The inhibition curves (Figure 5) showed that the fifth mouse had the lowest IC50 value (45.0 g/L). curve was established, and the sensitivity and specificity of the ZEN mAbs were determined under the optimized icELISA conditions. ZEN-BSA was successfully synthesized at a conjugation ratio of 17.2:1 (ZEN: BSA). Three hybridoma cell lines, 2D7, 3C2, and 4A10, were filtered, and their mAbs corresponded to an IgG1 isotype with a light chain. The mAbs titers were between (2.56 to 5.12) 102 in supernatants and (1.28 to 5.12) 105 in the ascites. Besides, the 50% inhibitive concentration (IC50) values were from 18.65 to 31.92 g/L in the supernatants and 18.12 to 31.46 g/L in the ascites. The affinity constant (and spp.-infected cereals contain ZEN, -ZEL, and -ZEL. Notably, ZEN is metabolized into -ZEL, -ZEL, and ZAN. -ZAL is metabolized into -ZAL and ZAN, indicating that both ZEN and its metabolites are toxic to the human body. However, -ZEL has the highest toxicity, 10C20 times higher than ZEN toxicity [13,14]. Therefore, immense research on detecting total ZEN and its metabolites (TZEN) is necessary since single ZEN detection cannot meet food and feed industry requirements. Open in a separate window Figure 1 The chemical structure of zearalenone and its metabolites. Currently, physicochemical analysis and immunoassay are the two main methods used for TZEN detection. The major physicochemical analysis methods used in all countries include thin-layer chromatography (TLC) [15], high-performance liquid chromatography (HPLC) [16], gas chromatography-mass spectrometry (GC-MS) [17], and liquid Reparixin L-lysine salt chromatography/tandem mass spectrometry (LC-MS/MS) [18]. However, these techniques are expensive, time-consuming, and require complex sample pretreatment procedures, expensive instruments, and skilled technicians, making them Reparixin L-lysine salt unsuitable for high-throughput detection [19]. The immunoassay method that is based on the specificity and sensitivity antigen-antibody reaction is a hotspot in the TZEN detection research due to its strong selectivity and sensitivity, high speed, easy sample screening, and large scale on-site operation [20,21]. In recent years, different immunoassay methods based on ZEN monoclonal antibodies (mAbs) Reparixin L-lysine salt with a high affinity and broad class specificity have been established to rapidly detect TZEN. These include an enzyme-linked immunosorbent assay (ELISA) [22,23,24], a gold immunochromatographic assay (GICA) [25], and a fluorescence polarization immunoassay (FPIA) [26]. However, Reparixin L-lysine salt these immunoassay methods have some drawbacks, such as poor specificity and sensitivity to TZEN, possibly due to a low quality mAb, thus not meeting the actual detection Reparixin L-lysine salt needs. Notably, high-quality mAbs are integral to immunoassay methods since immunoassay efficacy depends on the affinity and specificity of the mAbs used. Recent reports have shown that while most mAbs can specifically recognize ZEN, they only recognize some ZEN metabolites [27,28]. However, mAb sensitivity should be further improved, despite the broad specificity [22,23]. The preparation of broad spectrum class specific antibodies is essential for the development of a multiple analogues immunoassay [29,30]. In recent years scholars have researched antigen design and synthesis to broaden the recognition spectrum of antibodies. Thus far, the following three approaches have been used to obtain a broad spectrum of specific antibodies via antigen design and synthesis: (1) The development of a general structural immunogen, with the general structure of the analytes as the detection target, such as aflatoxins (AFs) [31], sulfonamides (SAs) [32], organophosphorus (OPs) [33], and fluoroquinolones (FQs) [34]. (2) The development of a multi-hapten immunogen by simultaneously coupling several different haptens to one carrier protein, such as avermectins (AVMs) [35], microcystins (MCs) [36], and pesticides, including chlorpyrifos, triazophos, carbofuran, and parathion methyl [29]. (3) The development of several mixed immunogens by individually coupling each hapten to a carrier protein. The mixed immunogens, such as tetracycline (TCS) [37] and (BTS), are then used for simultaneous immunization [38]. However, similar to most mycotoxins, antibiotics, and synthetic drugs, ZEN and its structural analogues are CACNA1C too small to elicit an immune response in the host animal. Therefore,.