上排列的抗体的双格式免疫测定的值相当。作为未标记毒素检测的竞争测定的潜力的证明，我们得出结论，与一个简单的多路复用测定的分化和鉴定的两个天然S。加标稀释血清样品中的金黄色葡萄球菌肠毒素B和破伤风毒素C片段。(c)2005年爱思唯尔公司All rights reserved.
We have developed antibody-based microarray techniques for the multiplexed detection of cholera toxin beta-subunit, diphtheria toxin, anthrax lethal factor and protective antigen, Staphylococcus aurelts enterotoxin B, and tetanus toxin C fragment in spiked samples. Two detection schemes were investigated: (i) a direct assay in which fluorescently labeled toxins were captured directly by the antibody array and (ii) it competition assay that employed unlabeled toxins as reporters for the quantification of native toxin in solution. In the direct assay, fluorescence measured at each array element is correlated with labeled toxin concentration to yield baseline binding information (Langmuir isotherms and affinity constants). Extending front the direct assay, the competition assay yields information on the presence, identity, and concentration of toxins. A significant advantage of the competition assay over reported profiling assays is the minimal sample preparation required prior to analysis because the competition assay obviates the need to fluorescently label native proteins in the sample of interest. Sigmoidal calibration curves and detection limits were established for both assay formats. Although the sensitivity of the direct assay is superior to that of the competition assay, detection limits for unmodified toxins in the competition assay are comparable to values reported previously for sandwich-format immunoassays of antibodies arrayed on planar substrates. As a demonstration of the potential of the competition assay for unlabeled toxin detection, we conclude with a straightforward multiplexed assay for the differentiation and identification of both native S. aureus enterotoxin B and tetanus toxin C fragment in spiked dilute serum samples. (c) 2005 Elsevier Inc. All rights reserved.