**1. Introduction**

Mycotoxins are toxic secondary metabolites produced naturally by many fungi under certain growth conditions. These toxins affect metabolic processes and cause disease and death in humans and animals [1]. Toxicological actions of mycotoxins are recognized but few of these compounds or their derivatives are identified as antibiotics, growth promoters, or other drugs. Major mycotoxins include aflatoxins, gliotoxin, citrinin, ergot alkaloids, fumonisins, ochratoxin, and patulin [2].

Humans do not produce antibodies to mycotoxins and cannot be immunized against their toxicity. Nearly 25% of food becomes inedible due to contamination with mycotoxins; aflatoxins are the most serious source of contamination [3]. In 1993, aflatoxin is classified as a class one carcinogen by the World Health Organization Cancer Research Institute.

**Citation:** Hafez, E.; Abd El-Aziz, N.M.; Darwish, A.M.G.; Shehata, M.G.; Ibrahim, A.A.; Elframawy, A.M.; Badr, A.N. Validation of New ELISA Technique for Detection of Aflatoxin B1 Contamination in Food Products versus HPLC and VICAM. *Toxins* **2021**, *13*, 747. https://doi.org/ 10.3390/toxins13110747

Received: 16 July 2021 Accepted: 8 October 2021 Published: 21 October 2021

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Aflatoxin causes hepatotoxicity in both humans and animals. Exposure to this toxin can lead to liver cancer and death. The chemical is a bifuran toxoid produced by strains of *Aspergillus flavus* and *Aspergillus parasiticus*. About 20 derivatives are recognized, for example, B1, B2, G1, G2, M1, and M2. Aflatoxin B1 (AFB1) is the most potent and carcinogenic [4]. Aflatoxins M1 and M2 are hydroxylated metabolites of aflatoxin B1 produced by animals and commonly exist in milk and dairy products. The toxins are not common in grains. AFB1 is detected on the surfaces of maize and peanuts.

Toxicity of and exposure to AFB1 has been extensively investigated. The toxin may result in severe disease, including carcinogenesis, mutagenesis, growth retardation, and immune suppression [5]. The aflatoxin-producing fungus, *P*. *flavus,* grows and produces aflatoxins on preharvest maize and on maize in storage [6]. Peanuts are also susceptible to *Aspergillus* infection in the field or during storage. Both maize and peanuts are rich nutrient sources for these fungi [7]. Aflatoxin-contaminated agricultural products may pose serious health risks to humans and animals and negatively affect international trade [8]. According to the Food and Drug Administration (FDA) in the USA, an acceptable aflatoxin level in food is 0 ppb.

Typically, high-performance liquid chromatography (HPLC) and liquid chromatography mass spectrometry (LC-MS) are used for quantification of aflatoxins. LC-MS/MS can detect trace levels, but some limits exist. Chemical detection is slower than spectral detection (hours vs. seconds) and tedious. Professional analysts and precise chemical instruments are required [9]. Thus, accurate, rapid, full-scale detection of AFB1 is important in assessing human health and economic impacts. Evaluating contaminated food directly for specific fungi using genes that control aflatoxin is a promising strategy [10]. Gallo et al. [11] reported such genes in the genome of aflatoxin-producing fungi. However, the authors' method required costly instruments, amplification, isolation, and quantification along with trained personnel [12]. The method is quite complex and costly for routine use. This study aimed to develop a new cost- and time-effective quantitative method using modified recombinant *AflR* gene antiserum enzyme-linked immunoassay (ELISA) for aflatoxin B1 detection in contaminated food products.

#### **2. Results and Discussion**

#### *2.1. The Aflatoxin B1 Detection with HPLC and VICAM*

VICAM was less sensitive than HPLC analysis for aflatoxin AFB1 detection in several samples—peanut 2, flours 2 and 3, and milk-powder 3. HPLC is widely used for the analysis of aflatoxins for sensitivity and accuracy [13] (Table 1). HPLC is an excellent quantitative method in detection of aflatoxins [14], although it requires skilled operators, extensive sample preparation, and is a high-cost equipment [15].


**Table 1.** HPLC and VICAM screening analysis of 36 food samples for aflatoxin detection.

+: refers to low aflatoxin contamination level; ++: refers to moderate aflatoxin contamination level; +++: refers to high aflatoxin contamination level.

#### *2.2. Molecular Detection and SDS-PAGE*

A unique band at about 760 bp was observed in all positive samples (Figure 1A). Negative amplification was observed in flour contaminated with aflatoxin (50, 75, and 100 mg). Amplified DNA was cloned and in vitro transcribed protein was separated on SDS-PAGE (Figure 1B). SDS-PAGE analysis revealed a protein of about 28 kDa. Molecular weight determination regarded as first characterization step of protein was further used in toxin detection.

**Figure 1.** (**A**) PCR product amplified using the specific primers of the afIR gene (Aflatoxin B1). M: DNA marker and afIR the amplified gene in molecular size about 760 bp. (**B**) The recombinant protein of the in vitro transcribed afIR gene (Aflatoxin B1) with molecular size about 28 kDa.
