**1. Introduction**

PAHs, composed of two or more aromatic rings, are the most ubiquitous persistent organic hydrocarbons in the world [1]. The chemistry properties of PAHs (hydrophobicity, stability and carcinogenicity) will change as the ring number of PAHs increases [2,3]. The main source of PAHs is the incomplete combustion of organic materials such as fossil fuels, natural gas and wood, as well as human activities, including food processing, garbage incineration and automobile exhaust fumes [4–6]. PAHs can migrate with air and water for a long distance and accumulate in organisms, causing extensive and serious harm to human fitness and the ecological environment. Exposure to PAHs can cause a variety of adverse effects in humans and animals, including carcinogenicity, DNA damage, teratogenicity, mutagenicity and immunotoxicity [7–10]. Therefore, 16 PAHs have been classified as significant pollutants by the European Scientific Committee for Food (ECSCF) [11].

The pollution of PAHs in water environments is mainly caused by wastewater discharge and oil spills. For example, in 2010, the famous oil spill occurred in the Gulf of Mexico, and millions of tons of oil were released into the Gulf of Mexico [12]. China is a large aquaculture country, and its aquaculture production makes up more than 69% of the total global production. However, monitoring of major pollution sources in the national water environment in 2018 showed that PAHs were found in water, sediment and aquatic organisms. With the significant increase in industrial production and demand, the level of PAH-based pollutants in aquatic ecosystems has become strikingly high [13]. Owing to PAHs lipophilicity, they can easily accumulate in aquatic organisms and cause serious harm because their membranes are easily penetrated by PAHs [14]. It was reported that PAHs tend to accumulate in fatty aquatic products such as fish, shrimp and crabs. PAHs

**Citation:** Wu, S.; Li, H.; Yin, X.; Si, Y.; Qin, L.; Yang, H.; Xiao, J.; Peng, D. Preparation of Monoclonal Antibody against Pyrene and Benzo [a]pyrene and Development of Enzyme-Linked Immunosorbent Assay for Fish, Shrimp and Crab Samples. *Foods* **2022**, *11*, 3220. https://doi.org/ 10.3390/foods11203220

Academic Editor: Thierry Noguer

Received: 29 August 2022 Accepted: 12 October 2022 Published: 15 October 2022

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can interfere with estrogen signaling pathways (especially 4- and 5-ring) [15,16]. PYR and BaP (Figure S1) are the main tetracyclic and pentacyclic compounds in PAHs, with strong persistence and harmfulness in the water environment. The residues in aquatic products are important factors affecting the quality and safety of aquaculture [17]. BaP is a residue marker of PAH pollution exposure due to its high carcinogenicity [18]. The European Commission has set limits of 2.0 μg/kg for fish, 5.0 μg/kg for crustaceans and cephalopods, and 10.0 μg/kg for shellfish [19].

Many chromatographic methods have been developed to monitor PAHs in different matrices, including GC–MS, LC-MS, and HPLC [20–22]. Although chromatographic methods have some advantages, the sample pretreatment is complicated and requires professional operators, making it unsuitable for on-site testing of a large number of samples. Immunoassays are more desirable because of their high sensitivity, time savings and low requirements for operators [23]. At present, ELISA is a widely used detection method in immunoassay, which is simple, fast and greatly improves the efficiency of residue analysis [24]. However, there are few studies on the ELISA methods of both PYR and BaP, and they mainly focus on the detection of environmental samples, such as water (lake water, tap water, drinking water) and air [25]. As far as we know, there is no reported immunoassay method for the PAHs residues assay in aquatic products.

In this work, a highly sensitivity mAb was prepared and an ELISA method was developed to detect PYR and BaP residues in fish, shrimp and crab without complex sample pretreatment. The effect of organic solvents on the sensitivity of ELISA was investigated. The accuracy of the ELISA is reliable compared to HPLC-FLD.
