Multi-residue colloidal gold lateral flow immunoassays (CG-LFIAs) play a critical role in high-throughput, rapid, and customized food safety testing. In this study, a systematic strategy for constructing multi-residue CG-LFIAs was developed, using chlorantraniliprole (CTP), emamectin benzoate (EMB), and fipronil (FEN) as model analytes. A stepwise workflow, progressing from single-strip to multiple T lines, was employed to systematically optimize key factors affecting assay performance, including antibody labeling, T line spatial arrangement, membrane treatment, absorbent pad length, buffer composition, pH, reaction time, and chromatographic time, in order to balance multi-analyte interactions and reduce cross-interference. Matrix-specific sample pretreatment further enhanced assay robustness and reproducibility. The resulting multi-residue LFIA demonstrated satisfactory sensitivity, precision, and matrix tolerance, enabling simultaneous detection of multiple chemical pesticides in complex plant- and animal-derived matrices, including cowpea, chicken, egg, and milk. The proposed strategy provides a general framework for developing multi‑residue LFIA platforms, improving analytical efficiency and offering a promising, systematically validated method that could serve as a practical on‑site tool for rapid pesticide residue monitoring in complex food systems.

The feed additive monensin (MON) poses consumer health risks, requiring sensitive detection. Thus, we developed a two-step endoplasmic reticulum-directed antibody-secreting cell selection (ER-DAS) platform for rapid isolation of high-affinity monoclonal antibodies, overcoming the key limitations of the lengthy screening cycles of hybridoma technology and low yield of conventional single B-cell approaches. The ER-DAS achieves 48-h isolation of MON-specific antibody-secreting cells (ASCs) through integrated fluorescence-activated cell sorting (FACS) enrichment and nanopore-array imaging technology. Using this platform, a novel high-affinity recombinant rabbit monoclonal antibody (RmAb) was successfully produced. Molecular docking showed that RmAb 3G interacts with MON primarily through π-π stacking and hydrogen bonding. A convenient and cost-effective immunoassay based on RmAb 3G exhibited an IC50 of 0.55 ng mL-1, lower than previously reported antibodies, exceptional salt and organic solvent tolerance, and 88.7-97.1% spike recoveries in raw milk. This work provides a streamlined workflow for efficient antibody development in food safety monitoring.

To address the challenge of detecting monensin (MON) residues, this study combined FACS-based enrichment of MON-specific memory B cells (MBCs) with rabbit single-cell BCR sequencing (scBCR-seq) to characterize the rabbit BCR immune repertoire. By analyzing somatic hypermutation (SHM) rates, germline gene usage bias, and CDR3 features, we identified 12 dominant rabbit monoclonal antibodies (RmAbs) from 15 paired heavy- and light-chain sequences, achieving an 80% positive screening rate. Requiring only one week from cell sorting to antibody production, this two-step single B-cell sorting strategy significantly improves screening efficiency and positive clone rates compared with traditional hybridoma technology and existing single-cell approaches. Molecular dynamics simulations showed that RmAb T40-Fv-MON complex maintains structural stability under high ionic strength and extreme pH conditions. Using RmAb T40, we developed a sensitive, efficient, and fielddeployable time-resolved fluorescence immunoassay (TRFIA). The method achieved spiked recoveries of 88.1%-107.1% and coefficients of variation below 15% in bovine tissues and raw milk samples. This work not only provides a key reagent for MON residue detection but also establishes a universal platform for antibody development against small-molecule contaminants.

This study was performed to investigate and compare the pharmacokinetic characteristics of tylvalosin tartrate in broiler chickens following oral administration of a nanocrystal suspension (PO-NM) or a soluble powder formulation (PO-SP), with intravenous administration (IV) of tylvalosin tartrate serving as the reference standard. A total of 30 healthy broiler chickens were randomly allocated into three groups (PO-NM, PO-SP, and IV; n = 10). Tylvalosin was administered at a dose of 25 mg/kg body weight (BW), and blood samples were collected at multiple time points from 0 to 24 h post-administration. Plasma concentrations of tylvalosin were quantified using a validated ultra-performance liquid chromatography–tandem mass spectrometry (UPLC-MS/MS) method, and pharmacokinetic parameters were calculated using non-compartmental analysis. The results showed no significant differences in the terminal elimination half-life (t1/2λz) and mean residence time (MRT) between the two oral formulations. However, the time to maximum concentration (Tmax) of PO-NM (0.71 ± 0.09 h) was significantly shorter than that of PO-SP (1.42 ± 0.18 h) (p < 0.05), while the maximum plasma concentration (Cmax) of PO-NM (255.52 ± 111.88 ng/mL) was markedly higher than that of PO-SP (120.45 ± 45.82 ng/mL) (p < 0.05). Furthermore, the absolute bioavailability (F) of PO-NM (15.73 ± 4.29%) showed a modest increase compared with PO-SP (11.45 ± 4.66%); however, this difference did not reach statistical significance. Collectively, these findings demonstrate that the PO-NM formulation achieved faster absorption, higher peak plasma levels, and greater systemic exposure compared with PO-SP, without significantly altering the elimination process. Overall, nanoparticle formulation appears to enhance the oral pharmacokinetic performance of tylvalosin in broiler chickens, potentially reducing residue risks and offering substantial application value in poultry medicine.

