4 research outputs found

    Development of an indirect competitive ELISA based on immunomagnetic beads’ clean-up for detection of maduramicin in three chicken tissues

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    <p>An indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) based on immunomagnetic beads' (IMBs) clean-up was developed for detection of the residues of maduramicin (MD) in different chicken tissues. IMBs coated with a specific monoclonal antibody (MAb) against MD named MAb 2D6 were applied to enrich the residues of MD in chicken tissues. The specificities of these IMBs were well maintained and the reversibility remained at more than 73% of the original capability after being used for three times. After elution, enriched MD was detected by a conventional ic-ELISA. The limits of detection of MD were 72, 74 and 173 μg/kg in chicken muscle, skin and fat, and liver, respectively. Recoveries ranged from 80.0% to 115.8% with coefficients of variation being less than 11.3%. These results indicated that a rapid, robust clean-up of IMBs combining ELISA provides a simple, time-saving and environmentally friendly method to detect MD in chicken tissues.</p

    Presentation_1_Mucoid Acinetobacter baumannii enhances anti-phagocytosis through reducing C3b deposition.pdf

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    BackgroundMultidrug resistant (MDR) Acinetobacter baumannii causes serious infections in intensive care units and is hard to be eradicated by antibiotics. Many A. baumannii isolates are identified as the mucoid type recently, but the biological characteristics of mucoid A. baumannii and their interactions with host cells remains unclear.MethodsThe mucoid phenotype, antimicrobial susceptibility, biofilm-forming ability, acid resistance ability, peroxide tolerance, and in vivo toxicity of clinical ICUs derived A. baumannii isolates were first investigated. Secondly, the phagocytic resistance and invasive capacity of A. baumannii isolates to macrophages (MH-S, RAW264.7) and epithelial cells (A549) were analyzed. Furthermore, the abundance of C3b (complement factor C3 degradation product) deposition on the surface of A. baumannii was investigated. Last, the relationship between C3b deposition and the abundance of capsule in A. baumannii isolates were analyzed.ResultsThese A. baumannii strains showed different mucoid phenotypes including hyper mucoid (HM), medium mucoid (MM), and low mucoid (LM). All tested strains were MDR with high tolerance to either acid or hydrogen peroxide exposure. Notably, these mucoid strains showed the increase of mortality in the Galleria mellonella infection models. Besides, the HM strain exhibited less biofilm abundance, higher molecular weight (MW) of capsule, and greater anti-phagocytic activity to macrophages than the LM strain. Together with the increased abundance of capsule, high expression of tuf gene (associated with the hydrolysis of C3b), the HM strain effectively inhibits C3b deposition on bacterial surface, resulting in the low-opsonization phenotype.ConclusionCapsular characteristics facilitate the anti-phagocytic activity in hyper mucoid A. baumannii through the reduction of C3b deposition. Mucoid A. baumannii exhibits high phagocytosis resistance to both macrophages and epithelial cells.</p

    Production of Monoclonal Antibody and Development of a New Immunoassay for Apramycin in Food

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    Apramycin (APR) residue in food of animal origin can cause harmful effects on human health. In this study, a monoclonal antibody (mAb) was successfully produced using APR–BSA as immunogen, which was prepared by using the glutaraldehyde method. mAb 2A2 showed low cross-reactivity (<0.1%) with other aminoglycoside antibiotics, and its IC<sub>50</sub> value was 0.35 ng/mL. On the basis of this mAb, a novel immunoassay in the format of an immunoaffinity test column (IATC) was developed. An immunoaffinity column filled with anti-APR antibody–Sepharose 4B gel was used as solid phase. APR in sample and HRP–APR conjugate compete with each other for the limited antibody on the column. The approach was able to give a naked-eye color signal for the detection of analyte. A blue color appears for negative results and no color for positive. The method was then successfully applied to the detection of APR in animal-origin food. To further evaluate the assay, direct competitive ELISA (dcELISA) based on the same antibody was developed for comparison in different aspects. Compared to the dcELISA, the detection time of IATC is shortened to 20 min, whereas a similar sensitivity for various samples was observed. The limits of detection (LOD) for raw milk, muscles, and livers are 3 ng/mL, 3 μg/kg, and 10 μg/kg, respectively

    Untargeted Metabolomic Profiling of Amphenicol-Resistant <i>Campylobacter jejuni</i> by Ultra-High-Performance Liquid Chromatography–Mass Spectrometry

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    <i>Campylobacter jejuni</i>, an important foodborne microorganism, poses severe and emergent threats to human health as antibiotic resistance becomes increasingly prevalent. The mechanisms of drug resistance are hard to decipher, and little is known at the metabolic level. Here we apply metabolomic profiling to discover metabolic changes associated with amphenicol (chloramphenicol and florfenicol) resistance mutations of <i>Campylobacter jejuni.</i> An optimized sample preparation method was combined with ultra-high-performance liquid chromatography–time-of-flight mass spectrometry (UHPLC–TOF/MS) and pattern recognition for the analysis of small-molecule biomarkers of drug resistance. UHPLC–triple quadrupole MS operated in multiple reaction monitoring mode was used for quantitative analysis of metabolic features from UHPLC–TOF/MS profiling. Up to 41 differential metabolites involved in glycerophospholipid metabolism, sphingolipid metabolism, and fatty acid metabolism were observed in a chloramphenicol-resistant mutant strain of <i>Campylobacter jejuni</i>. A panel of 40 features was identified in florfenicol-resistant mutants, demonstrating changes in glycerophospholipid metabolism, sphingolipid metabolism, and tryptophan metabolism. This study shows that the UHPLC–MS-based metabolomics platform is a promising and valuable tool to generate new insights into the drug-resistant mechanism of <i>Campylobacter jejuni</i>
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