Investigation of several parameters influencing signal generation in flow-through membrane-based enzyme immunoassay
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- 作者：Anna Yu. Kolosova ; Sarah De Saeger ; Sergei A. Eremin and Carlos Van Peteghem
- 关键词：Membrane ; based enzyme immunoassay ; Signal generation ; Horseradish peroxidase ; Alkaline phosphatase ; Ochratoxin A
- 刊名：Analytical and Bioanalytical Chemistry
- 出版年：2007
- 出版时间：February, 2007
- 年：2007
- 卷：387
- 期：3
- 页码：1095-1104
- 全文大小：353 KB
- 刊物类别：Chemistry and Materials Science
- 刊物主题：Chemistry
Analytical Chemistry
Food Science
Inorganic Chemistry
Physical Chemistry
Monitoring, Environmental Analysis and Environmental Ecotoxicology - 出版者：Springer Berlin / Heidelberg
- ISSN：1618-2650
文摘
Rapid-response analytical tests that can be performed at the point of sampling are based on a visual detection system. The influence of different factors on the signal generation in a membrane-based enzyme immunoassay was investigated. The research was applied to a flow-through immunoassay for the detection of ochratoxin A (OTA). This assay format is a very convenient, simple and fast qualitative screening tool. Conjugates of OTA with horseradish peroxidase (HRP) and alkaline phosphatase (AP) were used as enzyme tracers. A new conjugate OTA-AP has been synthesized in our laboratory and its performance in the assay was compared with that of OTA-HRP. Different substrate systems for HRP and AP were compared. Several reagents, including polymers and surfactants, were tested for their possible effect on signal generation with the use of OTA-HRP conjugate. Polymers such as poly(vinyl alcohol) (PVA) and poly(ethylene glycol) (PEG) 6000 exerted a favourable effect on signal amplification, whereas surfactants negatively affected assay performance. The highest signal amplification (30–70% compared to the standard assay procedure) was achieved using 0.5% PVA in tetramethylbenzidine (TMB) Colorburst substrate solution and phosphate-buffered saline (PBS) for the washing step. It allowed more reliable visual estimation of the results from OTA-HRP assay. Exclusion of the detergent (Tween 20) from the washing solution exerted a favourable effect on assay performance using both enzyme tracers. The assay using OTA-HRP was more susceptible to matrix interferences than the assay with OTA-AP. Signal development in the matrix was better for the OTA-AP assay and visual estimation of the results was easier to perform in this case. For the analysis of spiked wheat samples, OTA-AP conjugate gave a more sensitive, stable and reproducible assay with a cut-off level of 4 μg kg−1 for OTA. The application of the new OTA-AP conjugate resulted in improved assay performance for the food samples.