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AbstractImmunoassay based on the antibodies specific for targets has advantages of high sensitivity, simplicity and low cost, therefore it has received more attention in recent years, especially for the rapid detection of small molecule chemicals present in foods, diagnostics and environments. However, limited by low molecular weight and only one antigenic determinant existed, immunoassays for these small molecule chemicals, namely hapten substances, were commonly performed in a competitive immunoassay format, whose sensitivities were obviously lower than the sandwich enzyme-linked immunosorbent assay generally adaptable for the protein targets. In order to break through the bottleneck of detection format, researchers have designed and established several novel noncompetitive immunoassays for the haptens in the past few years. In this review, we focused on the four representative types of noncompetitive immunoassay formats and described their characteristics and applications in rapid detection of small molecules. Meanwhile, a systematic discussion on the current technologies challenges and the possible solutions were also summarized. This review aims to provide an updated overview of the current state-of-the-art in noncompetitive immunoassay for small molecules, and inspire the development of novel designs for small molecule detection. [ABSTRACT FROM AUTHOR]

The tadalafil‐like compounds have appeared recently as adulterants in drinks and healthcare dietary supplements sourced from medicinal and edible food, which may cause illness and even death. In this work, the rationality of haptens was explored by computational chemistry and molecular simulation theories such as frontier molecular orbital (FMO)‐based softness (S), three‐dimensional (3D) structure, surface electrostatic potential (ESP), and lipophilic potential (LP). An antiserum from hapten H5 with the highest softness and maintaining the appropriate three‐dimensional (3D) structure showed the optimal immunoassay performance, indicating an increasing softness was a critical factor for effective hapten. Based on the antibody induced by hapten H5, an indirect competitive enzyme‐linked immunosorbent assay (icELISA) method for detecting multiple tadalafil‐like adulterants was established. The icELISA showed a limit of detection (LOD), 50% inhibition concentration (IC50), and a working range of 0.004–0.396, 0.89–4.27, and 0.094–16.71 ng/ml for tadalafil, amino tadalafil, acetamino tadalafil, nortadalafil, and N‐desmethyl ent‐tadalafil, respectively. The spiked recoveries of tadalafil‐like adulterants in samples ranged from 84.9% to 116.2%. The results of the icELISA and HPLC‐MS/MS methods had a good correlation for real samples with the R2 of 0.9955. Specially, this work not only provided a convenient immunoassay method for measuring tadalafil‐like adulterants in spirit drinks and dietary supplements in group‐screening manner, but also suggested that softness was likely to be a general theory for rational hapten design. Practical Application: Rapid monitoring of tadalafil‐like adulterants in food samples is very necessary and important for consumers, regulatory agencies, and the food industry. [ABSTRACT FROM AUTHOR]

Histamine (HA) is a biogenic amine that can accumulate to high concentration levels in food as a result of microbial activity and can cause toxic effects in consumers. In this work, a portable electrochemical immunosensor capable of detecting HA with high sensitivity and selectivity was developed. Prussian blue-chitosan-gold nanoparticle (PB-CS-AuNP) nanocomposite films with excellent biocompatibility were synthesized and characterized by scanning electron microscopy and energy dispersive X-ray analysis. The PB-CS-AuNP were coated onto a screen-printed electrode by one-step electrodeposition and used to conjugate the HA ovalbumin conjugate (HA-Ag). HA was determined by a competition between the coating HA-Ag and the HRP labeled HA antibody (HRP-HA-Ab). After careful optimization of assay conditions and Box-Behnken analysis, the developed immunosensor showed a linear range from 0.01 to 100 μg/mL for HA in fish samples. The average recoveries from spiked samples ranged from 97.25% to 105%. The biosensor also showed good specificity, reproducibility, and stability, indicating its potential application in monitoring HA in a simple and low cost manner. [ABSTRACT FROM AUTHOR]

Purpose: To evaluate the clinical efficacy of bladder hydrodistention and intravesical sodium hyaluronate in the treatment of interstitial cystitis (IC).Materials and Methods: Twenty-one patients with IC received intravesical sodium hyaluronate therapy under nerve block or intravenous anesthesia. Bladders were perfused with 100 cmH2O perfusion pressure and expanded for 10 min and were later injected with 40 mg/50 mL sodium hyaluronate through the catheter. After 1 h, the perfusion fluid was released. Perfusion was applied once per week, 4 to 6 times as a course of treatment.Results: Under anesthesia, the average bladder capacity was 191.62 ± 88.67 mL, and after bladder expansion, the bladder capacity reached 425.33 ± 79.83 mL (P = .000). There were 2 suspected bladder ruptures after bladder expansion at 6.5 min and 7.2 min. After 10 min of bladder expansion, there were 19 cases of significantly gross hematuria. After treatment, the catheters of 17 patients were removed at 24 h; for the 2 cases of hematuria, catheters were removed at 72 h and for the 2 cases of suspected bladder rupture, catheters were removed after 4 days. After catheter removal, the pain threshold significantly decreased, and the maximum urinary output increased slightly. Compared with values before treatment, the day before the second injection of sodium hyaluronate, the frequency of urination decreased significantly (32.8 vs. 18.5 times/24 h), the maximum urinary output increased significantly (86.7 vs. 151.9 mL), the pain decreased significantly (8.7 vs. 3.0), and the O'Leary-Sant IC score and quality of life score were significantly decreased (30.0 vs. 17.0 and 5.9 vs. 2.4, respectively) (P = .000).Conclusion: Bladder hydrodistention under anesthesia for patients with severe intractable IC produces immediate effectiveness; sodium hyaluronic infusion can alleviate frequent urination and pain, and the efficacy was positively correlated with the duration of treatment. [ABSTRACT FROM AUTHOR]