Monensin is widely used in livestock and poultry for disease prevention and growth promotion. Still, its improper use can lead to residues in animal-derived foods, posing risks to human health. In immunoassays, monoclonal antibodies (mAbs) remain a preferred choice due to their high sensitivity and specificity. However, traditional hybridoma technology often suffers from a lengthy screening cycle and the risk of losing effective clones. To address these limitations, this study employed fluorescently labeled monensin antigens combined with fluorescence-activated cell sorting (FACS) for rapid screening of hybridoma cells secreting anti-monensin mAbs. Compared to the conventional limiting dilution method, this approach increased the yield of specific hybridoma cells by tenfold and reduced the screening cycle from four weeks to one week. Based on the selected mAb (4E6), a rapid visual lateral flow immunoassay (LFIA) was developed for on-site detection of monensin, with a total detection time of 10 min. The assay exhibited a half-maximal inhibitory concentration (IC50) of 2.32 μg/kg and a linear detection range of 0.32-10.3 μg/kg. This study provides a promising strategy for efficient hybridoma cell screening and practical monitoring of monensin residues in environmental and food samples.

A colloidal gold immunochromatographic assay (CGIA) based on single-chain variable fragments (scFvs) has been successfully developed for the detection of monensin (MON). Colloidal gold probes were conjugated to antiMON scFvs through electrostatic interaction, with the conjugated objects serving as the visual signals. The detection lines were formed by capturing the antibody with MON-OVA. This assay offers a rapid detection time of 15 min, a wide linear range from 2.19 to 10.76 ng mL- 1, and boasts high accuracy, precision, and an absence of cross-reactivity. By homology modeling and molecular docking, we predicted the interaction patterns between the scFv and monensin, and the amino acid residues involved in the recognition of MON by the antibody were analyzed. These key amino acid sites are presumed integral to ligand recognition per current interaction models. This hypothesis was confirmed by computer-aided alanine scanning mutation, MM/P(G)BSA molecular dynamics simulation, and in vitro binding experiments. In this study, we successfully developed the scFvs-based CGIA system for rapid and easy quantification of monensin, providing a simple, efficient routine detection of chicken muscle samples.

Maduramicin (MAD) and salinomycin (SAL) are the widely used poly(ether ionophore) antibiotics to control coccidiosis in animals. Due to their strong cytotoxicity, strict control over their dosage and residue in animal food is necessary. To improve the detection efficiency of the existing single-residue detection methods, a tetraploid tumor hybrid system was constructed using drug mutagenesis, and the bispecific monoclonal antibody (BsMAb) against MAD and SAL was obtained by hybridization-hybridoma technology. By optimizing the optimal working concentration of the tracer and antibody, a multiresidue fluorescence polarization immunoassay method based on BsMAb was successfully established. The whole detection process takes 10 min, and the LOD values of MAD and SAL were 4.71 and 3.49 ng·g-1, respectively. IC50 values were 6.45 and 6.24 ng·mL-1, respectively. There was no cross-reactivity with other polyether ionophore antibiotics. Finally, a breakthrough in detection was achieved: bispecific monoclonal antibody prepared by the hybridization-hybridoma technology was used to detect maduramicin and salinomycin.

