Topramezone is a highly selective herbicide that inhibits 4-hydroxyphenylpyruvate dioxygenase (HPPD). Because residues can pollute environments and contaminate foods, rapidly, quantitative detection is essential. Here we report a fast method for detecting topramezone in food and environmental matrices. Guided by computer simulations, we designed a hapten (TOP-Hapten-1) with high structural and electrostatic similarity among five candidates, and then generated a high-affinity, monospecific monoclonal antibody (mAb) and a single-chain variable fragment (scFv). Homology modeling followed by molecular docking was used to map the scFv-topramezone interface, and identified five key residues. And we developed an indirect competitive ELISA (icELISA) and colloidal gold immunochromatographic assay (CGIA). The ic-ELISA achieved an IC50 of 0.605 mu g/L; the CGIA provided a 0.5 mu g/L visual limit of detection and a 1 mu g/L cutoff. This work delivers two immunoassays and validates computationally assisted antibody design, offering a practical framework for biosensor development and risk monitoring.

Thiamethoxam (TMX) is a widely used second-generation neonicotinoid insecticide. However, improper application could result in the accumulation of residue in edible products, posing risks to consumers. As a result, accurately identifying TMX residues in foods is essential. In this research, monoclonal antibody (mAb) that specifically recognize TMX was developed. To improve detection sensitivity, a heterologous coating strategy was utilized in TMX detection. Based on the developed monoclonal antibody, ic-ELISA and GICA methods were established. Using the established optimal conditions for ic-ELISA, the IC50 was 0.85 mu g/L. Additionally, a sample extraction method was developed, which can handle both vegetables and animal-derived food at the same time. The limits of detection (LODs) were 0.099-0.117 mu g/kg, quantification (LOQs) were 0.143-0.194 mu g/kg, and the recovery rates were 81.5%-121.3% in lake water, pork, beef, eggs, and spinach. Furthermore, we also optimized key parameters of GICA; the VLOD for eggs was 10 mu g/kg, while for spinach, beef, and pork, it was 5 mu g/kg. These two detection methods established in this research can more sensitively and accurately detect TMX in various samples during on-site rapid testing.

Enzymes as labeling agents enhance lateral flow immunoassays (LFIA). However, their biological instability and reliance on labor-intensive chemical conjugation hinder practical high-throughput screening of food contaminants. To address this, we report a rapid, label-free luciferase-based platform for quantifying lomefloxacin (LOM) in food. The assay uses an engineered immunoprobe: a high-affinity single-chain variable fragment antibody (3G9-scFv) specific to LOM, combined with Nanoluciferase (NLuc) as an intrinsic signal generator without covalent labeling. NLuc catalyzes furimazine oxidation, producing stable bioluminescence inversely proportional to LOM concentration. This label-free design eliminates conjugation variability and simplifies protocol execution. Smartphone-based luminescence measurement enables sensitive field detection, with a limit of quantification of 0.12 ng/mL. Furthermore, homology modeling and molecular docking elucidated the structural basis for the dual functionality of 3G9-scFv, validating the rational use of unlabeled enzymatic tracers. The visual detection limit was determined to be 1 ng/mL. Recovery rates ranged from 79.0% to 113.0%, with coefficients of variation (CVs) between 2.3% and 10.9%, indicating high accuracy and reproducibility. This system offers a novel rapid detection platform for monitoring hazardous residues in food safety.

Porcine reproductive and respiratory syndrome virus (PRRSV) causes significant economic losses to the global swine industry. In this study, the inhibitory effect of recombinant porcine interferon alpha (rPoIFN-α) against PRRSV was evaluated using a MARC-145 cell model. Cytotoxicity assays confirmed that rPoIFN-α dilutions that were 100-fold (corresponding to a titer of 2 × 10⁵ IU/mL) greater were nontoxic to MARC-145 cells. Treatment with rPoIFN-α at concentrations of 4 × 10⁴ IU/mL and 2 × 10⁴ IU/mL (corresponding to 500-fold and 1000-fold dilutions, respectively) significantly reduced the PRRSV-induced cytopathic effect (CPE), viral nucleocapsid (N) gene expression, and viral titers (TCID₅₀) compared with those in the infected, untreated controls. Notably, compared with short-term co-incubation during viral adsorption, continuous treatment with rPoIFN-α post-infection was significantly more effective at inhibiting PRRSV. These findings demonstrate that rPoIFN-α possesses potent anti-PRRSV activity in vitro, with prolonged exposure post-infection being the more effective treatment regimen. This study provides a foundational basis for the design of treatment regimens and dose-ranging studies for rPoIFN-α in future in vivo experiments aimed at controlling PRRS.

