Quantitative assessment of factors affecting the sensitivity of a competitive immunomicroarray for pesticide detection
Quantitative assessment of factors affecting the sensitivity of a competitive immunomicroarray for pesticide detection
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DOI：
10.2144/03355rr04
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发表时间：
2003-11-01
期刊：
影响因子：
2.7
通讯作者：
Christensen, CBV
中科院分区：
文献类型：
--
作者：
Belleville, E;Dufva, M;Christensen, CBV
分析蛋白质微阵列提供高度平行的分析，可以成为一个非常宝贵的工具，广泛的免疫诊断应用。在这里，我们描述了影响竞争性免疫微阵列，定量小分子的灵敏度的因素;在这种情况下，农药氯苄腈代谢物2，6-二氯苯甲酰胺（BAM）和莠去津。溶液中的游离农药浓度通过荧光缀合的单克隆抗体与表面固定的农药半抗原-蛋白质缀合物或溶液中的农药的竞争性结合来定量。我们研究了抗体标记技术，微阵列基板，点样和孵育缓冲液的影响。结果表明，当阵列与稀释在补充有0.05%牛血清白蛋白（BSA）和Tween（R）20的Tris缓冲液中的样品/抗体一起孵育时，固定在EasySpot或内部制造的用Genetix胺点样溶液打印的琼脂糖基底上的微阵列产生最佳结果。此外，与二次多克隆抗体定量相比，直接标记的一抗的应用允许更好的灵敏度。
Analytical protein microarrays offering highly parallel analysis can become an invaluable tool for a wide range of immunodiagnostic applications. Here we describe factors that influence the sensitivity of a competitive immunomicroarray that quantifies small molecules; in this case, the pesticides dichlobenil metabolite 2,6-dichlorobenzamide (BAM) and atrazine. Free pesticide concentrations in solution are quantified by the competitive binding of fluorescence-conjugated monoclonal antibodies to either surface-immobilized pesticide hapten-protein conjugates or pesticides in solution. We investigated the influence of antibody labeling techniques, microarray substrates, and spotting and incubation buffers. The results showed that microarrays immobilized on EasySpot or in-house fabricated agarose substrates printed with Genetix Amine Spotting Solution resulted in optimum results when the arrays were incubated with the sample/antibodies diluted in a Tris buffer supplemented with 0.05% each bovine serum albumin (BSA) and Tween(R) 20. Furthermore, the application of directly labeled primary antibodies allowed for better sensitivity compared to secondary polyclonal antibody quantification.