Mycotoxin contamination affects approximately 60–80% of food crops globally, potentially leading to “mycotoxicosis” when the contaminated food is consumed by humans as components in livestock feed[1–2]. Aflatoxins, which are one of the most hazardous mycotoxins, are toxic secondary metabolites produced by certain Aspergillus spp. under appropriate conditions in various agricultural and food products [3–6]. These toxins cause health and economic issues affecting consumers and farmers worldwide. Approximately 20 AF types have been identified so far, of which aflatoxin B1 (AFB1) is the most potent. AFB1 is carcinogenic, genotoxic, mutagenic, and teratogenic and is categorized as a group 1 carcinogen in the International Agency for Research on Cancer (IARC) classification of carcinogenic substances [7–10]. When animals consume AFB1–contaminated feed, the toxin is metabolized to monohydroxy derivate AFM1 in their liver. This reaction is catalyzed by the hepatic cytochrome P 450 (CYP450) enzyme system, and the product is eliminated in blood, milk, tissues, and biological fluids [11–14]. As AFM1 binds to milk proteins, especially casein, it is often detected in dairy products (e.g., milk, yogurt, cheese, butter, and infant formula) [15–17]. Approximately 0.3–6.2% of AFB1 is converted into AFM1, but the extent of transformation depends on the diet type, ingestion and digestion rates, animal health, hepatic biotransformation capacity, and milk yield [18–19]. Several in vitro experiments have established that AFM1 is cytotoxic to human hepatocytes and results in hepatocyte degeneration, necrosis, cirrhosis, etc. Moreover, AFM1 is closely linked to the occurrence of various cancers, such as hepatocellular carcinoma, breast cancer, and colon cancer [20–21]. Furthermore, AFM1 may cause DNA damage and mutation, resulting in abnormal cell growth and function as well as negatively impacting gene stability and genetic information transmission [22]. AFM1 is less hazardous than AFB1; nevertheless, it can cause growth suppression, immunological problems, and cancer in both humans and animals [23]. Therefore, AFM1 is also classified as a group I carcinogen by IARC [24].
Milk and dairy products are excellent sources of fat, protein, and trace elements for humans and are often added to various processed foods as a technical aid [25]. Unfortunately, owing to the high thermal stability of AFM1, it is not degraded or destroyed during sterilization, storage, and processing [26–27]. Hence, this toxin undoubtedly poses severe health risks for consumers, especially in immunologically vulnerable age groups. To avert the adverse health impacts caused by AFM1, many countries have set the maximum residue level (MRL) for AFM1 in dairy products. In the European Union, the concentration of AFM1 in raw milk, heat-treated milk, and milk for the manufacture of dairy products should not exceed 0.05 µg/kg for adult consumption, and 0.025 µg/kg for infants and young children [28]. In the United States and several Asian countries (including China), the permissible limit for AFM1 residues in dairy products and milk-containing foods has been set at 0.5 µg/kg [29–31]. These strict limits make AFM1 surveillance critical. The current gold standard methods for AFM1 detection are instrumental methods, such as high-performance liquid chromatography (HPLC) and liquid chromatography–tandem mass spectroscopy (LC-MS/MS) [32–33]. Although these methods are sensitive, accurate, and reliable, they require expensive equipment, extensive sample pretreatment, and specialized personnel. Hence, developing a rapid, sensitive, and accurate method for monitoring AFM1 levels in milk and dairy products is of immense significance.
The enzyme-linked immunosorbent immunoassay (ELISA) is based on antigen–antibody reactions and is used commonly used in food industries and official food control agencies. This technique offers high selectivity and sensitivity, requires a small sample volume, is cost-effective, has a wide dynamic measuring range, and enables high-throughput parallel sample processing. Moreover, ELISA offers the advantages of simplicity of operation, and portability [34]. Over the past years, several ELISA-based methods have been introduced to detect AFM1 in milk and dairy products, most of which are competitive methods based on monoclonal antibodies (mAbs) [35–38]. The use of selective antibodies that do not cross–react with other comparable compounds is crucial for the immunoassay detection of tiny molecules [39]. However, mAbs have certain limitations, such as slow and expensive manufacturing and insufficient organic solvent stability to withstand the high concentrations used to extract the targeted toxin causing food contamination [40]. Nanobodies (Nbs), also referred to as Single–domain antibodies (SdAbs), are the variable domains of a heavy-chain antibody (VHH) obtained from Camelidae species and sharks. Nbs are the smallest available antibody fragment with functional antigen binding [41]. Compared to conventional antibodies, Nbs are characterized by small size (approximately 15kDa); high affinity, selectivity, solubility, and yield; low-cost generation, and increased temperature and organic–solvent stability [42–43]. These advantages make Nbs ideal reagents for the detection of small molecule contaminants [44–45].
In our previous study, a high-quality phage library was generated via the immunizations of a Bactrian camel with AFM1–BSA. Subsequently, using AFM1–OVA as the screening antigen, acidic elution, and competitive panning strategies were screened to obtain six AFM1–specific nanobodies named Nb M1-M6 [46]. This research aimed to express these six Nbs to screen the candidate whose IC50 met the detection criteria and evaluate its thermal stability and affinity. Furthermore, an indirect competitive enzyme-linked immunosorbent assay (icELISA) was used to create an accurate, sensitive, and selective AFM1 detection tool for dairy products.