Generation of broad-spectrum recombinant antibody and construction of colorimetric immunoassay for tropane alkaloids: Recognition mechanism and application.

Tropane alkaloids (TAs) have emerged as plant toxins, related to poisoning events. The development of stable antibodies is crucial to ensure the effectiveness of immunological methods in quickly and accurately monitoring these alkaloids. In this study, based on hybridoma, the variable region gene of monoclonal antibody (mAb) was amplified, and the recombinant antibody (rAb) gene sequence (VH-Linker-VL) was successfully constructed and expressed in HEK293F. The obtained rAb has kept the same performance as mAb, and the IC50 of 29 TAs ranged from 0.12 to 2642.78 ng/mL. In the recognition mechanism, the docking and dynamics model identified hydrophobic interaction as the most critical force. Substituent will impact recognition by influencing the spatial structure and hydrophobic properties. Then, a colorimetric immunoassay based on rAb was established, five types of water and thirty-nine nectars of honey were tested. The results demonstrated the absence of TAs in environmental water, whereas atropine was detected in more than 13.47% of honey samples at concentrations exceeding 1 µg/kg. The results show a good correlation with UHPLC-MS/MS, suggesting that the immunoassay has excellent screening ability. The data on TAs in honey and water could serve as a foundation for developing relevant policies.

NLRP6 Serves as a Negative Regulator of Neutrophil Recruitment and Function During Streptococcus pneumoniae Infection.

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.

Site-directed mutations of anti-amantadine scFv antibody by molecular dynamics simulation: prediction and validation.

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.

Label-free gold nanoclusters as quenchable fluorescent probes for sensing olaquindox assisted by glucose oxidase-triggered Fenton reaction.

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.

Quantitative and rapid detection of amantadine and chloramphenicol based on various quantum dots with the same excitations.

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. Graphical abstract ᅟ.

Application of quantitative structure-activity relationship analysis on an antibody and alternariol-like compounds interaction study.

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.

Magnetic-assisted biotinylated single-chain variable fragment antibody-based immunoassay for amantadine detection in chicken.

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.

A fluorometric clenbuterol immunoassay based on the use of organic/inorganic hybrid nanoflowers modified with gold nanoclusters and artificial antigen.

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 <11.6%. Spiked swine urine samples were analyzed by this method, and the results correlated well with data obtained by LC-MS/MS. Graphical abstract Fluorescent hybrid nanoflowers were fabricated with gold nanoclusters (BSA-AuNCs) and antigens. A fluorometric immunoassay based on the use of such nanoflowers and based on immunomagnetic separation was developed to detect clenbuterol residues in swine urine with satisfactory recoveries and acceptable accuracy.

Selection of Avibacterium paragallinarum Page serovar B strains for an infectious coryza vaccine.

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.

Development of an immunoaffinity column for the highly sensitive analysis of bisphenol A in 14 kinds of foodstuffs using ultra-high-performance liquid chromatography tandem mass spectrometry.

An immunoaffinity clean-up material based on a monoclonal antibody (mAb) has been prepared for concentrating and purifying bisphenol A (BPA) in 14 kinds of foodstuffs at trace level. Haptens and immunogen of bisphenol A have been synthesized and comprehensively characterized. An mAb towards BPA was prepared and cross-reactivities with 14 BPA analogues were below 5%. The prepared antibody was coupled to N-hydroxysuccinimide-activated Sepharose 4B to manufacture an immunoaffinity column (IAC), which was applied to purify BPA in 14 kinds of foodstuffs. The analyte was then detected by means of ultra-high-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS). Under the optimized conditions, compared with two traditional SPE clean-up methods, the IAC showed better selectivity (matrix effect <16.8%) and higher sensitivity. The limits of detection for BPA in 14 kinds of foodstuffs ranged from 0.001 µg L-1 to 0.01 µg kg-1, and the limits of quantification were in the range from 0.003 µg L-1 to 0.04 µg kg-1. The recoveries of BPA from spiked samples ranged from 82.0% to 104.9%, with RSDs below 13.8%. Besides, the IAC exhibited good reusability, with 40% column capacity remaining and no significant loss of recovery after 25 application cycles in real sample detection. These results demonstrated that the developed IAC-UPLC-MS/MS approach has wide applicability for purifying and detecting BPA in various foodstuffs.

Curcumin Attenuates Colistin-Induced Neurotoxicity in N2a Cells via Anti-inflammatory Activity, Suppression of Oxidative Stress, and Apoptosis.

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-1ß/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.

A novel multiplexed fluorescence polarisation immunoassay based on a recombinant bi-specific single-chain diabody for simultaneous detection of fluoroquinolones and sulfonamides in milk.

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.