Thiram (TMTD), a widely used dithiocarbamate fungicide, provokes oxidative hepatotoxicity, yet practical strategies for its detection and immunoprotection remain scarce. Here we develop an antibody-enabled framework integrating detection, therapy, and prevention against TMTD. In the murine model, we characterized the dose-dependent hepatic injury induced by TMTD. Guided by rational C3/C7 hapten design, we then generate a monoclonal antibody with high affinity. Building on this reagent, we create a portable immunodetection platform that couples Au nanoflowers with an Au/Ir@Zn/Cu-MOF probe, achieving an IC50 of 0.246 µg/mL. To evaluate protective efficacy, we demonstrated that passive immunization conferred a clear time-dependent therapeutic benefit: mortality decreased from 37.5% in the TMTD-poisoned group to 0% with treatment at 15 or 30 min and to 12.5% with treatment at 60 min, while improving body weight, liver function, and oxidative-stress markers. Complementarily, active immunization with TMTD-BSA elicited high antibody titers and conferred complete survival with graded histopathological protection. Together, this work offers an integrated detection-to-protection strategy that effectively bridges immunodetection with immune protection against TMTD-induced toxicity.

Sulfamethoxazole (SMX) is a widely used sulfonamide antibiotic, and its residues in animal-derived foods pose significant health risks. In this study, a high-affinity monoclonal antibody (2.65 × 1010 L/mol) against SMX was developed and used to construct an Au/Ir@Zn/Cu-MOF-based lateral flow immunoassay (LFIA). The probe was thoroughly characterized with good stability. The LFIA achieved a visual detection limit of 2.5 μg/mL, with an IC50 of 565.84 ng/mL and a linear detection range of 6.8 ng/mL-47.06 μg/mL. No cross-reactivity to structural analogues or matrix interference was observed in real food samples. Recovery rates (97.92-108%) were consistent with LC-MS results, confirming the accuracy of the method. This LFIA provides a sensitive and reliable tool for rapid SMX screening in food products.

Acifluorfen (AFF), a widely used diphenyl ether herbicide, raises significant environmental concerns due to its persistence and ecotoxicological risks, but the absence of immunoassay methods hinder efficient monitoring. A hybridoma cell line secreting a monoclonal antibody against AFF was developed via hybridoma technology to address this need. Using this antibody, we developed two lateral flow immunoassays (LFIAs) based on gold nanoparticles (AuNP-LFIA) and gold nanoflowers (AuNF-LFIA). The AuNP-LFIA exhibited a limit of detection (LOD) of 75.01 ng/mL. In contrast, the AuNF-LFIA demonstrated significantly enhanced sensitivity, achieving an LOD as low as 7.79 ng/mL, which represents a 9.6-fold improvement over the AuNP-LFIA. When applied to fruit and vegetable samples, both methods showed spike recovery rates ranging from 92.58% to 108.41%, and the results were in good agreement with those obtained by LC-MS. These findings suggest that the developed assays hold strong potential as reliable tools for the on-site detection of AFF.

