Investigation of several parameters influencing signal generation in flow-through membrane-based enzyme immunoassay |
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Authors: | Anna Yu Kolosova Sarah De Saeger Sergei A Eremin Carlos Van Peteghem |
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Institution: | (1) Laboratory of Food Analysis, Faculty of Pharmaceutical Sciences, Ghent University, Harelbekestraat 72, 9000 Ghent, Belgium;(2) Division of Chemical Enzymology, Faculty of Chemistry, M.V. Lomonosov Moscow State University, 119992 Moscow, Russia |
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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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Keywords: | Membrane-based enzyme immunoassay Signal generation Horseradish peroxidase Alkaline phosphatase Ochratoxin A |
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