Production of a monoclonal antibody against aflatoxin M1 and its application for detection of aflatoxin M1 in fortified milk

Abstract

Aflatoxin M1 (AFM1) is a toxic metabolite of the fungal product aflatoxin found in milk. For food safety concern, maximum residual limits of AFM1 in milk and dairy products have been differently enforced in many countries. A suitable detection method is required to screen a large number of product samples for the AFM1 contamination. In this study, monoclonal antibodies (MAbs) against AFM1 were generated using a conventional somatic cell fusion technique. After screening, five MAbs (AFM1-1, AFM1-3, AFM1-9, AFM1-11, and AFM1-17) were obtained that showed cross-reactivity with aflatoxin B1 (AFB1) and aflatoxin G1 (AFG1) but with no other tested compounds. An indirect competitive enzyme-linked immunosorbent assay (ELISA) using a partially purified MAb and antigen-coated plates yielded the best sensitivity with the 50% inhibition concentration (IC) and the limit of detection (LOD) values of 0.13 ng/mL and 0.04 ng/mL, respectively. This indirect competitive ELISA was used to quantify the amount of fortified AFM1 in raw milk. The precision and accuracy in terms of % coefficient of variation (CV) and % recovery of the detection was investigated for both intra- (n = 6) and inter- (n = 12) variation assays. The % CV was found in the range of 3.50-15.8% and 1.32-7.98%, respectively, while the % recovery was in the range of 92-104% and 100-103%, respectively. In addition, the indirect ELISA was also used to detect AFM1 fortified in processed milk samples. The % CV and % recovery values were in the ranges of 0.1-33.0% and 91-109%, respectively. Comparison analysis between the indirect ELISA and high performance liquid chromatography was also performed and showed a good correlation with the R of 0.992 for the concentration of 0.2-5.0 ng/mL. These results indicated that the developed MAb and ELISA could be used for detection of AFM1 in milk samples.

Determination of aflatoxin M1 in bovine milk from the Alagoas/Brazil State dairy belt by high performance liquid chromatography (HPLC)

“…This study was consistent with a study where pAb-based cdELISA was used to analyze milk (Wang et al, 2011). Chadseesuwan et al (2016) also observed in their research for aflatoxin M1 in fortified raw milk that the samples were within the acceptable range of detection.…”

Detection of Mycobacterium tuberculosis complex infection in Asian elephants (Elephas maximus) using an interferon gamma release assay in a captive elephant herd

“…Twelve replicates of each concentration were performed by using the sandwich ELISA. The percentage of intra-assay and inter-assay CV (% CV) was calculated using the following formula 27 : …”

Development and validation of a lateral flow immunoassay for the detection of aflatoxin M1 in raw and commercialised milks

“…The monoclonal antibody (mAb) against AFM1 (no. 9) was obtained from the Institute of Biotechnology and Genetic Engineering, Chulalongkorn University (Chadseesuwan et al 2016). To identify the optimum concentration ratio of mAb to GNPs, 25 µL of mAb at different concentrations (0-500 µg/ mL) was added to a 96-well plate.…”

“…In this article, a monoclonal antibody (mAb) against AFM1, which was produced by our research group (Chadseesuwan et al . 2016), was used in the development of an LFIA based on a competitive principle using a cut‐off value of 0.5 μg/L. The LFIA was validated using raw milk samples and used to screen for AFM1 contamination in various types of commercial milk samples from local supermarkets.…”