INTRODUCTION:Sulfamethazine (SM2) antibiotic residues in food pose a significant risk to human health, including pathogenic bacterial resistance, allergic reactions, and carcinogenicity. Current lateral flow immunoassay (LFIA) typically employing colloidal gold (AuNP) as a signal reporter exhibits unsatisfactory sensitivity. Recently, metal-organic frameworks (MOFs) nanomaterial as a supporting matrix for loading nanoparticles provides a powerful tool to improve the performance of LFIA. OBJECTIVES:This study aims to develop an enhanced immunosensor based on antibody labeled Au/Ir@Cu/Zn-MOF signal amplification nano-composite for sensitive and point-of-care monitoring of SM2 antibiotic residues. METHODS:Firstly, the highly specific monoclonal antibody (mAb) against SM2 belonging to the IgG1 subclass was screened. Subsequently, the Au/Ir@Cu/Zn-MOF nanocomposite was synthesized by incorporating Au/Ir bimetallic nanoparticles into the previously prepared Cu/Zn-MOF composite. The anti-SM2 mAb was then conjugated to Au/Ir@Cu/Zn-MOF nanocomposite via a co-precipitation method to fabricate the mAb-Au/Ir@Cu/Zn-MOF probe. Finally, a lateral flow immunoassay was constructed for the detection of SM2 residues. RESULTS:The vLOD the Au/Ir@Cu/Zn-MOF-based LFIA for SM2 detection was approximately 2.5 ng/mL, which was at least 600 times greater than that of the AuNP-based LFIA (with the vLOD of 1563 ng/mL). Average recovery rates were 80.08-90.8% in milk sample, 88-89.3% in eel sample and 85.3-95.2% in pork sample, respectively. CONCLUSION:The Au/Ir@Cu/Zn-MOF nanocomposite utilized as signal amplification probe demonstrated remarkable sensitivity for the detection of SM2. The developed Au/Ir@Cu/Zn-MOF-based LFIA exhibits significant potential for the rapid and sensitive detection of contaminants in environmental monitoring and food safety.

Mad2, a conserved core component of the spindle assembly checkpoint (SAC) in eukaryotes, delays anaphase onset in case of incorrect kinetochore-microtubule attachment. However, its functions in plant-pathogenic fungi remain largely unknown. Here, we identified the Mad2 homologue in rice blast fungus Magnaporthe oryzae (MoMad2), which shows high similarity with Mad2 in fission yeast. When expressed in fission yeast, MoMad2 associated with native SpMad1 and SpCdc20, and successfully rescued the ΔSpmad2 mutant's defect in arresting anaphase onset upon damaged spindle, indicating the conserved SAC function of MoMad2. Moreover, MoMad2 interacted with MoMad1 and depends on MoMad1 for its nuclear envelope-localisation. Although it plays a dispensable role in M. oryzae growth, MoMad2 is required for tolerance to the microtubule depolymerising agent treatment. ΔMomad2 mutants exhibited shorter hyphal compartments and earlier conidial germination and appressorium formation, suggesting that MoMad2 deletion shortens M. oryzae's mitotic cell cycle due to defective SAC arrest. Additionally, knockout of MoMAD2 decreased the appressorial turgor pressure, impaired appressorium penetration and compromised M. oryzae pathogenicity. Taken together, our findings revealed that MoMad2, as a conserved component in SAC signalling, is essential for full pathogenicity of rice blast fungus.

Flumequine (Flu) is a fluoroquinolone veterinary antibiotic, which is easy to accumulate in animals, and Flu residues may cause a latent risk to human physiological health. In this study, a high-affinity monoclonal antibody (2.09 × 109 M-1) to specifically recognize Flu (anti-Flu mAb) was prepared, and then the lateral flow immunochromatographic strips (LFIS) with excellent sensitivity and specificity were developed by combining mAbs with nanoparticles. The linear detection ranges of LFIS based on gold particles (AuNP-LFIS) and gold nanoflowers (AuNF-LFIS) were 1.95-250 ng/mL and 0.39-100 ng/mL, respectively, and two types of LFIS showed high sensitivity and accuracy in actual sample detection. It is noteworthy that the AuNF-LFIS exhibited a higher sensitivity compared to that of the AuNP-LFIS. The LFIS developed in this study were considered to have great application potential in monitoring food safety because of its superiorities including simple operation, high sensitivity, prompted speed and good specificity.

