Enzyme-linked immunosorbent assay and colloidal gold immunoassay for ochratoxin A: investigation of analytical conditions and sample matrix on assay performance

Abstract

A polyclonal antibody against ochratoxin A (OTA) was produced from rabbits immunized with the OTA-BSA conjugate. A competitive direct enzyme-linked immunosorbent assay (cdELISA) and a membrane-base colloidal gold immunoassay in flow-through format were developed for the rapid detection of OTA in various food matrices. In the cdELISA, the concentration causing 50% inhibition was 0.07 ng mL(-1), and the effects of different chemical conditions (ionic strength, pH value, and organic solvent) were studied. The sensitivity of the assay was higher than those previously reported. A simple, rapid, and efficient extraction method was developed and 74-110% recoveries of spiked samples were obtained. Fifty percent methanol extracts of some food samples such as barley, wheat, oat, corn, rice, and raisins could be analyzed directly by immunoassay after dilution in PBS; grape juice and beer samples could be analyzed directly after dilution with PBS; for coffee samples, a more complex method was used to remove the matrix effect effectively. Membrane-based colloidal gold immunoassays had a visual detection limit of 1.0 ng mL(-1) for OTA with a detection time of less than 10 min. For the validation of the cdELISA and membrane-based colloidal gold immunoassay, samples were analyzed by high-performance liquid chromatography. The correlation between data obtained using the microwell assay and HPLC was good (R2 = 0.984). The developed immunoassay methods are suitable for the rapid quantitative or qualitative determination of OTA in food samples.

Increased sensitivity of lateral flow immunoassay for ochratoxin A through silver enhancement

“…Effective advantages in terms of gain on sensitivity were demonstrated on a competitive LFIA for measuring ochratoxin A in wine and grape must, previously developed on a gold-based model. The IC 50 for the "ultrasensitive" silver enhanced-LFIA was more than 10-folds lower compared to the one of the gold-based LFIA, and, based on our knowledge, the developed method is the most sensitive LFIA for measuring ochratoxin A [23][24][25][26][27]. This assay exceeded requirements for measuring OTA in beverages according to the legislation in force.…”

“…However, immunochemical analyses are largely employed as screening methods, thanks to their simplicity, rapidity and cost effectiveness [16,[20][21][22]. LFIAs aimed at measuring OTA in food, beverages and feed have been reported, as well [23][24][25][26][27].…”

Aflatoxin Measurement and Analysis

“…requiring only a sample extraction step before use; 2. simplicity of procedure with single step, e.g., only adding test solution to the sample pad on the strip; 3. rapid on-site detection within a few minutes (5-15 min); 4. concentration levels of target analytes can be observed directly with the naked eyes; 5. user-friendly format no need for skill personnel; 6. less interference due to chromatographic separation; and 7. low cost Because of these advantages, lateral flow strip assay has become one of the commercial and widely-used immunoassays for rapid determination of mycotoxins, such as ochratoxin A (Lai et al, 2009;Liu, Tsao, Wang, & Yu, 2008;Wang, Liu, Xu, Zhang, & Wang, 2007;Cho et al, 2005), deoxynivalenol (Kolosova, De Saeger, Sibanda, Verheijen, & Van Peteghem, 2007;Xu et al, 2010;Kolosova et al, 2008), T-2 Toxin (Molinelli et al, 2008), zearalenone (Kolosova, De Saeger, Sibanda, Verheijen, & Van Peteghem, 2007), fumonisin B1 (Wang, Quan, Lee, & Kennedy, 2006), aflatoxins (Sun, Zhao, Tang, Zhou, & Chu, 2005;Sheibani, Tabrizchi, & Ghaziaskar, 2008) and so on. The visual detection limit (VDL), defined as the minimum concentration producing the color on the test line significantly different or weaker to that on the test line of negative control strip without aflatoxin (Li, Wei, Yang, Li, & Deng, 2009;Zhou et al, 2009), was used to express the sensitivity of the lateral flow strip assay.…”

A lateral flow immunoassay for measuring ochratoxin A: Development of a single system for maize, wheat and durum wheat

“…Wang et al (2007) made membranes which were able to detect OTA in cereals, beer and coffee with a visual detection limit of 1 mg kg À1 , but sample extraction required the use of methanol or chloroform, an ultrasonic system and a cooled centrifuge, with a total extraction time of over 30 min Shim, Dzantiev, Eremin, and Chung (2009) prepared strips for the multi-analysis of OTA and Zearalenone with a visual cut-off of 5 mg kg À1 of OTA in spiked corn samples, after the use of methanol as an extracting solvent, and centrifugation. The use of hazardous chemicals and laboratory equipment strongly frustrates the main advantages of the immunochromatographic assay's being point-ofuse test, i.e.…”