The ultra-performance liquid chromatography with tandem mass spectrometry (UPLC-MS/MS) detection method was developed for the residues of 10 NSAIDs (salicylic acid, acetylsalicylic acid, acetaminophen, diclofenac, tolfenamic acid, antipyrine, flunixin meglumine, aminophenazone, meloxicam, metamizole sodium) in swine muscle, liver, kidney, and fat. Swine tissue samples were extracted by phosphorylated acetonitrile with the addition of an appropriate amount of internal standard working solution, defatted with acetonitrile-saturated n-hexane, and purified by Hydrophile-Lipophile Balance (HLB) solid-phase extraction column, then separated by UPLC BEH shield RP18 column with 0.1% formic acid in water/0.1% formic acid in acetonitrile with gradient elution, which was detected in the multiple reaction monitoring (MRM) modes. The correlation coefficient of the standard curve equation is greater than 0.99, and the coefficient of variation within and between batches is less than 14.4%. We evaluated the analytical method using two green assessment tools. The method established in this study met the requirements of NSAID residue analysis and provides analytical tools for determining and confirming NSAIDs in swine tissue samples. This is the first report on the simultaneous determination of 10 NSAIDs in four swine tissues by the UPLC-MS/MS method and accurate quantification using deuterated internal standards.

Maduramicin (MAD) and salinomycin (SAL) are widely used polyether ionophore antibiotics (PIAs) to control coccidiosis in animals. Due to their strong cytotoxicity, strict control over their dosage and residue in animal food is necessary. To improve the detection efficiency of existing single residue detection methods, a tetraploid tumor hybrid system was constructed using drug mutagenesis, and the bi-specific monoclonal antibody (BsMAb) against MAD and SAL was obtained by hybridization-hybridoma technology. By optimizing the optimal working concentration of tracer and antibody, a multi-residue fluorescence polarization immunoassay (FPIA) method based on BsMAb was successfully established. The detection limit (LOD) of MAD and SAL were 4.71 ng·mL-1 and 3.49 ng·mL-1, respectively. IC50 were 6.45 ng·mL-1 and 6.24 ng·mL-1, respectively. There was no cross-reactivity with other PIAs. To our knowledge, this was the first time to detect maduramicin and salinomycin in chicken by hybridization-hybridoma technique.

A rapid, reliable, sensitive, and multiplex colloidal gold immunochromatographic assay (CGIA) was developed for the simultaneous detection of three polyether ionophore antibiotics (PEs) in chicken muscle. The IC50 values of the optimized CGIA were 1.63, 1.94, and 1.18 ng/mL with a linear range of 0.87-3.04 ng/mL, 0.85-4.41 ng/mL, and 0.57-2.44 ng/mL for maduramicin (MAD), monensin (MON), and salinomycin (SAL), respectively. The cut-off values were 10, 5, and 5 ng/mL, respectively. The whole testing process was completed within 15 min. A parallel analysis in blind chicken muscle samples was conducted by liquid chromatography-tandem mass spectrometry (LC-MS/MS), the developed CGIA did not appear false-positive and false-negative. The results show that the established method could provide a rapid and effective approach for on-site screening and determination of PEs residues in chicken muscle samples.

Ractopamine (RAC) is a feed additive that regulates protein synthesis. In this study, an indirect enzyme-linked immunosorbent assay (icELISA) for the detection of RAC residues in the porcine liver was developed based on our specific immunomagnetic bead (IMB) coated with ractopamine-single chain variable fragment (RAC-scFv). The intact RAC-scFv was constructed, and its key amino acid sites were analyzed. Finally, an icELISA based on immunomagnetic beads separation techniques (IMBs) of RAC from the porcine liver was established with an IC50 of 9.15 ng/mL and a linear range from 4.8 to 17.4 ng / mL. The IC50 of the prepared RAC-scFv was 8.7 ng/mL with a linear range of 3.8 to 19.6 ng/mL. The limit of detection (LOD) in the porcine liver of RAC was 4.57 mu g/kg, and the recoveries ranged from 72.65 to 89.41% (CV < 15%).

莱克多巴胺(Ractopamine,RAC)是一种人工合成的β-肾上腺素能受体激动剂类化合物,属于第二代"瘦肉精".分别从传统仪器检测法和快速检测法及生物传感器技术三个方面综述了当前RAC在动物性产品中残留检测技术的研究进展,并对RAC检测技术的发展趋势进行了展望,旨在为RAC的残留监控提供方法学上的参考,为检测新方法的建立提供思路.

