Elsevier

Food Control

Volume 60, February 2016, Pages 588-595
Food Control

An ultra-sensitive monoclonal antibody-based fluorescent microsphere immunochromatographic test strip assay for detecting aflatoxin M1 in milk

https://doi.org/10.1016/j.foodcont.2015.08.040Get rights and content

Highlights

  • An ultra-sensitive anti-AFM1 monoclonal antibody was produced.

  • The moderate hapten-to-protein coupling ratio (˜2) in the coating antigen could improve the immunoassay sensitivity.

  • The sensitivity of the FM-ICTS assay was comparable to the ciELISA and superior to the CG-ICTS assay.

Abstract

A rapid lateral flow fluorescent microsphere immunochromatographic test strip (FM-ICTS) assay has been developed for the detection of aflatoxin M1 (AFM1) residues in milk. For this purpose, an ultra-sensitive anti-AFM1 monoclonal antibody (MAb) 1D3 was prepared and identified. The IC50 value of the MAb 1D3 for AFM1 was 23 ng/L, and the cross-reactivity (CR) of the MAb with aflatoxin M2, aflatoxin B1, aflatoxin B2, aflatoxin G1 and aflatoxin G2 was 18%, 9%, <0.15%, 43% and <0.3%, respectively. The MAb was covalently conjugated with carboxylate-modified FMs, and the FMs were used as the label in a competitive immunochromatography assay. Using a moderate hapten-to-protein coupling ratio (˜2) in the coating antigen resulted in improved immunoassay sensitivity. Under optimal conditions, the IC50 value of the assay was 36.3 ng/L with the limit of detection (LOD) of 4.4 ng/L in milk samples using a fluorescence reader, and the recoveries of AFM1 in spiked milk samples ranged from 76.6 to 110.8% with coefficient of variation (CV) of 4–14.7%. The whole procedure could be completed within 30 min. The results demonstrated that the FM-ICTS assay is easy to conduct, rapid, highly sensitive and specific for the detection of AFM1 residues in milk.

Introduction

An immunochromatographic test strip (ICTS) assay is a popular screening tool for conducting onsite testing because of its sensitivity, rapidity (5–10 min), and user-friendliness (Xu et al., 2013). Colloidal gold, a metal nanoparticle-based probe, has been commonly used in the fields of agriculture (Putalun, Morinaga, Tanaka, & Shoyama, 2004), food safety (Li et al., 2011, Sun et al., 2014), and medical diagnostics (Wangoo, Kaushal, Bhasin, Mehta, & Suri, 2010). However, due to lack of sensitivity, this label has mainly been applied to the detection of compounds at high concentrations. The use of fluorescent signals has attracted recent attention to enhance the detection signal and improve the sensitivity of the ICTS. Fluorescent microspheres (FMs), which have a stable configuration, a high fluorescence intensity, are multicolored and safe, and have already been employed in food safety (Chen et al., 2013, Zhou et al., 2014, Zhou et al., 2014) and in medical diagnostics (Nankoberanyi et al., 2014). It has been reported that a fluorescent microsphere-immunochromatographic test strip (FM-ICTS) assay had eight times higher sensitivity than that of a colloidal gold based immunochromatographic test strip (CG-ICTS) assay for the detection of Escherichia coli O157:H7 using the sandwich format (Xie et al., 2014). Thus FMs used as labels can provide an excellent tool for improving the sensitivity of the ICTS assay.

Aflatoxins are highly toxic and carcinogenic mycotoxins produced by Aspergillus flavus and Aspergillus parasiticus. The four major naturally occurring aflatoxins are aflatoxin B1 (AFB1), aflatoxin B2 (AFB2), aflatoxin G1 (AFG1) and aflatoxin G2 (AFG2) (Fig. 1). Aflatoxin M1 (AFM1), the hydroxylated metabolite of AFB1, is often found in milk from animals fed with AFB1-contaminated feeds (Yu et al., 2004). Most countries have regulated the levels of AFM1 in milk, which vary from 50 ng/L established by the EU to 500 ng/L established by China and the US FDA.