Moniliformin (MON) is a toxic secondary metabolite from Fusarium species. The natural contamination of MON in cereals and cereal by-products, poses a risk of exposure to MON. However, so far, no immunoassay method has been reported to detect MON in field samples. In this study, anti-MON monoclonal antibody (mAb) with high affinity (1 × 108) and isotype of IgG1 was subjected to establish indirect competitive ELISA (ic-ELISA) and immunochromatographic test strips (ICTS). The optimized ic-ELISA resulted in the lowest detection limit (LOD) of 1.55 μg/mL. The LOD value for gold nanoparticles (AuNP) -based test strip was 0.83 μg/mL. The multi branched gold nano-flower particles (AuNFs) reported higher sensitivity with LOD of 0.38 μg/mL. Moreover, the developed test strips exhibited a high specificity without cross-reactivity to related competitive toxins. In conclusion, immunoassay methods were successfully developed to detect MON in field samples with a short detection time to ensure food security.

Due to the severe hazard of aflatoxins (AFs) to humans, it is of great significance to detect the key aflatoxins, aflatoxin B1 (AFB1) and aflatoxin G1 (AFG1), in food and feed in simple, rapid, and semi-quantitative ways. The hybridoma clone 3A1 was prepared in this study, and anti-AFB1 monoclonal antibody (mAb) with high specificity and affinity (9.38 × 108 L/mol) from 3A1 was purified. The indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) demonstrated that the linear detection range for AFB1 was 0.029–1.526 ng/mL with a limits of determination (LOD) of 0.023 ng/mL. A latex microsphere-based immunochromatographic test strip (LM-ICTS) was constructed based on 3A1, which showed that the strip could detect AFB1 (LOD: lower than 1.79 ng/mL) and AFG1 (LOD: lower than 8.08 ng/mL), and the linear detection ranges for AFB1 and AFG1 are 1.79–48.46 ng/mL and 8.08–107.40 ng/mL, respectively. The average recoveries of intra-assay and inter-assay for peanuts were (98.4 ± 4.7)% and (92.6 ± 7.6)%, and the average coefficient of variation (CVs) were 4.38% and 8.15%, respectively. For sunflower seeds, the intra-assay and inter-assay recoveries were (94.4 ± 7.2)% and (89.2 ± 4.3)%, and the average CVs were 6.6% and 4.9%, respectively. In summary, the developed LM-ICTS exhibited excellent sensitivity and specificity, which provided a rapidly stable on-site detection choice for AFB1 and AFG1 to contaminated agricultural samples, including grain and feed.

Highly selective herbicide quinclorac (Qui) is a type of quinoline carboxylic acid hormone herbicide, which has the characteristics of long half-life and difficulty for degradation, causing high risk to the environmental safety. In this study, anti-Qui 8A3 monoclonal antibody (mAb) with good specificity and high affinity (3.89 × 109 L/mol) was prepared, and two kinds of lateral flow immunochromatographic strips (LFICS) including nano-flower nanoparticles (AuNF) - and latex microsphere (LM)- based LFICS were established based on the antibody and signal amplification. The linear range of the AuNF- and LM- based LFICS were 5.31-345.48 ng/mL and 2.52-257.92 ng/mL, respectively. The limit of detection (LOD) of the AuNF- and LM- based LFICS were determined to be 5.31 ng/mL and 2.52 ng/mL, respectively. In summary, the developed LFICS using AuNF and LM as signal amplification reporters exhibited excellent sensitivity and provided the rapid on-site screening of Qui and other analytes in food safety field.

Bisphenol A has been reported to be a ubiquitous contaminant, and exposure to this compound can lead to adverse effects in human health. In the study, monoclonal antibody against BPA (anti-BPA mAb) with high affinity (3.74 × 109 L/mol) secreted by cell line 2E3 was successfully screened. Inspired by the signal amplification of nanoparticles, anti-BPA mAbs were labeled with nano-materials including colloidal gold (AuNP) and gold nanoflowers (AuNF) for preparation of immunoprobes and AuNP-/AuNF-based test strips. The developed AuNP- and AuNF-based test strips achieved the rapid and sensitive detection of BPA within 10 min, with the limit of detection (LOD) of 25 μg/mL and 3.125 μg/mL, respectively. The detection result in BPA spiked samples measured by the proposed methods was consistent with that detected by LC-MS method. The preparation process of as-prepared test strip is time-saving and considered as ideal candidates method for rapid screening BPA in real samples.

