Sandwich electrochemical immunoassay for the detection of Staphylococcal enterotoxin B based on immobilized thiolated antibodies

被引:56
作者
Chatrathi, Madhu Prakash
Wang, Joseph
Collins, Greg E.
机构
[1] USN, Res Lab, Div Chem, Washington, DC 20375 USA
[2] Arizona State Univ, Tempe, AZ 85287 USA
关键词
Staphylococcal enterotoxin B; sandwich electrochemical immunoassay; thiolated antibody;
D O I
10.1016/j.bios.2006.12.013
中图分类号
Q6 [生物物理学];
学科分类号
071011 ;
摘要
A new approach for the sensitive detection of Staphylococcal enterotoxin B (SEB) is presented based upon an electrochemical enzymatic immunoassay that utilizes thiolated antibodies immobilized on a gold surface. This method relies on the use of amine- or sulfhydryl-reactive heterobifunctional cross-tinkers for the introduction of 2-pyridyl-disulfide groups to the antibody. The disulfide-containing linkages are subsequently cleaved with a suitable reducing agent, such as dithiothreitol (DTT), and the thiolated antibody-gold bond is covalently formed on a gold working electrode. Various cross-linking agents for immobilization of the capture antibody onto the gold electrode were investigated and compared. Factors influencing the thiolation and immobilization were investigated and optimized. The feasibility of such antibody immobilization and the subsequent sandwich enzyme immunoassay is demonstrated for the sensitive detection of SEB. The detection limit estimated from a representative dose-response curve is 1 ng/mL, corresponding to 5 pg in a 5-mu L sample. Coupling the specificity of immurroassays with the sensitivity and low detection limits of electrochemical detection shows real promise for future sensing technology in enabling the development of single-use disposable devices. (c) 2006 Elsevier B.V. All rights reserved.
引用
收藏
页码:2932 / 2938
页数:7
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