A high-throughput, precipitating colorimetric sandwich ELISA microarray for Shiga toxins.
A high-throughput, precipitating colorimetric sandwich ELISA microarray for Shiga toxins.
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DOI：
10.3390/toxins6061855
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发表时间：
2014-06-11
期刊：
影响因子：
4.2
通讯作者：
Harris D
中科院分区：
文献类型：
--
作者：
Gehring A;He X;Fratamico P;Lee J;Bagi L;Brewster J;Paoli G;He Y;Xie Y;Skinner C;Barnett C;Harris D
使用新开发的高通量抗体微阵列平台同时检测来自产志贺毒素大肠杆菌 (STEC) 细菌的志贺毒素 1 和 2（Stx1 和 Stx2）。蛋白质毒素被固定并夹在生物识别元件（单克隆抗体）和混合的辣根过氧化物酶（HRP）结合的单克隆抗体之间。 HRP 与沉淀显色底物（金属增强的 3,3-二氨基联苯胺四盐酸盐或 DAB）反应后，使用廉价的平板扫描仪定量测量有色产物的形成。经证明，比色 ELISA 微阵列可在约 2 小时的总测定时间内检测低至约 4.5 ng/mL 的 Stx1 和 Stx2，线性动态范围窄，约 1-2 个数量级，饱和度水平远高于背景。用丝裂霉素 C（一种毒素诱导抗生素）和/或 B-PER（一种细胞破坏性蛋白质提取试剂）处理培养细胞后，还检测到由各种 STEC 菌株产生的 Stx1 和/或 Stx2。与丝裂霉素 C 一起孵育后，志贺毒素的半定量检测被证明在各种 STEC 菌株中是零星的；然而，与 B-PER 的进一步反应通常会导致 Stx1 的检测或相对于 Stx2 的检测增加，Stx1 是由接种到无菌肉汤培养物或含有碎牛肉的培养肉汤中的 STEC 产生的。
Shiga toxins 1 and 2 (Stx1 and Stx2) from Shiga toxin-producing E. coli (STEC) bacteria were simultaneously detected with a newly developed, high-throughput antibody microarray platform. The proteinaceous toxins were immobilized and sandwiched between biorecognition elements (monoclonal antibodies) and pooled horseradish peroxidase (HRP)-conjugated monoclonal antibodies. Following the reaction of HRP with the precipitating chromogenic substrate (metal enhanced 3,3-diaminobenzidine tetrahydrochloride or DAB), the formation of a colored product was quantitatively measured with an inexpensive flatbed page scanner. The colorimetric ELISA microarray was demonstrated to detect Stx1 and Stx2 at levels as low as ~4.5 ng/mL within ~2 h of total assay time with a narrow linear dynamic range of ~1–2 orders of magnitude and saturation levels well above background. Stx1 and/or Stx2 produced by various strains of STEC were also detected following the treatment of cultured cells with mitomycin C (a toxin-inducing antibiotic) and/or B-PER (a cell-disrupting, protein extraction reagent). Semi-quantitative detection of Shiga toxins was demonstrated to be sporadic among various STEC strains following incubation with mitomycin C; however, further reaction with B-PER generally resulted in the detection of or increased detection of Stx1, relative to Stx2, produced by STECs inoculated into either axenic broth culture or culture broth containing ground beef.
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