The spindle assembly checkpoint (SAC) proteins are conserved among eukaryotes safeguarding chromosome segregation fidelity during mitosis. However, their biological functions in plant-pathogenic fungi remain largely unknown. In this study, we found that the SAC protein MoMad1 in rice blast fungus (Magnaporthe oryzae) localizes on the nuclear envelope and is dispensable for M. oryzae vegetative growth and tolerance to microtubule depolymerizing agent treatment. MoMad1 plays an important role in M. oryzae infection-related development and pathogenicity. The monopolar spindle 1 homologue in M. oryzae (MoMps1) interacts with MoMad1 through its N-terminal domain and phosphorylates MoMad1 at Ser-18, which is conserved within the extended N termini of Mad1s from fungal plant pathogens. This phosphorylation is required for maintaining MoMad1 protein abundance and M. oryzae full virulence. Similar to the deletion of MoMad1, treatment with Mps1-IN-1 (an Mps1 inhibitor) caused compromised appressorium formation and decreased M. oryzae virulence, and these defects were dependent on its attenuating MoMad1 Ser-18 phosphorylation. Therefore, our study indicates the function of Mad1 in rice blast fungal pathogenicity and sheds light on the potential of blocking Mad1 phosphorylation by Mps1 to control crop fungal diseases.

Tetrodotoxin (TTX) could result in serious diseases due to its extremely high neurotoxicity. Thus, it is of great importance to measure TTX for food safety. In this study, an anti-TTX monoclonal antibody with good specificity and high affinity was used to develop the immunochromatographic test strips (ICTS). Gold nanoflower (AuNF) with multiple branches and latex microsphere (LM) with large particle size as signal reporters were employed for improving the sensitivity of test strips. Both AuNF and LM probes are stable, and the developed ICTS were specific to TTX, demonstrating no cross-reactivity with other marine toxins. The linear range of AuNF- and LM-based strips for TTX was 9.49-330.98 ng/mL and 5.40-443.19 ng/mL, respectively. The limit of detection (LOD) of AuNF- and LM-based strips was determined to be 9.49 ng/mL and 5.40 ng/mL, respectively. In summary, the developed ICTS based on AuNF and LM signal probes displayed enhancement of sensitivity and provided rapid and specific detection of TTX.

Background The use of beneficial microorganisms as an alternative for pest control has gained increasing attention. The objective of this study was to screen beneficial rhizosphere bacteria with the ability to enhance tomato anti-herbivore resistance. Results Rhizosphere bacteria in tomato field from Fuqing, one of the four locations where rhizosphere bacteria were collected in Fujian, China, enhanced tomato resistance against the tobacco cutworm Spodoptera litura, an important polyphagous pest. Inoculation with the isolate T6-4 obtained from the rhizosphere of tomato field in Fuqing reduced leaf damage and weight gain of S. litura larvae fed on the leaves of inoculated tomato plants by 27% in relative to control. Analysis of 16S rRNA gene sequence identities indicated that the isolate T6-4 was closely related to Stenotrophomonas rhizophila supported with 99.37% sequence similarity. In the presence of S. litura infestation, inoculation with the bacterium led to increases by a 66.9% increase in protease inhibitor activity, 53% in peroxidase activity and 80% in polyphenol oxidase activity in the leaves of inoculated plants as compared to the un-inoculated control. Moreover, the expression levels of defense-related genes encoding allene oxide cyclase (AOC), allene oxide synthase (AOS), lipoxygenase D (LOXD) and proteinase inhibitor (PI-II) in tomato leaves were induced 2.2-, 1.7-, 1.4- and 2.7-fold, respectively by T6-4 inoculation. Conclusion These results showed that the tomato rhizosphere soils harbor beneficial bacteria that can systemically induce jasmonate-dependent anti-herbivore resistance in tomato plants.