Histamine (HA) is one of the key causative agents associated with allergies and food poisoning. The accurate and convenient detection of HA is highly required to avert HA toxicity and ensure food safety. Herein, a multicolorimetric and fluorometric dual-readout immunosensor for HA detection was developed based on an alkaline phosphatase (ALP) enabled multicolor change reaction and fluorescence quenching of carbon dots (CDs) resulting from the metallization of gold nanorods (Au NRs). In this immunosensor, ALP is employed as labeling enzyme which can efficiently catalyze the hydrolysis of ascorbic acid 2-phosphate into ascorbic acid (AA). Then, the resultant AA can reduce Ag+ into silver monomer, and a silver shell is deposited on the surface of Au NRs to form Au/Ag core-shell nanorods (Au NRs@Ag). This leads to the blue-shift of the longitudinal local surface plasmon resonance peak of Au NRs and a vivid multicolor variation. Meanwhile, the fluorescence of CDs can be gradually quenched via inner-filter effect by the resultant Au NRs@Ag. Multicolorimetric assay allows a semiquantitative detection with a visible detection limit of 0.25 mg/L, and fluorometric assay provides a sensitive and accurate quantitative detection with a detection limit of 0.08 mg/L. The immunosensor not only fulfills the reliable and accurate detection requirements but also fits different testing needs for both laboratory and on-site detection. • We developed a dual-mode sensor for rapid and accurate histamine detection in foods. • Tunable metallization of gold nanorods was used for multicolorimetric sensing. • Samples with excess histamine can be easily discriminated with the naked eye. • A carbon dots was employed as fluorescent signal source for fluorescent sensing. • The carbon dots quenching mechanism was confirmed to be inner filter effect. [ABSTRACT FROM AUTHOR]

A simple and rapid method for determination of carbofuran was developed using the fluorescence polarisation immunoassay (FPIA). Three tracers with different lengths of bridge (0-, 2- and 6-carbon bridge) between the hapten molecule 4-[[(2,3-dihydro-2,2-dimethyl-7-benzofuranyloxy)carbonyl]-amino]-butanoic acid (BFNB) and 5-aminofluorescein (AF), fluoresceinthiocarbamyl ethylenediamine (EDF), fluoresceinthiocarbamyl hexylenediamine (HDF), were synthesised and their binding response with anti-carbofuran-specific antibody were evaluated. The physicochemical parameters were optimised for the FPIA. The AF-labelled BFNB conjugate (BFNB-AF) was found to be the optimal tracer for FPIA of carbofuran. The detection limit of carbofuran, IC50value and the working range were 2.3, 48.8 and 7.4−202.2 µg/L, respectively; and the reaction time was only 10 min. The average recovery from spiked water and vegetable samples was 86.9−95.4% and the mean coefficient of variation was 6.2% for inter-assay and 8.7% for intra-assay, which showed good reproducibility for FPIA. Thus, the developed FPIA method exhibited the potential for the rapid and accurate determination of carbofuran in agricultural and environmental samples. [ABSTRACT FROM AUTHOR]

An indirect competitive chemiluminescent enzyme immunoassay method was developed for screening malachite green (MG). Assay conditions, including the concentration of coating, antibody dilution, incubation time, dilution buffer, ionic strength and pH, were optimised. Under the optimised conditions, coating antigen concentration was 125 ng/mL; and dilution fold of antibody was 40,000. The 50% inhibition values of 0.22 ng/mL for MG were achieved with a limit of detection of 0.01 ng/mL, the linear range was from 0.03 ng/mL to 3.27 ng/mL. The assay showed a little cross-reactivity of 3.4%, 2.7% and 1.0% with methylene blue, brilliant green and crystal violet, respectively, and negligible cross-reactivity with other analogues of MG. The developed method has been used to quantify MG in seafood samples. The recovery of MG ranged from 82.43% to 108.0% at four concentrations (0.1, 1, 3 and 5 ng/mL), and the coefficients of variation were less than 13%. A comparison results between the high-performance liquid chromatography and the developed assay showed good relativity. The results indicated that the assay was a sensitive and stable method for screening MG. [ABSTRACT FROM PUBLISHER]

Parkinson's disease (PD) is a common neurodegenerative disease. The main pathological feature is the degeneration and loss of dopaminergic neurons in the substantia nigra, which leads to the significant decrease of dopamine content in the striatum. Our recent studies have shown that scorpion venom heat-resistant synthetic peptide (SVHRSP) have protective effects on neuroinflammation. In this study, using C. elegans induced by 6-hydroxydopamine (6-OHDA) as neurodegenerative model, we investigated the effect of SVHRSP on dopaminergic neurons neurotoxicity. Our results implied that SVHRSP treatment could improve the motor capacity in 6-OHDA-induced C. elegans and improve dopaminergic neuron mediated food sensitivity behavior. After SVHRSP treatment, dopaminergic neuron degeneration induced by 6-OHDA was significantly prevented along with a decreased α-synuclein aggregation and restored lipid deposition in C. elegans induced by 6-OHDA. We also observed the reduced levels of reactive oxygen species (ROS) after SVHRSP treatment in model-building C. elegans. In addition, the genes related to apoptosis, oxidative stress, like ctl-1 , egl-1 and cat-2 in C. elegans induced by 6-OHDA upregulated after treatment with SVHRSP. In conclusion, SVHRSP may impose anti-PD effect through its neuroprotective action on dopaminergic neurons. This study elucidates the effect and related mechanism of SVHRSP on PD and provides evidences for the therapeutic treatment of PD. • SVHRSP ameliorate the dyskinesia in C. elegans induced by 6-OHDA. • SVHRSP can improve 6-OHDA-induced dopaminergic neuron damage in C. elegans. • SVHRSP can reduce the aggregation of α-synuclein in C. elegans. • SVHRSP increased expression of neuroprotection-related genes in C. elegans. [ABSTRACT FROM AUTHOR]

