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

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 microg kg(-1) for OTA. The application of the new OTA-AP conjugate resulted in improved assay performance for the food samples.

Immunochemical methods for rapid mycotoxin detection in food and feed

“…Polymers such as poly(vinyl alcohol) (PVA) and poly(ethylene glycol) (PEG) 6000 exerted favourable effects on signal amplification, whereas surfactants negatively affected the assay performance. The highest signal amplification was achieved with the use of 0.5% (w/w) PVA in TMB Colorburst solution (Kolosova et al, 2007a). These authors also compared two enzymes as labels for OTA detection, HRP and AP, both in combination with different substrate systems.…”

Non-instrumental immunochemical tests for rapid ochratoxin A detection in red wine

“…The OTA-horseradish peroxidase (OTA-HRP) conjugate was supplied by Eurodiagnostica, the Netherlands. OTA-alkaline phosphatase (OTA-AP) conjugate was prepared in the laboratory [20]. The substrate chromogenic solution used for the HRP-enzyme was Colorburst TM Blue TMB/Peroxide (ALerCHEK, Inc., USA).…”

“…Two different formats were tested including gel-based [18,19] and membrane-based flow-through immunoassays [20]. Optimal conditions for the assays and a suitable, simple and rapid clean-up method for red wine were selected.…”

Immunoaffinity pre-concentration combined with on-column visual detection as a tool for rapid aflatoxin M1 screening in milk

“…For flow-through test kits 25-600 lL of sample solution are usually used (Kolosova, De Saeger, Eremin, & Van Peteghem, 2007;Saha, Acharya, Roy, Shrestha, & Dhar, 2007). For clean-up tandem immunoassay columns for ochratoxin A detection volumes of 1.5 mL and 0.5 mL were applied for a cut-off at 6 lg/kg in roasted coffee (Lobeau et al, 2005) and 10 lg/kg in spices (Goryacheva et al, 2006), respectively.…”