Mercury ion (Hg2+) as a major environmental pollutant threatens human health even at very low concentrations, so it is essential to monitor mercury residues in food. In this study, Hg2+ was conjugated with protein carrier using 1-(4-Isothiocyanobenzyl) ethylenediamine N, N, N ', N '-tetraacetic acid (ITCBE) as a bifunctional chelator. 7A1 monoclonal antibody (mAb) against Hg2+-ITCBE with high affinity (7.3 x 109 L/moL) and good specificity was obtained by cell fusion technology and performed to establish immunosensors. Immunochromatographic test strip using colloidal gold nanoparticles (AuNP with an average diameter of 18 nm) as signal reporter showed low sensitivity. Signal amplification probes including larger multi-branched gold nanoflowers (AuNF) and latex microspheres (LM) were employed to enhance the sensitivity of immunosensors. The visible limit of detection (vLOD) of the AuNF-and LM-based strip were determined to be 50 ng/mL and 25 ng/mL respectively, showing more sensitive than that of AuNP-based strip (200 ng/mL). Quantitative analysis showed that AuNF-based strip exhibited lower quantitative limit of detection (qLOD) (0.44 ng/mL) which was 20-fold lower than that of AuNP-based strip (8.92 ng/mL) for determination of Hg2+, and LM-based strip (0.49 ng/mL) was 18 times as sensitive as AuNP-based strip. In summary, the developed immunosensors using AuNF and LM as signal amplification probes exhibited excellent sensitivity and provided portable, on-site detection for Hg2+.

Tenuazonic acid (TA) is a highly toxic mycotoxin mainly generated by the fungi of Alternaria genus and widely contaminates agricultural by-products. Given the threat of TA to food-security, it is very important to develop rapid and sensitive detection methods for TA monitoring. In this study, gold nano-particles (AuNP) with average diameter near 17.25 nm were prepared, and the developed AuNP-based strip has an assay time of 15 min with visual limit of detection (LOD) of 12.5 ng/ml and threshold of 100 ng/ml. To further improve sensitivity, multi-branched gold nano-flowers (AuNF) with average diameter near 50 nm were prepared and characterized by UV-VIS and TEM, and the established AuNF-based strip has visual LOD of 0.78 ng/ml and threshold of 50 ng/ml within 15 min. Both assays were applied to determine TA in apple juice and tomato ketchup, and the results were consistent with that of UHPLC-MS/MS. Thus, these assays could be applied for rapid determination of trace TA in real samples.

Given the application of αB-VxXXIVA-conotoxin (αB-CTX) in analgesics and cancer chemotherapeutics, and its threat to humans, it is urgent to develop a rapid, effective and accurate method for the analysis and detection of αB-CTX in real shellfish and medicine drug samples. In the present study, two different immunochromatographic strips were established for αB-CTX detection, based on the monoclonal antibody 5E4 against αB-CTX, and the visual limits of detection (vLOD) for the colloidal gold nanoparticles-based strip (AuNPs-based strip) and nanoflowers-based strip (AuNFs-based strip) were 4 μg/mL and 1.5 μg/mL, respectively. The developed AuNPs-/AuNFs-based strips have good specificity and accuracy, and the detection results were analyzed in less than 10 min, without using an instrument. In view of the excellent repeatability and usability, the established methods could be applied to detect and analyze the content of αB-CTX in real samples.

Lead (Pb) threatens public health due to its toxicity and nonbiodegradable characteristics. It is of significance to develop a sensitive and rapid method for Pb detection. In this study, monoclonal antibodies against Pb were screened with a high affinity constant (Kaff) of 3.56 × 109 L/mol. Au nanosphere particles (AuNS) and Au nanoflower particles (AuNF) were synthesized with a diameter of 15 nm and 60 nm, respectively. The specific anti-Pb antibodies were then immobilized on AuNS and AuNF for probe development. At last, AuNS- and AuNF-based strips were successfully assembled for comparative study, which were able to effectively detect environmental Pb in 10 min. The limits of detection (LODs) were determined to be 3.91 ng/ml and 0.2 ng/ml, respectively. Thus the developed method provides a feasible solution for sensitive and rapid detection of Pb on site, which is beneficial to food safety and pollution control.