The widespread occurrence of ethyl carbamate (EC, 89.09 Da), a group 2A carcinogen, in fermented foods and alcoholic beverages has raised worldwide public health concern. Immunoassay for EC is unavailable due to the simple and small structure of EC. In this work, an initial attempt to produce antibody specific for EC, by using 4-((ethoxycarbonyl)amino)butanoic acid as hapten, was made but failed. However, since EC can easily react with 9-xanthydrol to form xanthyl ethyl carbamate (XEC), two haptens based on XEC structure were designed and synthesized. Polyclonal antibody against XEC, instead of EC was obtained and then used to develop a competitive indirect ELISA for EC via a pre-analysis derivatization. After optimization, the ciELISA was applied in analyzing Chinese rice wine with detection limit of 166 μg/L, and negligible cross-reactivity with EC analogs. Recoveries of EC in fortified samples were from 84.4% to 100.9%, with coefficients of variation below 10%. Results for analysis of real samples by the ci-ELISA correlated well with that by reference method GC-MS, suggesting the good accuracy and reproducibility of the proposed method. This is the first report of an immunoassay capable of detecting EC, which is suitable for monitoring EC in a large amount of samples. [ABSTRACT FROM AUTHOR]

The accumulation of quinalphos pesticide in agricultural products have detrimental effects to human body and environment, hence, an efficient and sensitive detection approach is desirable. In this study, a label-free electrochemical immunosensor based on the directly immobilizing nanobody onto functionalized polyvinyl alcohol/gelatin-gold nanoparticles nanofiber membrane (PVA/G-AuNPs NFM) was fabricated. First, a homogeneous PVA/G-AuNPs NFM with multiple-porosity structure and appropriate hydrophilicity was prepared. Then, the electrochemical immunosensor was assembled and under the optimized conditions, the prepared biosensor exhibited good detection performance towards quinalphos with the linear range and limit of detection being 0.06–1000 ng/mL and 50.74 pg/mL (S/N = 3), respectively. Moreover, the sensor presented good specificity, regenerability and stability, maintaining more than 90% activity after 5 times of regeneration and about 90% activity after 6 weeks of storage. Finally, the average recoveries of quinalphos from food samples were 89.68%–110.88% with the coefficient of variation being 1.65%–7.81%, showing good correlation with UPLC-MS/MS (R2 = 0.9859), indicating the accuracy of the proposed electrochemical immunosensor assay. Overall, the label-free electrochemical immunosensor presents a novel approach for the rapid and sensitive detection of quinalphos and other pesticide residues. • A label-free biosensor platform based on nanofiber-coupled nanobody was developed. • Nanobody was immobilized onto the nanofiber's surface based on the Schiff reaction. • Nanofiber's microstructure and functionalization facilitated detection sensitivity. • The biosensor exhibited good specificity, reproducibility and long-term stability. [ABSTRACT FROM AUTHOR]

Steroid receptor coactivator 1 (SRC-1) is one of the coactivators recruited by the nuclear receptors (NRs) when NRs are activated by steroid hormones, such as glucocorticoid. SRC-1 is abundant in hippocampus and hypothalamus and is also related to some major risk factors for depression, implicated by its reduced expression after stress and its effect on hypothalamus-pituitary-adrenal gland axis function. However, whether SRC-1 is involved in the formation of depression remains unclear. In this study, we firstly established chronic unpredictable stress (CUS) to induce depressive-like behaviors in mice and found that SRC-1 expression was reduced by CUS. A large number of studies have shown that neuroinflammation is associated with stress-induced depression and lipopolysaccharide (LPS) injection can lead to neuroinflammation and depressive-like behaviors in mice. Our result indicated that LPS treatment also decreased SRC-1 expression in mouse brain, implying the involvement of SRC-1 in the process of inflammation and depression. Next, we showed that the chronic unpredictable mild stress (CUMS) failed to elicit the depressive-like behaviors and dramatically promoted the expression of SRC-1 in brain of wild type mice. What's more, the SRC-1 knockout mice were more susceptible to CUMS to develop depressive-like behaviors and presented the changed expression of glucocorticoid receptor. However, SRC-1 deficiency did not affect the microglia activation induced by CUMS. Altogether, these results indicate a correlation between SRC-1 level and depressive-like behaviors, suggesting that SRC-1 might be involved in the development of depression induced by stress. [ABSTRACT FROM AUTHOR]

G protein-coupled receptor 50 (GPR50) belongs to the G protein-coupled receptor which is highly homologous with the sequence of melatonin receptor MT1 and MT2. GPR50 expression has previously been reported in many brain regions, like cortex, midbrain, pons, amygdala. But, the distribution of GPR50 in the hippocampus and cortex and the cell types expressing GPR50 is not yet clear. In this study, we examined the distribution of GPR50 in adult male mice by immunofluorescence. Our results showed that GPR50 was localized in the CA1-3 pyramidal cells and the granule cells of the dentate gyrus. GPR50 was also expressed in excitatory and inhibitory neurons. As inhibitory neurons also contain many types, we found that GPR50 was localized in some interneurons in which it was co-expressed with the calcium-binding proteins calbindin, calretinin, and parvalbumin. Besides, similar results were seen in the cortex. The widespread expression of GPR50 in the hippocampus and cortex suggests that GPR50 may be associated with synaptic plasticity and cognitive function. [ABSTRACT FROM AUTHOR]

