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Investigation of several parameters influencing signal generation in flow-through membrane-based enzyme immunoassay

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Abstract

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.

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Acknowledgement

This research was supported by Belgian Federal Science Policy Office, S&T Cooperation Belgium-Central and Eastern Europe: Research Fellowships.

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Authors and Affiliations

  1. Laboratory of Food Analysis, Faculty of Pharmaceutical Sciences, Ghent University, Harelbekestraat 72, 9000, Ghent, Belgium

    Anna Yu. Kolosova, Sarah De Saeger & Carlos Van Peteghem

  2. Division of Chemical Enzymology, Faculty of Chemistry, M.V. Lomonosov Moscow State University, 119992, Moscow, Russia

    Sergei A. Eremin

Authors
  1. Anna Yu. Kolosova
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  2. Sarah De Saeger
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  3. Sergei A. Eremin
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  4. Carlos Van Peteghem
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Correspondence to Anna Yu. Kolosova.

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Yu. Kolosova, A., De Saeger, S., Eremin, S.A. et al. Investigation of several parameters influencing signal generation in flow-through membrane-based enzyme immunoassay. Anal Bioanal Chem 387, 1095–1104 (2007). https://doi.org/10.1007/s00216-006-0991-3

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  • DOI: https://doi.org/10.1007/s00216-006-0991-3

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