The residues of veterinary drugs in the food chain are a global concern for food safety, including questions about the origin of these residues, exposure pathways, health impacts, methods for their dissolution, and accurate monitoring methods. In recent years, numerous professional studies have addressed the above concerns from various perspectives. However, these studies are relatively scattered and cannot provide a systematic and comprehensive understanding of recent developments. In this systematic review, we aim to provide a comprehensive synthesis of the current state of knowledge concerning the residues of veterinary drugs in the food chain through critical examination of their origins, exposure pathways, and associated health/environmental hazards. Investigating creative mitigation techniques to lower such residues in food products is given special attention. In summary, this research proposes a paradigm that balances the development of animal production with strict food safety governance to address productivity, consumer health, and international standards.

Masked mycotoxins pose a persistent analytical challenge because their conjugated moieties are often weakly immunogenic and poorly captured by antibodies raised against parent toxins, leading to inadequate selectivity in rapid assays. Here, we introduce a computation-guided hapten engineering strategy for selectivity-by-design toward a masked toxin epitope, integrating conformational alignment, electrostatic potential mapping, and electronic structure descriptors to prioritize epitope presentation during immunization. Using zearalenone-14-glucoside (ZEN-14G) as a model analyte, this workflow enabled the generation of mAb-1C1, a monoclonal antibody elicited directly against a masked mycotoxin. The antibody exhibits sub-ng mL-1 competitive performance (IC50 = 0.093 ng mL-1) and a cross-reactivity profile consistent with masked-epitope preference. Docking and alanine-scanning mutagenesis establish a dual-interaction architecture in which hydrophobic contacts stabilize the conserved toxin core, while polar hotspot residues interact with the glucoside moiety, providing a mechanistic basis for selectivity. We further translate the recognition element into an indirect competitive enzyme-linked immunosorbent assay (ELISA), a rapid competitive lateral flow assay, and a smartphone-based quantitative readout that normalizes strip variability by using a C/T metric. Accuracy in multiple cereal matrices is validated against liquid chromatography-mass spectrometry/MS (LC-MS/MS). Collectively, this work demonstrates a generalizable selectivity engineering framework that links in silico hapten design, mechanistic paratope mapping, and deployable measurement formats for analytically elusive conjugated small molecules.

Simultaneous detection of carbofuran (CBF) and 3-hydroxy carbofuran (3-OH-CBF) in fruits and vegetables is important due to their high toxicity and widespread use in pest control. However, most lateral flow immunoassay (LFA) approaches only detection CBF. To overcome this limitation, two haptens, 6-((2,2-dimethyl-2,3-dihydrobenzofuran-7-yl)oxy)hexanoic acid and 6-((((2,2-dimethyl-2,3-dihydroben zofuran-7-yl)oxy)carbonyl)amino) hexanoic acid, named H1 and H2, were designed for broad-spectrum antibody detection of CBF and 3-OH-CBF. The highly specific monoclonal antibody (mAb) 3F4 based on hapten H1 only recognized CBF, while broadspectrum mAbs 1D3, 6B7 and 6E8 based on hapten H2 recognized both compounds. The carbon bond to the hydroxyl group of 3-OH-CBF adopts a 28.55 degrees angle relative to the same carbon bond of CBF, explaining the specificity of the H1-based mAb against CBF. After optimizing the antigen-antibody combination, a sensitive colloidal gold-LFA (CG-LFA) strip was constructed based on mAb 1D3 and H2-BSA for CBF and 3-OH-CBF detection following simple sample pretreatment. The established LFA had a limit of detection of 0.49-6.63 ng/mL, and adequate recoveries (69.3-105.7 %) for CBF and 3-OH-CBF in 26 fruit and vegetable samples.

Altrenogest is one of the most commonly used steroid hormones; however, there are currently no relevant reports on monospecific molecular recognition elements and immunoassay methods for altrenogest. Herein, a computer-aided precise hapten design strategy was proposed for monospecific monoclonal antibodies (mAb) preparation. Based on this strategy, a monospecific and sensitive mAb-D7 was prepared for the first time. The mAb-D7 has the 50 % inhibitory concentration (IC50) of 0.12 ng/mL for altrenogest and does not cross-react with other common steroid hormones. Additionally, a single-chain variable fragment (scFv) for altrenogest was constructed for the first time, which exhibits an IC50 of 1.7 ng/mL for altrenogest. The molecular recognition mechanism studies showed the monospecific mAb-D7 to altrenogest originated from the amino acids PHE-94 and LEU-237, demonstrating the reliability of this strategy. Finally, two monospecific, rapid, and sensitive immunoassays were established for altrenogest in pork and pork liver for the first time.