In this work, a monoclonal antibody-based indirect competitive enzyme-linked immunosorbent assay (icELISA) was established to detect tylosin and tilmicosin in milk and water samples. A sensitive and specific monoclonal antibody was prepared by rational designed hapten, which was achieved by directly oxidizing the aldehyde group on the side chain of tylosin to the carboxyl group. Under the optimized conditions, the linear range of icELISA for tylosin and tilmicosin were 1.3 to 17.7 ng/mL and 2.0 to 47.4 ng/mL, with half-maximal inhibition concentration (IC50) values of 4.7 and 9.6 ng/mL, respectively. The cross-reactivity with other analogues of icELISA was less than 0.1%. The average recoveries of icELISA for tylosin and tilmicosin ranged from 76.4% to 109.5% in milk and water samples. Besides, the detection results of icELISA showed good correlations with HPLC-MS/MS. The proposed icELISA was satisfied for rapid and specific screening of tylosin and tilmicosin residues in milk and water samples. [ABSTRACT FROM AUTHOR]

Highlights • A novel hapten was first designed for uniform immune induction effect. • A sensitive ciCLEIA method was developed for detecting Alternariol. • The detection results for ciCLEIA are consistent with those of HPLC-MS/MS. Abstract Alternariol (AOH), a secondary metabolites of the genus Alternaria , is a genotoxic mycotoxin. Because the previous "Mannich antigen" for AOH readily underwent unstable induction, a new AOH hapten was designed for inducing a high-quality antibody against AOH using a conjugate of the AOH carboxymethyl derivative with a carrier protein as the immunogen. A competitive indirect chemiluminescence enzyme immunoassay (ciCLEIA) for AOH was optimized and showed good sensitivity (limit of detection 0.068 ng/mL), a linear range (0.11–1.23 ng/mL), and negligible cross-reactivity with analogues. Average rates of recovery from spiked samples (juice and cereal) were between 72.7% and 115.8%, with coefficients of variation <14.3%. Results from ciCLEIA correlated well with those of high-performance liquid chromatography coupled with tandem mass spectrometry, meaning the proposed method might be an effective screening tool for detection of AOH in food samples. [ABSTRACT FROM AUTHOR]

Abstract An electrochemical immunosensor for ultrasensitive detection of acrylamide (AA) in water and food samples was developed. SnO 2 -SiC hollow sphere nanochains with high surface area and gold nanoparticles with good electroconductivity were fabricated onto the surface of a glassy carbon electrode pre-coated with chitosan. The coating antigen (AA-4-mercaptophenylacetic acid-ovalbumin conjugate, AA-4-MPA-OVA) was immobilized on the electrode. Polyclonal antibody specific for AA-4-MPA was conjugated to gold nanorod (AuNR) as primary antibody (AuNR-Ab 1). Horseradish peroxidase labelled anti-rabbit antibody produced in goat was conjugated to AuNR as secondary antibody (HRP-AuNR-Ab 2). For detection, the analyte (AA-4-MPA) in sample competed with coating antigen for binding with AuNR-Ab 1. After washing, HRP-AuNR-Ab 2 was added to capture the AuNR-Ab 1 , and the electrical signal was obtained by addition of hydroquinone and H 2 O 2. After investigation of the binding ability on nanomaterials and optimization of competitive immunoassay conditions, the proposed immunosensor exhibited a sensitive response to AA with a detection limit of 45.9 ± 2.7 ng kg−1, and working range of 187 ± 12.3 ng kg−1 to 104 ± 8.2 μg kg−1 for drinking water samples. Recoveries of AA from spiked samples were ranged from 86.0% to 115.0%. The specificity, repeatability and stability of the immunosensor were also proved to be acceptable, indicating its potential application in AA monitoring. Graphical abstract Image 1 Highlights • An electrochemical immunosensor capable of detecting acrylamide sensitively and selectively was developed. • Signal amplification strategy based on chitosan/SnO2-SiC HSNC/gold nanomaterials was used. • The immunosensor was able to detect acrylamide with a LOD of 45.9 ± 2.7 ng kg−1 for drink water samples. [ABSTRACT FROM AUTHOR]

• An electrochemical immunosensor for parathion based on nanomaterial was prepared. • Nanofiber was favorable for electrochemical response compared to casting film. • The established sensor showed low limit detection of 2.26 pg/mL. • The sensor had good specificity and long-term stability. • The sensor was accurate to use in spiked food samples. For monitoring of the residual of parathion pesticide in food, herein, a sensitive and reliable electrochemical immunosensor based on cross-linked polyvinyl alcohol/citric acid nanofiber membrane (PVA/CA NFM) and horseradish peroxidase labeled anti-parathion nanobody was constructed. Firstly, the cross-linked PVA/CA NFM with extra-high surface area and uniform morphology was prepared and characterized. Then, the immunosensor was assembled and its analytical performances were evaluated. It exhibited high specificity and sensitivity to parathion with the liner range and limit of detection being 0.01–100 ng/mL and 2.26 pg/mL, respectively. Moreover, the biosensor kept almost 75% of its initial activity after regenerating 4 times, and remained 85% after 9 weeks of storage. Finally, the average recoveries from food samples were 96.20%–114.61% with the coefficient of variation being 1.06%–5.28%, which was correlate well with UPLC (R2 = 0.9964). Therefore, the sensor was demonstrated to be a feasible alternative for sensitive assay of parathion. [ABSTRACT FROM AUTHOR]

Immunoassay is an emerging technique that has been used in the fields of food and environmental analysis to monitor and control hazardous residues. Multianalyte immunoassays (MAIAs) are favored because they can simultaneously determine several targets in a single test. In this review, an overview of various formats for the MAIA is given. New trends on how to produce antibodies, how to improve antibody specificity and how to manipulate assay formats to obtain the desired multi-analysis schemes are summarized. Finally, the limitations and prospects of MAIAs are discussed. [ABSTRACT FROM AUTHOR]

