**1. Introduction**

Quinoxalines (Qx) are chemically synthesized antibacterial drugs that have a strong inhibitory effect on both gram-positive and gram-negative bacteria [1,2]. Due to the broad antimicrobial spectrum and obvious growth-promoting effects, Qx are used in excessive amounts by farmers as feed additives to obtain more economic benefits [3]. However, several studies have shown that Qx can cause toxic side effects such as trichothecation, photosensitivity, and adrenal cortical damage in livestock and poultry. More seriously, drug residues in animal-derived foods can pose a significant threat to the population directly consuming them [4–6]. According to the latest reports, the quinoxalines desoxymetabolites (desoxyquinoxalines, DQx) have the highest residues and have been identified as major toxicants. They will soon replace 3-methyl-quinoxaline-2-carboxylic acid (MQCA) and quinoxaline-2-carboxylic acid (QCA) as the new residue markers for Qx [1,7,8]. The chemical structures of Qx and the major DQx are shown in Figure 1. The Ministry of Agriculture and Rural Affairs of the People's Republic of China has stated that olaquindox (OLA), carbadox (CBX), and mequindox (MEQ) have been banned for use in food animals, and

**Citation:** Song, W.; Luo, M.; Li, H.; Xiao, J.; He, X.; Liang, J.; Peng, D. A Novel Metabolite as a Hapten to Prepare Monoclonal Antibodies for Rapid Screening of Quinoxaline Drug Residues. *Foods* **2022**, *11*, 3305. https://doi.org/10.3390/ foods11203305

Academic Editor: Thierry Noguer

Received: 20 August 2022 Accepted: 19 October 2022 Published: 21 October 2022

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the requirements for residue detection of quinoxalines have been increasing. In order to accurately monitor the residual hazards of Qx in animals and reduce the risk to human health, there is a need to develop a residue-monitoring method for DQx.

**Figure 1.** The chemical structures of Qx and the major DQx.

Currently, many detection assays have been reported but mainly for Qx prototypes or MQCA and QCA, which were previously identified as residue markers. Additionally, the detection of QX is mainly focused on instrumental detection assays, such as highperformance liquid chromatography (HPLC) [9,10] and liquid-chromatography tandem mass spectrometry (LC–MS/MS) [11,12]. Though the instrument-detection method has high sensitivity and precision, it requires rather expensive instruments, complicated technology, a long operation time, and low detection efficiency. In contrast, immunoassay methods, such as enzyme-linked immunosorbent assay (ELISA), are simple, convenient, sensitive, specific and inexpensive. ELISA has become an alternative analytical method or a meaningful complement, especially in the detection of large numbers of samples [13,14]. Current indirect competitive ELISAs (ic-ELISA) are all for Qx prototypes, MQCA, or QCA [15,16]. To the best of our knowledge, immunoassay assays for the residue detection of DQx have not been previously reported. The maximum residue limits (MRLs) for DQx are not legally standardized, leading to potential gaps in food safety. Therefore, it is important to develop a rapid and accurate detection method for DQx.

In this study, we propose to synthesize novel haptens against desoxymequindox (DMEQ) to fully expose the main part of DQx. The aim is to prepare monoclonal antibodies (mAb) against DQx and establish an ic-ELISA method to provide strong technical support for ensuring the food safety of animal origin and reducing the risk to human health.