Dexamethasone, a long-acting glucocorticoid, exhibits considerable structural similarity to other glucocorticoids. To avoid false-positive results in immunoassays due to cross-reactivity of antibodies with structural analogues, the preparation of highly specific antibodies is crucial. In this study, a novel hapten DEX-GA was designed and synthesized, and the monoclonal antibody (mAb) 3D1 was successfully prepared based on the hapten. The mAb 3D1 has the 50 % inhibitory concentration of 0.38 ng/mL for dexamethasone, with cross-reactivity below 7.45 % against twelve glucocorticoids including betamethasone, prednisone, triamcinolone and beclomethasone. Based on this antibody, indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) and colloidal gold immunochromatography assay (GICA) were developed for accurate detection of dexamethasone in milk and animal tissues. The limits of detection of ic-ELISA in milk and animal tissues ranged from 0.132 to 0.215 mu g/kg, which was a 1-3-fold increase in sensitivity compared with the previously reported ic-ELISA method. The GICA method established in this study had a visual limit of detection of 0.3 mu g/L in milk. Notably, this constitutes the first application of GICA in animal tissues, demonstrating a visual detection limit of 0.2 mu g/kg. The accuracy and reliability of both ic-ELISA and GICA were validated using liquid chromatography-tandem mass spectrometry.

The fluorescence quenching Lateral flow immunoassay (FQ-FLIA) with a "turn on" pattern has shown considerable superiority. However, unclear fluorescence quenching mechanisms limit the development of FQ-FLIA. Herein, four distinct shapes of gold nanoparticles were designed and synthesized. Experimental measurements and finite-difference time-domain (FDTD) calculations revealed the fluorescence quenching mechanism. Gold nanoflowers (GNFs) possess a large molar extinction coefficient and effectively quench the fluorescence of time-resolved fluorescent microspheres (TRFMs) through a powerful inner filter effect (IFE). Notably, the multiple tip structures on the GNFs surface generated stronger localized electric fields, enhancing the localized surface plasmon resonance (LSPR) effect and thereby endowing GNFs with superior nanometal surface energy transfer (NSET)-based fluorescence quenching capability. With the synergistic effect of fluorescence resonance energy transfer and inner filter effect, achieving a dual-enhanced quenching efficiency of up to 95 %. A highly sensitive FQ-LFIA for detecting the target molecule lomefloxacin (LOM) was constructed. The visual detection limit (vLOD) was 0.1 ng/mL, and the sensitivity was 20 times higher than that of the traditional AuNPs-LFIA. The proposed GNFs serve as dual-mechanism quenchers to realize high-sensitivity FQ-LFIA, offering innovative insights for designing efficient quenchers in fluorescence quenching biosensors. Overall, this dual-enhanced fluorescence quenching FQ-LFIA based on GNFs enables practical detection of LOM in food samples.

The porcine enteric coronaviruses (PECs) currently reported include porcine epidemic diarrhea virus (PEDV), porcine deltacoronavirus (PDCoV), transmissible gastroenteritis virus (TGEV), and swine acute diarrhea syndrome coronavirus (SADS-CoV). In the absence of effective treatment, they can cause similar clinical characteristics including weight loss, sleepiness, vomiting, anorexia and fatal diarrhea in neonatal piglets, resulting in significant economic losses to the global pig industry. Although many studies on drugs for treating and combating PECs have been issued. There are still no specific drug targeting PECs and used in clinical production. Therefore, it is necessary to sort out and summarize the research on the treatment and anti PECs drugs, and further development of low toxicity and high efficiency drugs is needed. Here, we review the latest progress of anti PECs drugs, focus on the mechanism of anti PECs reaction of drug components, and try to clarify new strategies for effective control and elimination of PECs. These comprehensive and profound insights will help to further investigate, prevent and control the transmission of PECs infection.