In view of the toxicological hazard and important applications in analgesics and cancer chemotherapeutics of αB-CTX, it is urgent to develop an accurate, effective and feasible immunoassay for the determination and analysis of αB-CTX in real samples. In this study, MBP-αB-CTX4 tandem fusion protein was used as an immunogen to elicit a strong immune response, and a hybridoma cell 5E4 secreting IgG2b against αB-CTX was successfully screened by hybridoma technology. The affinity of the purified 5E4 monoclonal antibody (mAb) was 1.02 × 108 L/mol, which showed high affinity and specificity to αB-CTX. Epitope 1 of αB-CTX is the major binding region for 5E4 mAb recongnization, and two amino acid residues (14L and 15F) in αB-CTX were critical sites for the interaction between αB-CTX and 5E4 mAb. Indirect competitive ELISA (ic-ELISA) based on 5E4 mAb was developed to detect and analyze αB-CTX in real samples, and the linear range of ic-ELISA to αB-CTX was 117–3798 ng/mL, with a limit of detection (LOD) of 81 ng/mL. All the above results indicated that the developed ic-ELISA had high accuracy and repeatability, and it could be applied for αB-CTX detection and drug analysis in real samples.

Due to the threat of tenuazonic acid (TA) to public health, it is urgent to establish rapidly effective and sensitive assay methods for TA. In this study, a TA-specific IgG monoclonal antibody (McAb) with high affinity (Kaff was 1.72 x 1010 L/mol) was screened, and the developed icELISA for TA detection has IC50 of 2.50 ng/mL and LOD of 0.17 ng/mL. Platinum-modified gold nanoparticle (Au@PtNP) was optimized as Au@Pt0.4NP, and the resulted Au@Pt0.4NP-McAb probe was designed to catalyze precipitation-type tetramethylbenzidine for visual detection of trace TA with visual LOD of 0.39 ng/mL. The sensitivity of this established Au@Pt0.4NP-McAb strip was highly increased when compared with the existing colloidal gold strip. The developed strip was used to detect trace TA in apple juice and tomato ketchup which were consistent with the results from UHPLC-MS/MS. Therefore, this developed strip could be used for rapid detection of trace TA in real samples.

The functions of ubiquitin-conjugating enzymes (E2) in plant immunity are not well understood. In this study, OsUBC26, a rice ubiquitin-conjugating enzyme, was characterized in the defence against Magnaporthe oryzae. The expression of OsUBC26 was induced by M. oryzae inoculation and methyl jasmonate treatment. Both RNA interference lines and CRISPR/Cas9 null mutants of OsUBC26 reduced rice resistance to M. oryzae. WRKY45 was down-regulated in OsUBC26 null mutants. In vitro E2 activity assay indicated that OsUBC26 is an active ubiquitin-conjugating enzyme. Yeast two-hybrid assays using OsUBC26 as bait identified the RING-type E3 ligase UCIP2 as an interacting protein. Coimmunoprecipitation assays confirmed the interaction between OsUBC26 and UCIP2. The CRISPR/Cas9 mutants of UCIP2 also showed compromised resistance to M. oryzae. Yeast two-hybrid screening using UCIP2 as bait revealed that APIP6 is a binding partner of UCIP2. Moreover, OsUBC26 working with APIP6 ubiquitinateds AvrPiz-t, an avirulence effector of M. oryzae, and OsUBC26 null mutation impaired the proteasome degradation of AvrPiz-t in rice cells. In summary, OsUBC26 plays important roles in rice disease resistance by regulating WRKY45 expression and working with E3 ligases such as APIP6 to counteract the effector protein AvrPiz-t from M. oryzae.