• Monoclonal antibody specific for tyramine was prepared based on a new hapten design. • Alkaline phosphatase enabled the growth of gold nanostars for multicolor generation. • A multicolor colorimetric immunoassay for tyramine was developed. • Smartphone was used for quantitative detection of tyramine. In this work, a specific monoclonal antibody against tyramine was produced based on a new hapten design. Then, we developed a high-resolution multicolor colorimetric immunoassay for tyramine based on this antibody by integrating enzyme-induced multicolor generation with smartphone-assistant signal readout. The multicolor generation is due to the shift of the local surface plasmon resonance band of gold nanostructure controlled by alkaline phosphatase-induced the growth of gold nanostars. Quantitative detection of tyramine was achieved via analyzing the red/blue channel values of assay solution's image taken by a smartphone with the support of a color recognizer application. The limit of detection of this immunoassay for tyramine detection in beef, pork and yoghurt was 19.7 mg/kg or L. The average recoveries were between 83 % and 103 %., and the results were validated by high performance liquid chromatography to be reliable. Overall, this developed immunoassay provides a promising platform for on-site detection of tyramine. [ABSTRACT FROM AUTHOR]

19-nortestosterone (19-NT), being regarded as the residual marker of banned synthetic steroid hormone nandrolone phenylpropionate, could cause adverse effects when consumed by human. To ensure food safety and minimize 19-NT toxicity, herein, an innovative electrochemical immunosensor for detection of 19-NT was constructed based on the modified polyvinyl alcohol/polyacrylic acid (PVA/PAA) nanofiber mat (NFM). The detection was achieved via a direct competition for binding to horse radish peroxidase-tagged anti-19-NT nanobody between free 19-NT and the 19-NT-ovalbumin covalently immobilized onto the NFM, and the amperometric response of immunosensor was analyzed using cyclic voltammetry. Under the optimal preparation conditions, the PVA/PAA NFM with appropriate hydrophilicity and ultra-high surface was obtained and characterized, and its electrochemical performance exhibited a 20-times increased response compared to the PVA/PAA casting membrane. The established immunosensor possessed high sensitivity towards 19-NT with a detection limit of 2.14 pg/mL, a 46-fold times lower compared to the traditional ic-ELISA of pure anti-19-NT nanobody. Recovery rates from the spiked animal urine samples ranged from 97.09 % to 117.32 %. Moreover, the sensor showed good reproducibility and stability over 6 weeks, suggesting that the nanofiber-based biosensor is a promising alternative for the fast and sensitive determination of 19-NT. [Display omitted] • A nanobody and PVA/PAA nanofibers based immunosensor for 19-NT was prepared. • The established biosensor possessed a detection limit of 2.14 pg/mL. • The special structure of nanofibers was favorable for the enhanced response. • The biosensor showed good reproducibility and stability over 6 weeks. [ABSTRACT FROM AUTHOR]

Acute stress exerts pleiotropic actions on learning behaviors. The induced negative effects are sometimes adopted to measure the efficacy of particular drugs. Until now, there are no detailed experimental data on the time-gradient effects of acute stress. Here, we developed the time gradient acute restraint stress (ARS) model to precisely assess the roles of different restrain times on inducing acute stress. Time gradient ARS facilitates escape behaviors and learning outcomes, peaking at 2 h-ARS and then declining to baseline at 3.5 h-ARS as confirmed by time gradient post-stress data. Furthermore, time gradient ARS activates glucocorticoid receptor (GR) phosphorylation site at Serine211 (P S221) as an inverted V-shaped pattern peaking at 2 h-ARS, whereas that of the GR phosphorylation site at Serine226 (P S226) from 2 h-ARS to 3.5 h-ARS. The 2 h-ARS but not 3.5 h-ARS enhances synaptic plasticity and genes transcription associated with learning and memory in the hippocampus of male mice. The Cdk5 inhibitor, roscovitine, blocks this facilitation effect by intervening in GR phosphorylation at Serine211 in the 2 h-ARS mice. Altogether, these findings show that the time gradient ARS selectively activates GR phospho-isoforms and differentially influences the behaviors along with maintaining a relationship between 2 h-ARS and Cdk5/GR P S211-mediated transcriptional activity. [Display omitted] • The active and passive escape behaviors in the duration of time gradient ARS • 2 h-ARS not 3.5 h-ARS enhances synaptic plasticity and synaptic molecules expression. • The GR P S211 mediates the promotion of escape behavior in 2 h-ARS mice. • Inhibition of Cdk5 attenuates the passive avoidance behaviors in 2 h-ARS mice. [ABSTRACT FROM AUTHOR]

Scorpion venom has been used in the Orient to treat central nervous system diseases for many years, and the protein/peptide toxins in Buthus martensii Karsch (BmK) venom are believed to be the effective components. Scorpion venom heat-resistant peptide (SVHRP) is an active component of the scorpion venom extracted from BmK. In a previous study, we found that SVHRP could inhibit the formation of a glial scar, which is characterized by enhanced glial fibrillary acidic protein (GFAP) expression, in the epileptic hippocampus. However, the cellular and molecular mechanisms underlying this process remain to be clarified. The results of the present study indicate that endogenous GFAP expression in primary rat astrocytes was attenuated by SVHRP. We further demonstrate that the suppression of GFAP was primarily mediated by inhibiting both c-Jun expression and its binding with AP-1 DNA binding site and other factors at the GFAP promoter. These results support that SVHRP contributes to reducing GFAP at least in part by decreasing the activity of the transcription factor AP-1. In conclusion, the effects of SVHRP on astrocytes with respect to the c-Jun/AP-1 signaling pathway in vitro provide a practical basis for studying astrocyte activation and inhibition and a scientific basis for further studies of traditional medicine. [ABSTRACT FROM AUTHOR]