Fumonisins (FBs) are mycotoxins primarily synthesized by Fusarium moniliformis. Among these, FB1 exhibits not only high toxicity towards humans and animals but also carcinogenic properties. The global prevalence of FB1 contamination in cereals and related products, particularly maize, is alarmingly significant. Consequently, the accurate determination of FB1 levels in cereals holds immense importance. In this study, highly sensitive monoclonal antibodies specifically targeting FB1 were prepared and utilized for the establishment of a time-resolved fluorescence immunochromatographic assay (TRFIC) to detect FB1. The parameters of antibody labeling with time-resolved fluorescent microspheres were optimized. The detection time was significantly reduced to 6 min. The limits of detection (LOD) for corn, rice, and feed were determined as 0.496-0.844 μg/kg, and the quantification (LOQ) was 0.788-1.322 μg/kg. In addition, an indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) was successfully developed. Under optimized conditions, the half inhibitory concentration (IC50) value for FB1 was determined as 2.137 μg/L. A strong correlation between the results obtained from these two methods and HPLC-MS/MS analysis was observed in the same samples tested. In conclusion, both immunological methods developed in this work are highly suitable for rapid FB1 detection in real field samples.

The lack of new drugs that are effective against antibiotic-resistant bacteria has caused increasing concern in global public health. Based on this study, we report development of a modified antimicrobial drug through structure-based drug design (SBDD) and modular synthesis. The optimal modified compound, F8, was identified, which demonstrated in vitro and in vivo broad-spectrum antibacterial activity against drug-resistant bacteria and effectively mitigated the development of resistance. F8 exhibits significant bactericidal activity against bacteria resistant to antibiotics such as methicillin, polymyxin B, florfenicol (FLO), doxycycline, ampicillin and sulfamethoxazole. In a mouse model of drug-resistant bacteremia, F8 was found to increase survival and significantly reduce bacterial load in infected mice. Multi-omics analysis (transcriptomics, proteomics, and metabolomics) have indicated that ornithine carbamoyl transferase (arcB) is a antimicrobial target of F8. Further molecular docking, Isothermal Titration Calorimetry (ITC), and Differential Scanning Fluorimetry (DSF) studies verified arcB as a effective target for F8. Finally, mechanistic studies suggest that F8 competitively binds to arcB, disrupting the bacterial cell membrane and inducing a certain degree of oxidative damage. Here, we report F8 as a promising candidate drug for the development of antibiotic formulations to combat antibiotic-resistant bacteria-associated infections.

Porcine epidemic diarrhea virus (PEDV) remains one of the major causative microorganisms of viral diarrhea in piglets worldwide, with no approved drugs for treatment. We identified a natural molecule, flavonol, which is widely found in tea, vegetables and herbs. Subsequently, the antiviral activity of compound flavonol was evaluated in Vero cells and IPEC-J2 cells, and its anti-PEDV mechanism was analyzed by molecular docking and molecular dynamics. The results showed that flavonol could effectively inhibit viral progeny production, RNA synthesis and protein expression of PEDV strains in a dose-dependent manner. When flavonol was added simultaneously with viral infection in Vero cells, it demonstrated potent anti-PEDV activity by affecting the viral attachment and internalization phases. Similarly, in IPEC-J2 cells, flavonol effectively inhibited PEDV infection at different stages of infection, except for the release phase. Moreover, flavonol mainly interacts with PEDV Mpro through hydrogen bonds and hydrophobic forces, and the complex formed by it has high stability. Importantly, flavonol also showed broad-spectrum activity against other porcine enteric coronaviruses such as TGEV and PDCoV in vitro. These findings suggest that flavonol may exert antiviral effects by interacting with viral Mpro, thereby affecting viral replication. This means that flavonol is expected to become a potential drug to prevent or treat porcine enteric coronavirus.

1-Aminohydantoin (AHD), the residual marker of nitrofurantoin, is usually detected after derivatisation using the derivatisation reagent 2-nitrobenzaldehyde. Avoiding the antibody recognition of the derivatisation reagent is essential for the accurate detection of AHD residues. In this paper, a novel hapten called hapten D was designed, and then, a monoclonal antibody that did not recognise 2-nitrobenzaldehyde was prepared based on this novel hapten. An ultra-sensitive indirect competitive enzyme linked-immunosorbent assay (icELISA) was established under optimal conditions. The 50% inhibition concentration and limit of detection of AHD were 0.056 and 0.0060 ng/mL, respectively, which improved the sensitivity by 9-37-fold compared with the previously reported icELISA methods. The average recovery rates were 88.1%-97.3%, and the coefficient of variation was <8.6%. The accuracy and reliability of the icELISA were verified using liquid chromatography-tandem mass spectrometry. These results demonstrated that the developed icELISA is a useful and reliable tool.