To reduce the risk of AFM1 exposure, it is essential to identify the sources of AFM1 contamination, which can be appropriately conducted by using a rapid, high-throughput and sensitive assay. Several analytical methods for detecting AFM1 have been reported, including high-performance liquid chromatography with fluorescence or mass spectrometry detection (Manetta et al., 2005, Muscarella et al., 2007, Solfrizzo et al., 2011). Enzyme-linked immunosorbent assays (ELISAs) (Magliulo et al., 2005, Radoi et al., 2008, Vdovenko et al., 2014) and colloidal gold immunoassays (Wang et al., 2011, Zhang et al., 2013, Zhang et al., 2013) have also been described and are widely employed as screening methods in routine analysis. A non-instrumental rapid test was developed for screening AFM1 in milk at the 40 ng/L level, but the author used an anti-AFB1 monoclonal antibody (MAb) with a 79% cross-reactivity for AFM1 (Goryacheva, Karagusheva, Peteghem, Sibanda, & Saeger, 2009). A broad-specific MAb 3C10 whose IC50 value was 0.13 μg/L for AFB1 and 0.16 μg/L for AFM1 was prepared; however, AFB1-O-carboxymethyl-oxime was used as the hapten (Jiang et al., 2012, Jiang et al., 2012). An ultra-sensitive electrochemiluminescent immunoassay (ECLIA) was reported for the detection of AFM1 in milk samples using magnetic Fe3O4-graphene oxides as the absorbent and antibody-labeled cadmium telluride quantum dots (CdTe QDs) as the signal tag, and the detection limit (LOD) was 0.3 ng/L (S/N = 3) (Gan et al., 2013). A rapid sensitive method for the detection of AFM1 by dynamic light scattering coupled with superparamagnetic beads and gold nanoprobes has also been reported, and the LOD was 27.5 ng/L in milk samples (Zhang, Lin, Zhang, & Vardhanabhuti, 2013). Thus a sensitive anti-AFM1 antibody was the principal limitation factor of these immunoassays, in addition, a new signal tag, such as QDs or FMs could potentially increase the immunoassay sensitivity to meet the high requirement necessary to satisfy the legislated concentration limits for AFM1. For this purpose, here we produced an ultra-sensitive anti-AFM1 MAb with high specificity and developed a FM-ICTS assay for detecting AFM1 residues in milk.

Section snippets

Chemicals and materials

AFM1, AFM2, AFB1, AFB2, AFG1, AFG2, hemocyanin from Megathura crenulata (KLH), bovine serum albumin (BSA), Freund's incomplete adjuvant (FIA), Freund's complete adjuvant (FCA), Poly(ethylene glycol) (PEG) 1500, hypoxanthine aminopterin thymidine (HAT), and a mouse MAb isotyping kit were obtained from Sigma–Aldrich (St. Louis, MO, USA). DMEM cell culture medium and fetal calf serum were obtained from Gibco Life Technologies (Grand Island, NY, USA). FluoSpheres® Carboxylate-Modified Microspheres

MAb production and characterization

The AFM1 is a small molecule hapten; therefore, it needs to be coupled with a carrier protein to elicit a specific immune response. By inserting CMO, the haptens of AFM1-CMO and AFB1-CMO having a free carboxyl group were coupled to the amino groups of the carrier protein KLH or BSA, and there was a four atom linker between AFM1 and the carrier protein. This linker was suitable for exposing the molecular structure and stimulating the animals to create specific antibodies. We also reacted

Conclusion

This paper demonstrates a methodology for preparation of an ultra-sensitive anti-AFM1 MAb 1D3 and a broad anti-aflatoxin MAb 2D4 by using AFM1-CMO-KLH as immunogen. The use of a moderate hapten-to-protein coupling ratio (˜2) in the coating antigen resulted in improved immunoassay sensitivity. Here also is reported a FM-ICTS assay for the rapid and sensitive detection of AFM1 residues in milk. Under optimal conditions, the linear dynamic range of the test was from 10 ng/L to 320 ng/L, the IC50

Acknowledgments

This work was supported by the International Science & Technology Cooperation Program of China (2009DFA32330) and the Special Fund for Agro-scientific Research in the Public Interest (No.201203040).

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