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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.

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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.

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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.

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Contamination in food and the environment with fluoroquinolones (FQs) has become a serious threat to the global ecological balance and public health safety. Ofloxacin (OFL) is one of the most widely utilized sterilization agents in FQs. In the process of monitoring OFL, broad-spectrum monoclonal antibodies (mAb) cannot meet the demand for monospecific detection. Here, a computational chemistry-assisted hapten screening strategy was proposed in this study. Differences in the properties of antigenic epitopes were precisely extracted through a comprehensive comparative study of 16 common FQs molecules and a monospecific and ultrasensitive mAb-3B4 for OFL was successfully prepared. The screened fleroxacin (FLE) hapten was applied in a heterologous competition strategy resulting in a 20-fold improvement in the half inhibitory concentration (IC50) of mAb-3B4 to 0.0375 µg L-1 and cross-reacted only with marbofloxacin (MAR) in regulated FQs. In addition, a single-chain variable fragment (scFv) for OFL was constructed for the first time with an IC50 of 0.378 µg L-1. Molecular recognition mechanism studies validated the reliability of this strategy and revealed the key amino acid sites responsible for OFL specificity and sensitivity. Finally, ic-ELISA and GICA were established for OFL in real samples. This work provides new ideas for the preparation of monospecific mAb and improves the monitoring system of FQs.

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Pymetrozine is a neonicotinoid insecticide with high efficacy against aphids and planthoppers, and has been used worldwide. To monitor its residue in food, a highly specific and sensitive monoclonal antibody (McAb) was prepared, and an indirect competitive enzyme-linked immunosorbent assay (icELISA) was developed to detect pymetrozine, with a 50% inhibition value (IC50) of 7.70 µg/L. The McAb showed little affinity for acetamiprid, hexazinone, metamitron, nitenpyram, metribuzin, and imidacloprid. The limits of detection (LOD) calculated from the analysis of broccoli, cabbage, wheat, maize, rice, chicken, fish, and crayfish samples were from 1.56 to 2.72 µg/kg and the average recoveries were from 81.25 to 103.19%. icELISA was confirmed using liquid chromatography-tandem mass spectrometry (LC-MS/MS). These results demonstrated that the optimised icELISA is a convenient and effective analytical tool for monitoring pymetrozine residues in food.

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Amantadine (AMD) is an antiviral drug that is prohibited for use in livestock and poultry. In this study, carboxyl-modified magnetic nanoparticles (MNPs) were synthesized using the solvothermal method in one step with harmless and inexpensive regents, and they were used to label monoclonal antibodies (mAbs) of AMD in microwells with electrostatic adsorption. Then, a magnetic immunochromatography assay (MICA) method was successfully established. Under optimal conditions, the MICA showed a good performance, with a linear range of 0.2~10.0 µg/L. The limit of detection (LOD) was 0.068 µg/L with the instrument, and the visual LOD (vLOD) was 0.5 µg/L. There was no cross-reaction with rimantadine and ribavirin. The vLOD in real samples was 1.0 µg/kg. The developed MICA has the advantages of convenience, speed, and sensitivity, which make it suitable for the on-site rapid detection of AMD residues in chicken tissues and eggs.

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Polycyclic aromatic hydrocarbons (PAHs) are significant environmental and food pollutants that can cause cancer. In this work, a specific monoclonal antibody (mAb) to identify pyrene (PYR) and benzo [a]pyrene (BaP) was prepared, and an indirect competitive enzyme-linked immunoassay (ic-ELISA) was established to detect PYR and BaP residues in living aquatic products for the first time. The effects of complete antigens with different coupling ratios on the production of high-sensitivity mAb was explored. Under the optimal conditions, the IC50 value was 3.73 ± 0.43 µg/L (n = 5). The limits of detection (LODs) for PYR and BaP in fish, shrimp, and crab ranged from 0.43 to 0.98 µg/L. The average recoveries of the spiked samples ranged from 81.5-101.9%, and the coefficient of variation (CV) was less than 11.7%. The validation of the HPLC-FLD method indicated that the ELISA method set up in this experiment provided a trustworthy tool for PAHs residues detection in aquatic products.