Purpose. The purpose of this study was to investigate the marginal fit of metal-free crowns made by three different computeraided design/computer-aided manufacturing (CAD/CAM) systems. Materials and Methods.The maxillary left first premolar of a dentiform was prepared for all-ceramic crown restoration. Thirty all-ceramic premolar crowns were made, ten each manufactured by the Lava system, Cercon, and Cerec. Ten metal ceramic gold (MCG) crowns served as control.The marginal gap of each sample was measured under a stereoscopic microscope at 75x magnification after cementation. One-way ANOVA and the Duncan's post hoc test were used for data analysis at the significance level of 0.05. Results. The mean (standard deviation) marginal gaps were 70.5 (34.4) μm for the MCG crowns, 87.2 (22.8) μm for Lava, 58.5 (17.6) μm for Cercon, and 72.3 (30.8) μm for Cerec.There were no significant differences in the marginal fit among the groups except that the Cercon crowns had significantly smaller marginal gaps than the Lava crowns (P < 0.001). Conclusions.Within the limitation of this study, all the metal-free restorations made by the digital CAD/CAM systems had clinically acceptable marginal accuracy. [ABSTRACT FROM AUTHOR]

Rapid and quantitative detection of paraquat is crucial because of its high toxicity. Here, we developed an ultrasensitive time-resolved fluorescence immunochromatographic assay (TRFICA) strip based on our synthesized variable domain of heavy chain antibody (VHH, also called Nanobody) for paraquat detection. Briefly, the specific immunogen selected from six designed antigens was employed to immunize alpaca, and a high-efficiency capacity of 1.6 × 1013 pfu mL−1 phage display nanobody library was established for biopanning against paraquat. The selected nanobody exhibited high sensitivity (limit of detection (LOD) was 0.0090 ng mL−1 and IC 50 was 0.0588 ng mL−1 in buffer) and stability to high temperatures and denaturants. The molecular docking results indicated that the π-π, cation-π, and hydrogen bond interactions between paraquat and the pocket-like structures of complementarity-determining regions (CDRs) in VHH played a critical role in the antibody-paraquat recognition, competition, and affinity processes. The constructed TRFICA recognized paraquat through a quantitative analysis using the strip reader, and showed no cross-reactivity with other herbicides, and a semi-quantitative analysis using the naked eye. Notably, the potential practical applications of the TRFICA evaluated by performing a quantitative analysis of paraquat in food samples (vegetables, fruits, and grain products) and biological samples (blood and urine) showed a recovery rate range between 76.7% and 133.3% with inter-assay coefficient variation lower than 18.5%. The nanobody from phage display libraries was effective for small molecule recognition and detection, and it is a vital tool for immunoassay. • The binding mode between the nanobody and paraquat was analysed through molecular simulation. •The ultrasensitive ICA strip was developed by nanobody-based time-resolved fluorescent microspheres as signal probe. •The test results of the developed TRFICA in real samples showed a good consistence with those of UPLC-MS/MS. [ABSTRACT FROM AUTHOR]

In this study, an indirect competitive chemiluminescent enzyme immunoassay (ic-CLEIA) for determining aflatoxin M1(AFM1) in milk products has been developed. A luminol–hydrogen peroxide chemiluminescence system catalysed by horseradish peroxidase (HRP) was used as the signal detecting system. The effects of several factors, including concentration and pH of phosphate buffer, dilution ratio of antibody and antigen and other relevant variables on the immunoassay, were studied and optimised by single-factor experiments. The developed method presented an IC50of 0.05 ng/mL, a detection limit of 0.01 ng/mL and a linear range from 0.018 to 0.13 ng/mL. This method has been successfully applied to the evaluation of AFM1in milk products, the recoveries ranging from 71.9% to 109.0%. A good correlation with the commercial available ELISA kit for AFM1(r= 0.9978) was obtained, indicating that the ic-CLEIA method developed can be used to determine AFM1in real samples. [ABSTRACT FROM PUBLISHER]

The expression efficiency was improved for the recombinant single-chain variable fragment (scFv) against clenbuterol (CBL) obtained from mouse and expressed in the methylotrophic yeast Pichia pastoris GS115, by redesigning and synthesizing the DNA sequence encoding for CBL-scFv based on the codon bias of P. pastoris. The codons enco4ding 124 amino acids were optimized, in which a total of 156 nucleotides were changed, and the G+C ratio was simultaneously decreased from 53 to 47.2 %. Under the optimized expression conditions, the yield of the recombinant CBL-scFv (41 kDa) antibodies was 0.223 g L in shake culture. Compared to the non-optimized control, the expression level of the optimized recombinant CBL-scFv based on preferred codons in P. pastoris demonstrated a 2.35-fold higher yield. Furthermore, the recombinant CBL-scFv was purified by Ni-NTA column chromatography, and the purity was 95 %. The purified CBL-scFv showed good CBL recognition by a competitive indirect enzyme-linked immunoassay. The average concentration required for 50 % inhibition of binding and the limit of detection for the assay were 5.82 and 0.77 ng mL, respectively. [ABSTRACT FROM AUTHOR]