Salinomycin (SAL) and lasalocid (LAS) are widely used as ionophore antibiotics for coccidiosis control. However, their common use as feed additives has led to the occurrence of feed cross-contamination, which has toxic effects on non-target animals. There have been few reports on multiple-residue detection for SAL and LAS in recent years. In this study, two single-chain antibody fragments (scFvs) capable of specifically recognizing SAL and LAS were constructed. Using LAS-scFv and SAL-scFv as parent antibodies, a complete bispecific single-chain diabody (scDb) against both LAS and SAL was built using splicing by overlap extension polymerase chain reaction (SOE-PCR). In addition, the key amino acid sites and interaction energy of antibody variable regions for small-molecule recognition were preliminarily studied by homology modeling and molecular docking. Finally, IC50 values of 12.9 and 8.6 ng/mL, with a linear range of 6.9–24.0 and 4.7–16.0 ng/mL, were obtained for LAS-scFv and SAL-scFv, respectively. An indirect competitive enzyme-linked immunosorbent assay (icELISA) method was established using scDb to obtain an IC50 of 3.5 ng/mL for LAS and 4.1 ng/mL for SAL, which showed better sensitivity and specificity than those of the parent scFv antibodies. The recoveries of LAS and SAL in chicken liver were 89.2–92.7%(CV<4.7%) and 88.6–90.2% (CV<6.8%)), respectively.

抗球虫药物在畜禽业应用广泛,可预防治疗球虫病,提高家禽饲料转化率,提升肉品质.但是,随即会出现饲料交叉污染,对非目标动物产生毒性作用,动物性食品中药物残留超标等问题.因此,建立简便、快速、灵敏度高的检测方法检测动物源性食品中的抗球虫药物残留极为重要.基于抗原-抗体特异性结合原理的免疫分析检测技术能够完成快速检测,高通量筛选,具有特异性强、灵敏度高、操作简便、成本低的优点.本文综述了不同基质中抗球虫药物残留的免疫检测技术进展,重点介绍了酶联免疫吸附检测法、免疫层析法、荧光偏振免疫分析检测法、时间分辨荧光免疫分析检测法和生物传感器检测法.并对免疫检测技术在残留检测方面的发展趋势进行了展望,旨在为抗球虫药物的残留监控提供方法学上的参考,为新方法的建立提供思路.

An indirect competitive enzyme-linked immunosorbent assay (icELISA) based on immunomagnetic bead clean-up for detection of Maduramicin (MAD) was developed using a single-chain antibody (scFv). The single-chain antibody synthesized by genetic engineering technology was immobilized on the surface of carboxylic acid magnetic beads with a diameter of 2.8 μm. After simple extraction, the residue in the sample extract was specifically adsorbed, and the supernatant was removed by magnetic separation. The half-maximal inhibitory concentration (IC50) of 15.43 ng·ml−1 with a limit of detection of 6.31 ng·ml−1 for MAD in chicken muscle. The recovery ranged from 72.93 to 89.51% with variation coefficients in intraday and interday less than 15%. We improved the inhibition efficiency and sensitivity of this method compared with traditional hydrophilic-lipophilic balance column clean up icELISA. The convenience and repeatability of the immunomagnetic clean-up render the new method for the analysis of drug residues in complex matrices.

Herein, we developed a sensitive and quantitative flow assay for simultaneous detection of amantadine (AMD) and chloramphenicol (CAP) in chicken samples based on different CdSe/ZnS quantum dots (QDs). In contrast to other reports, the QDs could be excited by the same excitations that lowered the requirements for the matching instruments. Under the optimal conditions, the strategy permitted sensitive detection of AMD and CAP in a linear range of 0.23 to 1.02 ng/g and 0.02 to 0.66 ng/g. The limits of detection were 0.18 ng/g and 0.016 ng/g, respectively. Moreover, the whole detection process could be completed within 20 min with no additional sophisticated instruments and complicated operations. Spiked samples were analyzed using both QD-based lateral flow immunoassay (QD-LFIA) and commercial ELISA kits with good correlation (R2 = 0.96). Moreover, this study laid the foundation and simplified the development of the requisite instrument.

