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Streptococcus pneumoniae is an invasive pathogen with high morbidity and mortality in the immunocompromised children and elderly. NOD-like receptor family pyrin domain containing 6 (NLRP6) plays an important role in the host innate immune response against pathogen infections. Our previous studies have shown that NLRP6 plays a negative regulatory role in host defense against S. pneumoniae, but the underlying mechanism is still unclear. The further negative regulatory role of NLRP6 in the host was investigated in this study. Our results showed that NLRP6−/− mice in the lung had lower bacterial burdens after S. pneumoniae infection and expressed higher level of tight junction (TJ) protein occludin compared to WT mice, indicating the detrimental role of NLRP6 in the host defense against S. pneumoniae infection. Transcriptome analysis showed that genes related to leukocytes migration and recruitment were differentially expressed between wild-type (WT) and NLRP6 knockout (NLRP6−/−) mice during S. pneumoniae infection. Also, NLRP6−/− mice showed higher expression of chemokines including C-X-C motif chemokine ligand 1 (CXCL1) and 2 (CXCL2) and lower gene expression of complement C3a receptor 1 (C3aR1) and P-selectin glycoprotein ligand-1 (PSGL-1) which are the factors that inhibit the recruitment of neutrophils. Furthermore, NLRP6−/− neutrophils showed increased intracellular bactericidal ability and the formation of neutrophil extracellular traps (NETs) during S. pneumoniae infection. Taken together, our study suggests that NLRP6 is a negative regulator of neutrophil recruitment and function during S. pneumoniae infection. Our study provides a new insight to develop novel strategies to treat invasive pneumococcal infection.

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.

A recombinant single-chain variable fragment (scFv) antibody was produced from a hybridoma cell strain secreting the monoclonal antibody for amantadine (AMD), and then its recognition mechanisms for AMD were studied using the molecular docking and molecular dynamics. Complex dockings revealed that three regions are involved in antibody recognition; framework 2 of the VL chain (LFR2) GLU40 and TYR42, complementarity-determining region of the VL chain (LCDR3) TYR116, and framework 2 of the VH chain (HFR2) HIS40 and TRP52 were the key amino acid residues. The results of molecular dynamics show that the most important amino acid residues in the interaction between AMD and scFv are HIS40 and TYR116. On the basis of the results of virtual mutation, the scFv antibody was evolved by directional mutagenesis of amino acid residue GLY107 to PHE. Indirect competitive ELISA (icELISA) results indicated that the scFv mutant had highly increased affinity for AMD with up to 3.9-fold improved sensitivity. Thus, the scFv antibody can be applied for mechanistic studies of intermolecular interactions, and our work offered affinity maturated antibodies by site mutations, which were beneficial for valuable anti-AMD antibody design and preparation in future.

Streptomycin (STR), used extensively in the treatment of bovine mastitis, may cause damage such as ototoxicity, allergic reaction, and increasing bacterial resistance to consumers on account of remnant in milk. A time-resolved fluoroimmunoassay (TRFIA) was developed to quantify STR for the first time to ensure food safety. Using secondary antibody labeled with europium (Eu3+) chelate as a tracer, the proposed TRFIA showed that the linear working range was 0.32–5.0 ng/mL under the optimal conditions. Milk samples were deproteinized by trichloroacetic acid and the limit of detection of STR in milk was 1.8 μg/kg. The recoveries of milk samples fortified with 4.0, 20, and 40 μg/kg of STR ranged from 86.2 to 96.3% with relative standard deviations less than 11%. Results of TRFIA for the authentic samples were coincided with those of UHPLC-MS/MS analyses. This study confirmed that the established TRFIA was sensitive as well as reliable and could be an alternative method to monitor STR residue in milk.

Neurotoxicity is an unwanted side-effect seen in patients receiving therapy with the "last-line" polymyxin antibiotics. This is the first study to show that colistin-induced neurotoxicity in neuroblastoma-2a (N2a) cells gives rise to an inflammatory response involving the IL-1β/p-IκB-α/NF-κB pathway. Pretreatment with curcumin at 5, 10, and 20 μM for 2 h prior to colistin (200 μM) exposure for 24 h, produced an anti-inflammatory effect by significantly down-regulating the expression of the pro-inflammatory mediators cyclooxygenase-2 (COX-2), phosphorylation of the inhibitor of nuclear factor-kappa B (NF-κB) (p-IκB)-α, and concomitantly NF-κB levels. Moreover, curcumin significantly decreased intracellular reactive oxygen species (ROS) production and increased the activities of the anti-ROS enzymes superoxide dismutase, catalase, and the intracellular levels of glutathione. Curcumin pretreatment also protected the cells from colistin-induced mitochondrial dysfunction, caspase activation, and subsequent apoptosis. Overall, our findings demonstrate for the first time, a potential role for curcumin for treating polymyxin-induced neurotoxicity through the modulation of NF-κB signaling and its potent anti-oxidative and anti-apoptotic effects.

