Dual-label time-resolved fluoroimmunoassay for simultaneous detection of aflatoxin B1 and ochratoxin A

Abstract

A dual-label time-resolved fluoroimmunoassay (TRFIA) for simultaneous quantification of aflatoxin B1 (AFB1) and ochratoxin A (OTA) is described. For this, microtitration wells were coated with AFB1-horse radish peroxidase (HRP) and OTA-bovine serum albumin. The standards and samples were loaded on the coated plates, and diluted antibodies and Eu3+- and Sm3+-labeled IgG were then added. Our results showed that the sensitivity of TRFIA for AFB1 was 0.02 microg/L (range 0.02-100 microg/L). The intra- and inter-batch coefficient of variation (CV) was 3.2 and 7.3%, respectively, and the average recovery rate was 88.1%. On the other hand, the sensitivity of OTA was 0.05 microg/L (range 0.05-50 microg/L), the intra- and inter-batch CV was 2.9 and 7.9%, respectively, and the average recovery rate was 89.9%. In the AFB1/OTA-TRFIA, AFB1 and OTA did not mutually interfere. The correlation coefficients between the dual-label AFB1/OTA-TRFIA and the single-label AFB1-TRFIA or OTA-TRFIA were 0.972 and 0.981, respectively, indicating that the results were consistent. Our study suggests that AFB1/OTA-TRFIA allows the simultaneous detection of AFB1 and OTA; is a simple, fast, and economic method for screening large quantities of samples, and has good prospects of application.

Eu3+‐labeled IgG‐based time‐resolved fluoroimmunoassay for highly sensitive detection of aflatoxin B1 in feed

“…Europium‐labeled IgG was prepared according to the modified method reported earlier . 0.4 mL of 2 mg mL −1 GAM‐IgG in 0.05 mol L −1 CBS containing 0.155 mol L −1 NaCl was added to 0.15 mg Eu 3+ ‐DTTA in a small amber bottle under stirring, and reacted for 18 h at r.t. After centrifugation at 17 000 × g for 10 min, the precipitation was dissolved with CBS (containing 1% BSA) to close the rest of the antibody‐combining sites.…”

Immunochemical Methods for Ochratoxin A Detection: A Review

“…In addition, when using two different labels, multi-analyte immunoassay is feasible. An example of mycotoxins detection has been given by Huang et al (2009) [92] who developed a TR-FIA for OTA and Aflatoxin B1 (AFL B1) using Sm and Eu as a label, respectively. In this format, antigen-protein was coated onto microtiter plates, then sample and antibody (MAb for OTA and PAb for AFL B1) were added, followed by differently labeled second antibody.…”

Impedimetric Aptasensor for Ochratoxin A Determination Based on Au Nanoparticles Stabilized with Hyper-Branched Polymer

“…From other methods, immunochemical detection with SPR [20,21], electrochemical [21–25] and fluorescence [26–28] sensing can be mentioned (see also review [29] on immunochemical OTA detection and references cited). Direct oxidation of OTA from alkaline solution on glassy carbon electrode was evaluated by square-wave voltammetry [30].…”