Diphacinone (DPN) is an extensively used anticoagulant rodenticide that is also considered a hazardous chemical, which poses a threat to nontarget species. DPN poisoning cases in humans or other species frequently occur, while rapid and sensitive detection methods are rarely reported. Thus, it is meaningful to develop an immunoassay for DPN detection with high sensitivity and specificity. In this study, a hapten was synthesized and then conjugated with carrier proteins to prepare the immunogens with different conjugation ratios for the preparation of antibody. After evaluation of the antisera using an indirect competitive enzyme-linked immunosorbent assay (icELISA) and statistical analysis, we found that the immunogen prepared using the N,N-dicyclohexylcarbodiimide (DCC) method with a conjugation ratio of 28.5 could elicit mice to generate antibodies with high performance. Using hybridoma technology, we obtained the specific monoclonal antibody (mAb) 4G5 with a half maximal inhibitory concentration (IC50) of 0.82 ng/mL in buffer solution. We initially explored the recognition mechanism of DPN/CLDPN and mAb from both conformational and electronic aspects. Then, mAb 4G5 was applied to develop icELISA for biological samples. The limits of detection (LODs) of icELISA were 0.28 μg/L, 0.32 μg/L, and 0.55 μg/kg for swine plasma, urine, and liver samples, respectively, and the recoveries ranged from 72.3 to 103.3% with a coefficient of variation (CV) of less than 12.3% in spiked samples. In summary, we developed a sensitive, specific, and accurate icELISA for the detection of DPN in biological samples, which showed potential in food safety analysis and clinical diagnosis.

In 2-(N-morpholino)ethanesulfonic acid buffer (0.1 M, pH 6.0), manganese dioxide-modified silicon dioxide (SiO2@MnO2) nanocomposites were facilely synthesized under ultrasonic conditions. Given their good bio-compatibility and evident brown color, SiO2@MnO2 nanocomposites were used as labels in immunochromatography sensor for high-throughput screening of antibiotic residues in milk within 10 min. Meanwhile, excessive amounts of antibiotic residues might cause certain diseases and inhibit the activities of glutathione reductase in human body through the food chain, altering glutathione (GSH) levels in the human serum. GSH can reduce MnO2 to Mn(II). This phenomenon can effectively quench the fluorescence of Au nanoclusters. Thus, a fluorescent platform based on SiO2@MnO2 nanocomposites and Au nanoclusters has been designed for sensitive detection of GSH in the human serum with a limit of detection of 1.23 nM. After the parameters were optimized, the developed detection platforms based on SiO2@MnO2 nanocomposites have been used to detect antibiotics and GSH in real samples (milk and human serum) with satisfactory results. Results demonstrated that the proposed SiO2@MnO2 nanocomposites hold great potential for applications in food safety and clinical diagnosis simultaneously.

<span id="ChDivSummary" name="ChDivSummary" class="abstract-text">为探索鸡组织中氢溴酸常山酮残留量及其消除规律,选取25～30日龄AA肉鸡100只,体重约1.0kg,按氢溴酸常山酮推荐剂量3mg/kg给药。于最后一次给药后第0d、6h、1d、2d、3d、5d和8d等每个时间点处死10只鸡,并采集每只鸡的胸肌、皮肤+脂肪、全肝和双肾,分别装入密封袋,做好标记-20℃保存。鸡组织试样经胰蛋白酶酶解,乙酸乙酯提取,正己烷脱脂,固相萃取净化,采用超高效液相色谱-串联质谱检测,外标法定量,并采用WT1.4软件处理,从而推算其休药期。结果表明:该方法检测限为1.5μg/kg,定量限为5.0μg/kg;4种组织的添加回收率为74.8%～91.1%,批内、批间变异系数均&lt;11.5%;残留检测结果表明肌肉和皮肤+脂肪组织的休药期为0d,肾脏组织的休药期为4d,肝脏组织的休药期为5d。因此,本研究中检测方法操作简单,灵敏度较高,可用于鸡组织中氢溴酸常山酮残留的检测和分析。考虑到饲养环境和个体差异等因素的影响,建议氢溴酸常山酮的休药期暂定为5d。</span>

Pyrenylbutyric acid and streptavidin were coupled to films of reduced graphene oxide (rGO) and then conjugated to a biotinylated broad-spectrum monoclonal antibody against aflatoxins (AFs). It is shown that such films can efficiently and selectively capture AFs inculding AFB1, AFB2, AFG1, AFG2, AFM1 and AFM2. The rGO films were characterized by using scanning electron microscopy, energy-dispersive spectroscopy, and raman spectroscopy. The selectivity and purification performance of the antibody-loaded rGO films were investigated. They were applied to the purification of extremely small samples (100 μL) of AFs-spiked rabbit serum after enzymatic hydrolysis. The AFs were analyzed by ultra-performance liquid chromatography coupled to tandem mass spectrometry. The limits of detection for the six AFs investigated ranged from 50 to 170 pg·mL−1. The average recoveries of AFs in spiked rabbit serum samples ranged from 55% to 75%, with relative standard deviations of less than 9.4%.