Glucose oxidase (GOx) catalyses oxidation of glucose accompanied with the generation of hydrogen peroxide. With the addition of Fe2+, hydroxyl radical produced by Fenton reaction between hydrogen peroxide and Fe2+ may quench the fluorescence of gold nanoclusters. In this work, a fluorescent enzyme-linked immunosorbent assay with gold nanoclusters was designed with a straightforward signal output, in which the fluorescence of gold nanoclusters was quenched by GOx-triggered Fenton reaction. Olaquindox was selected as a target analyte. Gold nanoclusters capped with bovine serum albumin and GOx-linked olaquindox conjugates were successfully prepared. Olaquindox in samples directly competed with the GOx-linked olaquindox conjugates for binding immobilized antibody. Consequently, the fluorescence signal increased with the amount of olaquindox. Under optimal conditions, the fluorescent enzyme-linked immunosorbent assay exhibited a favorable performance to detect olaquindox in swine feeds, demonstrating a good linear range from 1.0 µg kg−1 to 150 µg kg−1 with a reliable correlation coefficient (R2 = 0.9918); the limit of detection was 0.68 µg kg−1. Average recoveries in spiked samples were 85.3% to 113.5%. The proposed strategy is a promising approach for the detection of olaquindox and other harmful small molecules.

Tilmicosin has been widely used in the dairy industry, but this compound is hazardous to human health. Therefore, it is urgent to develop a rapid, convenient, sensitive, cost-effective, and user-friendly method to determine tilmicosin. A fluorescent microsphere-based immunochromatographic assay was developed for this analyte. A 200-nm red fluorescent microspheres were selected to be the label, and the mass of the labeled antibody was 4.48 µg/mL. 1-Ethyl-3-(3-dimethylaminopropyl)-carbodiimide was used to catalyze carboxyl groups on the surface of the microspheres. The optimum reaction time between the antibodies and fluorescent microspheres was 30 min, and under these conditions, high coupling ratios and stable conjugations were obtained. The linear dynamic range of the developed protocol was from 0.02 to 2.10 µg/L. The half maximal inhibitory concentration was 0.19 µg/L. The recoveries were from 87 to 98% with a coefficient of variation less than 7.14%. In comparison with a colloidal gold immunoch...

Organic/inorganic hybrid nanoflowers were synthesized from calcium phosphate and protein modified fluorescent gold nanoclusters and antigens. These nanoflowers are shown to be well suited labels for bioassay because they fulfill the functions of biological recognition and signal output. A fluorometric immunoassay was developed that was combined with immunomagnetic separation. In the detection system, the red fluorescence of the supernatant (measured at excitation/emission wavelengths of 360/640 nm) is found to be proportional to the clenbuterol (Clen) concentration after two immunomagnetic separations. The assay has a linear response in the 0.5 μg L−1 to 40 μg L−1 Clen concentration range, and 0.167 μg L−1 limit of detection. This makes it well suited for food safety monitoring. The average recoveries from spiked samples range from 92.7 to 109.1% (intra-assay) and 101.2 to 125.7% (inter-assay) with relative standard deviations of

A sensitive competitive immunoassay with simple operation was developed for the detection of the anti-virus drug amantadine (AMD). The single-chain variable fragment (scFv) antibody against AMD was site-specific biotinylated and overexpressed as a secreted body in Escherichia coli AVB101. Horseradish peroxidase-labeled streptavidin-biotinylated scFv antibody (HRP-SA-BIO-scFv) could specifically bind to AMD-functionalized magnetic beads (MBs) and then the immune complexes were separated from the matrix solution by magnet. The concentration of the AMD could be known by the measurement of the signal produced by the horseradish peroxidase. The newly established assay provides a significant improvement in comparison to the conventional ELISA without SA-BIO signal amplification and MBs separation. The limit of detection and assay time was 0.64 vs. 8.4 ng/mL and 50 vs. 150 min, respectively. The recoveries ranged from 77.8 to 112% with the coefficient of variation less than 13%. The immunoassay exhibited an obvious cross-reactivity to rimantadine (84%), 1-(1-adamantyl)ethylamine (72%), and somantadine (63%). These results demonstrated that the developed immunoassay provided a sensitive, rapid, and accurate approach for the detection of AMD in chicken by employing MBs as solid phase and SA-BIO as signal amplification. When applied in natural chicken samples, the newly established method provided results consistent with those from UPLC-MS/MS, suggesting that the proposed method could be used for rapid screening of the target of interest; the new immunoassay could also be extended to other small molecular contaminants and thus represents a universal strategy for food safety analysis. Graphical abstract ᅟ.