A Pichia pastoris (P. pastoris) cell surface display system of Bombyx mori acetylcholinesterase (BmAChE) was constructed and its bioactivity was studied. The modified Bombyx mori acetylcholinesterase gene (bmace) was fused with the anchor protein (AGα1) from Saccharomyces cerevisiae and transformed into P. pastoris strain GS115. The recombinant strain harboring the fusion gene bmace-AGα1 was induced to display BmAChE on the P. pastoris cell surface. Fluorescence microscopy and flow cytometry assays revealed that the BmAChE was successfully displayed on the cell surface of P. pastoris GS115. The enzyme activity of the displayed BmAChE was detected by the Ellman method at 787.7 U/g (wet cell weight). In addition, bioactivity of the displayed BmAChE was verified by inhibition tests conducted with eserine, and with carbamate and organophosphorus pesticides. The displayed BmAChE had an IC50 of 4.17×10−8 M and was highly sensitive to eserine and five carbamate pesticides, as well as seven organophosphorus pesticides. Results suggest that the displayed BmAChE had good bioactivity. [ABSTRACT FROM AUTHOR]

Abstract: A rapid, sensitive chemiluminescent enzyme immunoassay (CLEIA) for ractopamine (RAC) based on a single-chain variable fragment (scFv)-alkaline phosphatase (AP) fusion protein was developed. The scFv gene was prepared by cloning the heavy- and light-chain variable region genes (VH and VL) from hybridoma cell line AC2, which secretes antibodies against RAC, and assembling VH and VL genes with a linker by means of splicing overlap extension polymerase chain reaction. The resulting scFv gene was inserted into the expression vector pLIP6/GN containing AP to produce the fusion protein in Escherichia coli strain BL21. The purified scFv-AP fusion protein was used to develop a direct competitive CLEIA (dcCLEIA) protocol for detection of RAC. The average concentration required for 50% inhibition of binding and the limit of detection of the assay were 0.25±0.03 and 0.02±0.004ngmL−1, respectively, and the linear response range extended from 0.05 to 1.45ngmL−1. The assay was 10 times as sensitive as the corresponding enzyme-linked immunosorbent assay based on the same fusion protein. Cross-reactivity studies showed that the fusion protein did not cross react with RAC analogs. DcCLEIA was used to analyze RAC spiked pork samples, and the validation was confirmed by high-performance liquid chromatography–tandem mass spectrometry (HPLC–MS). The results showed a good correlation between the data of dc-CLEIA and HPLC–MS (R 2 >0.99), indicating that the assay was an efficient analytical method for monitoring food safety. [Copyright &y& Elsevier]

Abstract: A multi-analytes method for monitoring of organophosphorus pesticides (OPs) using a combination of broad-specificity direct competitive enzyme-linked immunosorbent assay (dcELISA) and high-performance liquid chromatography–tandem mass spectrometry (HPLC–MS/MS) was developed. The reaction formats of dcELISA as well as the matrix effects of vegetable samples by different treatments were studied. The dcELSIA based on horseradish peroxidase-labelled monoclonal antibody and solid-phase extraction can analyse 42 samples in duplicate simultaneously for 12 OPs with a limit of detection at 20μgL−1 within 40min, with good accuracy and reproducibility. For screening purpose, the dcELISA can distinguish positive samples from hundreds of negative samples at a rapid, high-throughput and low cost manner. The positive samples can be following confirmed by HPLC–MS/MS for the kinds and the relative amounts of OPs. The method is suitable for monitoring of OP contamination in vegetables samples with high-efficiency and low cost. [Copyright &y& Elsevier]

Abstract: A simple, rapid and high-throughput fluorescent polarization immunoassay (FPIA) for simultaneous determination of organophosphorus pesticides (OPs) using a broad-specificity monoclonal antibody was developed. The effects of tracer structure, tracer concentration, antibody dilution, methanol content and matrix effect on FPIA performance were studied. The FPIA can detect 5 OPs simultaneously with a limit of detection below 10ngmL−1. The time required for the equilibrium of antibody–antigen interaction was less than 10min. The recovery from spiked vegetable and environmental samples ranged from 71.3% to 126.8%, with the coefficient of variations ranging from 3.5% to 14.5%. The developed FPIA was applied to samples, followed by confirmation with high-performance liquid chromatography–tandem mass spectrometry (HPLC–MS/MS) analysis. The developed FPIA demonstrated good accuracy and reproducibility, and is suitable for rapid and high-throughput screening for OP contamination with high-efficiency and low cost. [Copyright &y& Elsevier]

Abstract: An immunizing hapten (4-(carboxymethoxy)phenyl)bis(4-(diethylamino)phenyl)methylium for brilliant green (BG), a triphenylmethane dye with a potential illegal use in fish feeding, was synthesized and used to produce polyclonal antibody (PcAb) against BG. Unexpectedly, the obtained PcAb showed high cross-reactivity (CR) to malachite green (MG) and crystal violet (CV) in an indirect competitive enzyme-linked immunosorbent assay (icELISA). After screening against three heterologous coating antigens, the icELISA exhibited good sensitivity and uniform response to BG (IC50 of 1.98ngmL−1 and CR of 100%), MG (IC50 of 1.61ngmL−1 and CR of 105%) and CV (IC50 of 1.34ngmL−1 and CR of 142%) when using (4-(carboxymethoxy)phenyl)bis(4-(dimethylamino)phenyl)methylium as the coating hapten. Therefore, a broad-specificity icELISA for simultaneous determination of BG, MG and CV was developed. The recoveries of single analyte and mixture of three analytes from spiked grass carp tissues were estimated ranging from 74.94% to 110.39%. A statistically significant correlation of results was obtained between the developed icELISA and previously established HPLC approaches with the food-relevant three triphenylmethane dyes concentration range 1.83–200ngmL−1 (R 2 =0.9224), indicating good accuracy of the icELISA and suitability for the broad-specific detection of the three triphenylmethane dyes in grass carp tissues. [Copyright &y& Elsevier]

