Development of a Monoclonal Antibody against Ochratoxin A and Its Application in Enzyme-Linked Immunosorbent Assay and Gold Nanoparticle Immunochromatographic Strip

Abstract

A monoclonal antibody (mAb) specific to ochratoxin A (OTA) was produced from a stable hybridoma cell line, 9C9H9, generated by the fusion of P3/NS1/1-AG4-1 myeloma cells with spleen cells isolated from a BALB/c mouse immunized with OTA-keyhole limpet hemocyanin. The 9C9H9 mAb belongs to the immunoglobulin G1 (kappa chain) isotype. A competitive direct enzyme-linked immunosorbent assay (cdELISA) and a competitive indirect ELISA were established for antibody characterization. The concentrations causing 50% inhibition of binding of OTA-horseradish peroxidase to the antibody by OTA, OTB, and OTC were found to be 0.32, 0.17, and 0.28 ng/mL, respectively, in the cdELISA. A sensitive and rapid mAb-based gold nanoparticle immunochromatographic strip was also developed using this mAb. This strip has a detection limit of 5 ng/mL for OTA and can be completed in 10 min. Analysis of OTA in coffee samples revealed that data obtained from immunochromatographic strip were in a good agreement with those obtained from cdELISA. The mAb-based cdELISA and immunochromatographic strip assay established in this study were sensitive and accurate for rapid screening of OTA in coffee samples.

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

“…: the molar conjugation rate, cr, between OTA and BSA of the OTA-BSA conjugate). All those factors were demonstrated to play some role on determining the sensitivity of the assay in our previous observations [30] and according to several other authors [31][32][33][34].…”

“…High sensitivity in indirect lateral flow immunoassays for haptens is promoted from having the opportunity of tuning some key-factors, such as the amount of specific antibodies and of immobilized antigens in the Test line [30][31][32][33][34]. Specifically, the lower the amount of one or both of those components, the higher the sensitivity reached.…”

“…As far as the conjugate used to form the Test line, according to previous observations [31][32], both the amount of OTA-BSA sprayed onto the membrane and the hapten density of the OTA-BSA itself were varied.…”

Detection of chemical pollutants in water using gold nanoparticles as sensors: a review

“…In any functionalization efforts with DNA and DNA hybridization, charge screening (salt-aging) was necessary to compensate for the electrostatic repulsion between the negatively charged DNA molecules (Hill and Mirkin 2006 ). Immunochromatographic strip assay 0.32 ng/ml, 2.5 ng/ml Bo et al 2007, Liu et al 2008, Shim et al 2009 Table 1 Summary of the detection sensitivity achieved by gold nanoparticle-based sensor platform.…”

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.…”