Olaquindox (OLA), used as a medicinal feed additive, has been put under ban due to hazard concerns over animal-derived food security. In this study, a simple, rapid, ultrasensitive, and quantitative gold immunochromatography assay (GICA) was established to analyze OLA in animal feed samples and surface water samples to monitor food security. Various trying has been experimented to improve the sensitivity. The IC50 of the optimized method is 3.35 μg L−1 for feedstuff and 0.35 μg L−1 for environmental water. The recoveries ranged from 77.33 to 86.91 % (CV

A fluorescent single-domain antibody (fluobody), consisting of a single-chain variable fragment antibody (scFv) and a green fluorescent protein extracted from aequorea coerulescens (AcGFP), was produced and used to develop a rapid and sensitive fluorescence-linked immunosorbent assay (FLISA) for the detection of avermectins (AVMs). The scFv gene was prepared by cloning VH and VL genes from a hybridoma cell line (2C11) and then fused to the C-terminus of AcGFP (fluobody) with a flexible peptide linker (Gly4Ser)2 between the two domains. After expression and purification, the functional fluobody was used to develop a one-step FLISA protocol for the determination of AVMs in milk samples. The 50% inhibition concentration (IC50) value and the limit of detection (LOD) of the optimized immunoassay for abamectin (ABM) were 2.13 and 1.07 ng mL−1, respectively. Cross-reactivity studies showed that the fluobody exhibited high affinity for the other four AVMs. The recoveries from the spiked milk samples ranged between 86.8 and 125.0%, with relative standard deviation lower than 10.2%. Moreover, the developed FLISA was applied to field samples, followed by confirmation with liquid chromatography-fluorescence detection (LC-FLD) analysis. The consistency of results between the immunoassay and instrumental techniques in detecting the presence of AVMs near the detection limit of the FLISA indicated that the newly developed method is suitable for rapid screening of AVM contamination in food samples prior to chromatographic analysis.

Infectious coryza is an important respiratory disease of chickens around the world and is caused by Avibacterium paragallinarum. Among the three Page serovars currently recognized for this bacterium, serovar B is a major circulating serovar in China nowadays. The cross-protection ability of the Page serovar B reference strain (0222) and five local isolates was evaluated by a vaccination-challenge trial in SPF chickens. The clinical signs seen in control birds challenged by strain 0222 and isolate HB 01 were significantly different, with isolate HB 01 giving more severe clinical signs. In terms of cross-protection, the protection in the groups vaccinated with isolate HB 01 and BJ 02 was significantly higher than that in the groups vaccinated with 0222 and the other three isolates. In addition, an experimental oil adjuvant trivalent vaccine, containing field isolate HB 01 antigen, was compared for immune efficacy with two commercial trivalent infectious coryza vaccines containing internationally recognized serovar B strains. The experimental oil adjuvant trivalent vaccine elicited best protection (80%) among the three trivalent vaccines. In conclusion, the oil adjuvant vaccine, containing field isolate HB 01 may be a better choice in control of current serovar B Av. paragallinarum outbreaks in China under current circumstances.

The antigen-antibody interaction determines the sensitivity and specificity of competitive immunoassay for hapten detection. In this paper, the specificity of a monoclonal antibody against alternariol-like compounds was evaluated through indirect competitive ELISA. The results showed that the antibody had cross-reactivity with 33 compounds with the binding affinity (expressed by IC50 ) ranging from 9.4 ng/mL to 12.0 μg/mL. All the 33 compounds contained a common moiety and similar substituents. To understand how this common moiety and substituents affected the recognition ability of the antibody, a three-dimensional quantitative structure-activity relationship (3D-QSAR) between the antibody and the 33 alternariol-like compounds was constructed using comparative molecular field analysis (CoMFA) and comparative molecular similarity indices analysis (CoMSIA) methods. The q2 values of the CoMFA and CoMSIA models were 0.785 and 0.782, respectively, and the r2 values were 0.911 and 0.988, respectively, indicating that the models had good predictive ability. The results of 3D-QSAR showed that the most important factor affecting antibody recognition was the hydrogen bond mainly formed by the hydroxyl group of alternariol, followed by the hydrophobic force mainly formed by the methyl group. This study provides a reference for the design of new hapten and the mechanisms for antibody recognition.

Major research efforts are focusing on the development of simultaneous multiplexed immunoassays. In this study, a novel dual-binding fluorescence polarisation immunoassay (DB-FPIA) using a broad-specificity bi-specific single-chain diabody (scDb) and two fluorescent-labelled tracers (sulfamethoxypyridazine-fluorescein isothiocyanate (SMP-FITC) and sarafloxacin-Texas Red (SAR-TR)) with different excitation and emission wavelengths was developed for simultaneous and high-throughput detection of 19 fluoroquinolones (FQs) and 13 sulfonamides (SAs) at the maximum residue limits in milk samples. Recoveries for spiked milk samples were from 76.4% to 128.4%, with a relative standard deviation lower than 13.9%. The developed DB-FPIA was then applied to field samples, followed by confirmation by LC-MS/MS. All three instances in which FQs and SAs were present at concentrations near or above the assay limit of detection were identified as positive by the developed DB-FPIA, demonstrating that the method is suitable for rapid screening of FQs and SAs contamination. The novel methodology combines the advantage of the FPIA and the broad sensitivity of scDb and shows great promise for fast multi-analyte screening of low-molecular weight chemical residues in food samples.