Metamizole (MET) is an antipyretic and analgesic drug, the illegal use of which can result in residues of MET metabolites in edible tissues of animals. In this study, a computational chemistry-assisted hapten screening strategy was used to screen for the optimal immunogenic hapten-A and the optimal coating antigen hapten-G-OVA. A monoclonal antibody capable of recognizing two pharmacologically active metabolites of MET, 4-methylamidinoantipyrine (MAA) and 4-aminoantipyrine (AA), was prepared from the hapten-A. The antibody showed excellent specificity for MAA and AA and almost no cross-reactivity with the pharmacologically inactive metabolites 4-formamidinoantipyrine (FAA) and 4-acetamidinoantipyrine (AAA). An ic-ELISA was developed for the simultaneous detection of MAA and AA in animal-derived food, the limits of detection for MAA ranged from 0.93 to 1.18 μg/kg, while those for AA ranged from 1.74 to 4.61 μg/kg. The recovery rate fell within the range of 82 %-110 %, with a coefficient of variation less than 16.39 %.

Some fruits after long-term fermentation have anti-obesity function, however, the mechanisms have not been systematically determined. This study aimed to screen the effective fermented fruits and explore the mechanisms. C57BL/6J male mice were fed with a high fat diet (HFD) to establish an obese model and intervened with nine kinds of fermented fruits individually. Fermented fruits significantly lowered body weight gain (BWG), average daily feed intake (ADFI) and adipose tissue coefficient, and attenuated the hepatic steatosis. Serum triglyceride (TG) and total cholesterol (T-CHO) were significantly reduced, but leptin was remarkably increased. While the expressions of PPARα and CPT1 were upregulated, that of PPARγ and aP2 were downregulated. Additionally, fermented fruits improved the gut microflora structures of HFD-fed mice. This study suggested that fermented fruits, especially fermented blueberry and fermented apple, could ameliorate obesity by controlling food intake and regulating lipid metabolism and gut microbiota dysbiosis, potentially replacing anti-obesity drugs.

Quinoxalines are a class of veterinary drugs with antibacterial and growth-promoting functions. They are often widely used to treat and prevent animal diseases and are illegally used as animal growth promoters to increase economic benefits. Quinoxalines could be easily metabolized in animals to various residue markers and remain in animal-derived foods, which would pose a serious threat to human health. Consequently, it is necessary to detect the residues of quinoxalines and their metabolites. This article reviewed and evaluated immunoassays for quinoxalines and their metabolites in animal-derived foods, mainly including enzyme-linked immunosorbent assays, fluorescence immunosorbent assays, immunochromatography, and surface plasmon resonance biosensors. In addition, we deeply explored the design of haptens for quinoxalines and their metabolites and analyzed the effect of haptens on antibody performance. This paper aims to provide guidance and references for their accurate and sensitive detection, thereby ensuring food safety and human public health.

This is the first report on the screening, expression, and recognition mechanism analysis of single-chain fragment variable (scFv) against phenylethanolamine A (PEAA), a newly emerged β-adrenergic agonist illegally used as a feed additive for growth promotion. The PEAA-specific scFv scFv, called scFv-32, was screened from hybridoma cell lines by phage display and was found to be optimally expressed in the E. coli system. The ic-ELISA results revealed an IC 50 value of 10.34 μg/L for scFv-32 and no cross-reactivity with other β-adrenergic agonists. Homology modeling and molecular docking revealed the key binding sites VAL178, TYP228, and ASP229. One hydrogen bond, two pi-sigma bonds, and one pi-pi bond maintain the formation of the antibody‒drug complex. Alanine scanning mutagenesis of the three predicted key binding sites showed that the mutants completely lost their recognition activity, which confirmed the accuracy of the theoretical analysis. These results are valuable for the preparation of scFvs and the analysis of the molecular recognition mechanism of antigen-antibodies. Graphical abstract

Diethylstilbestrol (DES), a synthetic non-steroid estrogen, it has been prohibited from being added to animal feed for any purposes. Herein, an indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) based on a specific monoclonal antibody (mAb) was developed for the rapid screening of DES. Primarily, conjugates of mono-O-3-carboxypropyl diethylstilbestrol with keyhole limpet hemocyanin were used to raise a specific mAb, 1B7, which had IC50 value for DES of 213.0 ng L-1. The limits of detection and limits of quantification value for DES in animal-derived foods ranged from 68.1 to 103.1 ng L-1 and 100.8-192.7 ng L-1, respectively. The DES recovery ranged from 70.1 % to 103.1 %, with coefficients of variation below 13.9 %. A positive correlation (R2 = 0.997) was observed between the results of ic-ELISA and HPLC-MS/MS for milk. In order to inspect its detection effect, milk and animal-derived foods were chosen as the testing object. The results showed that this ic-ELISA method (specific mAb, 1B7) can effectively examine for DES residues.