The hapten structure TS is presented inFig. indicating that nanobodies could be reliable reagents for the accurate detection of chemical compounds. Keywords:Nanobody, Ultra-specificity, Acknowledgement mechanism, Immunoassay, Accurate detection == 1. Introduction == Immunoassays for chemical compounds have been widely used in many fields such as environmental pollutants monitoring, food security analysis, and clinical diagnostics due to their inherent specificity, high sensitivity, easy operation, and short assay time (Wild, 2013). As the crucial recognition elements in immunoassays, antibodies were recognized with sufficient specificity to discriminate marginally different analytes DKFZp781H0392 from structurally comparable analogs, even as less as a single functional group or even chirality (Landsteiner, Dinaciclib (SCH 727965) 2013). However, the lack of specificity of antibodies with some analytes has always practically offered troubles (Prassas and Diamandis, 2014). There is accumulating evidence that many antibodies can inevitably bind to different structurally-related compounds with varied affinities, a phenomenon generally referred to as cross-reactivity (CR) (Jain and Salunke, 2019;Michel et al., 2009). Unexpected CRs will cause severe false positive results and an inexact quantification of the analyte of interest (Egelhofer et al., 2011;Baker, 2015;Peveler et al., 2016). CR occurs because the antibody is usually a mixture of immune globulins with multiple specificities, or simply because the antibody could bind to more than one analyte sharing a similar epitope. To avoid the former problem, a monoclonal antibody (mAb) should be produced as a real mAb, which should include the prevention of contaminations by other antibodies during the production phase in the abdominal cavity of animals (Bordeaux et al., 2010) and the avoiding of the expression of additional functional variable regions in hybridomas (Bradbury et al., 2018). Regarding the latter problem, employing an intentional counter selection step, such as a cautiously designed panning strategy to characterize and limit CR during antibody isolation, will be a prudent solution. It seems that the emerging nanobodies, the variable domain of naturally occurring camelid heavy-chain-only antibodies (VHHs), are more encouraging reagents produced by using an intentional counter selection step strategy (Hamers-Casterman et al., 1993;Ingram et al., 2018). Because nanobodies can be very easily produced as single clones based on their unique sequences and the phage display technology used to obtain nanobodies has higher efficiency and flexibility during the isolation process (Bradbury and Plckthun, 2015;Peltomaa et al., 2019). Besides, compared with other antibodies, nanobodies have the smallest size (1/10th the size of standard antibodies), and the highest stability, and they have novel structural conformations of paratopes that can bind to antigens in ways that cannot be accomplished by other antibodies (Muyldermans et al., 2009;Stijlemans et al., 2004;Genst et al., 2006;Zhang et al., 2019;He et al., 2020). Since chemical compounds belong to haptens and can only provide limited epitopes, nanobodies can be potentially more specific than standard antibodies derived from mice or rabbits with proper Dinaciclib (SCH 727965) selection and genetic Dinaciclib (SCH 727965) manipulation (Deckers et al., 2009;Li et al., 2018). Sulfadimethoxine (SDM) is a long-acting broad-spectrum antimicrobial agent of sulfonamide (SA) with a common structure of para-aminobenzenesulfonamide, which is widely used in livestock and aquacultures. Due to its large usage and hard-biodegradable, SDM has been reported to be detected in various environmental media and animal-derived food (Zhuang et al., 2019;Ben et al., 2020). Residual SDM can cause bacterial resistance, impact ecological balance, and endanger human health through food chain enrichment (Huang et al., 2020;Li et al., 2022)..