Investigation of several parameters influencing signal generation in flow-through membrane-based enzyme immunoassay.
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.
Kolosova
Anna Yu
AY
Laboratory of Food Analysis, Faculty of Pharmaceutical Sciences, Ghent University, Harelbekestraat 72, 9000, Ghent, Belgium. Anna.Kolosova@UGent.be
De Saeger
Sarah
S
Eremin
Sergei A
SA
Van Peteghem
Carlos
C
D000469:Alkaline Phosphatase / Q000378:metabolism; D015374:Biosensing Techniques / Q000295:instrumentation / Q000379:methods*; D002021:Buffers; D004797:Enzyme-Linked Immunosorbent Assay / Q000295:instrumentation / Q000379:methods*; D005506:Food Contamination / Q000032:analysis*; D006735:Horseradish Peroxidase / Q000378:metabolism; D009793:Ochratoxins / Q000032:analysis*; D011092:Polyethylene Glycols / Q000737:chemistry; D011136:Polysorbates / Q000737:chemistry; D011142:Polyvinyl Alcohol / Q000737:chemistry; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D012965:Sodium Chloride / Q000737:chemistry; D013501:Surface-Active Agents / Q000737:chemistry
D002021:Buffers; D009793:Ochratoxins; D011136:Polysorbates; D013501:Surface-Active Agents; C025589:ochratoxin A; D011092:Polyethylene Glycols; D012965:Sodium Chloride; D011142:Polyvinyl Alcohol; D006735:Horseradish Peroxidase; D000469:Alkaline Phosphatase