The type of hapten linkage to a carrier protein can play an important role in determining the nature of the resulting antibody response. Generic haptens using three types of linkers were synthesized (a monocarboxylic acid, an unsaturated hydrocarbon and a carboxamido spacer). These haptens were conjugated to bovine serum albumin (BSA) and used as immunogens to produce broad-specificity monoclonal antibodies (MAbs) to organophosphorus pesticides (OPs). Three-dimensional (3D) structures of hapten-lysine conjugates were optimized using molecular modeling (MM) to mimic conformations of hapten-BSA conjugates. The results from MM studies revealed a change of the 3D conformation and electrostatic potential of hapten 1 when the monocarboxylic acid linker was coupled to lysine. This result was consistent with the observed high-cross-reactivity of the corresponding MAb–H1 for the OPs. The competitive indirect enzyme-linked immunosorbent assay based on MAb–H1 is ideally suited to be used as a screening method for OP contaminants. [ABSTRACT FROM AUTHOR]

Abstract: Immunoassay for low molecular weight food contaminants, such as pesticides, veterinary drugs, and mycotoxins is now a well-established technique which meets the demand for a rapid, reliable, and cost-effective analytical method. However, due to limited understanding of the molecular structure of antibody binding sites and antigenic epitopes, as well as the intermolecular binding forces that come into play, the traditional ‘trial and error’ method used to develop antibodies still remains the method of choice. Therefore, development of enhanced immunochemical techniques for specific- and generic-assays, requires new approaches for antibody design that will improve affinity and specificity of the antibody in a more rapid and economic manner. Computer-assisted molecular modeling (CAMM) has been demonstrated to be a useful tool to help the immunochemist develop immunoassays. CAMM methods can be used to help direct improvements to important antibody features, and can provide insights into the effects of molecular structure on biological activity that are difficult or impossible to obtain in any other way. In this review, we briefly summarize applications of CAMM in immunoassay development, including assisting in hapten design, explaining cross-reactivity, modeling antibody–antigen interactions, and providing insights into the effects of the mouse body temperature on the three-dimensional conformation of a hapten during antibody production. The fundamentals and theory, programs and software, limitations, and prospects of CAMM in immunoassay development were also discussed. [Copyright &y& Elsevier]

Abstract: Objectives: To evaluate the effects of fluoridated bleaching agents and post-bleaching fluoridation treatment on the whitening efficiency and microhardness of bovine enamel. Methods: Twenty five freshly extracted bovine incisors were cut into halves, embedded and then divided into the following five groups: Group 1, untreated controls; Group 2, treatment with 10% carbamide peroxide (CP) bleaching agent; Group 3, treatment with 10% CP followed by a 0.9% sodium fluoride gel application, Group 4, treatment with 10% CP containing 0.11% fluoride; Group 5, treatment with an experimental bleaching agent consisting of 10% CP and 0.37% fluoride. Groups 2–5 were treated 8h per day for 14 days then immersed in saliva for 2 weeks. Enamel morphology changes were evaluated under SEM on Day 14. Changes in enamel color and microhardness were evaluated on Days 7 and 14, and compared with the baseline data. Additionally, microhardness was determined on post-bleaching Days 21 and 28. Results: After 2 weeks, an erosion pattern was noted on the specimens in Groups 2 and 3. Groups 4 and 5 showed a milder demineralized pattern. All the bleached enamel specimens revealed increased whiteness and overall color value. Groups 2 and 3 showed significantly decreased enamel microhardness compared to their baseline data. The specimens treated with fluoridated bleaching agents showed relatively less reduction in enamel microhardness than those treated with nonfluoridated agents during the bleaching treatment. Conclusions: The fluoridated bleaching agents produced less demineralization of surface morphology and microhardness. The addition of fluoride did not impede the whitening effect. [Copyright &y& Elsevier]

Filoviruses are enveloped viruses that cause sporadic outbreaks of severe hemorrhagic fever [CDC, MMWR Morb. Mortal. Wkly. Rep. 50:73-77, 2001; Colebunders and Borchert, J. Infect. 40:16-20, 2000; Colebunders et al., J. Infect. Dis. 196(Suppl. 2):S148-S153, 2007; Geisbert and Jahrling, Nat. Med. 10:S110-S121, 2004]. Previous studies revealed that endosomal cysteine proteases are host factors for ebolavirus Zaire (Chandran et al., Science 308:1643-1645, 2005; Schornberg et al., J. Virol. 80:4174-4178, 2006). In this report, we show that infection mediated by glycoproteins from other phylogenetically diverse filoviruses are also dependent on these proteases and provide additional evidence indicating that they cleave GP1 and expose the binding domain for the critical host factor Niemann-Pick C1. Using selective inhibitors and knockout-derived cell lines, we show that the ebolaviruses Zaire and Cote d'Ivoire are strongly dependent on cathepsin B, while the ebolaviruses Sudan and Reston and Marburg virus are not. Taking advantage of previous studies of cathepsin B inhibitor-resistant viruses (Wong et al., J. Virol. 84:163-175, 2010), we found that virus-specific differences in the requirement for cathepsin B are correlated with sequence polymorphisms at residues 47 in GP1 and 584 in GP2. We applied these findings to the analysis of additional ebolavirus isolates and correctly predicted that the newly identified ebolavirus species Bundibugyo, containing D47 and I584, is cathepsin B dependent and that ebolavirus Zaire-1995, the single known isolate of ebolavirus Zaire that lacks D47, is not. We also obtained evidence for virus-specific differences in the role of cathepsin L, including cooperation with cathepsin B. These studies strongly suggest that the use of endosomal cysteine proteases as host factors for entry is a general property of members of the family Filoviridae. [ABSTRACT FROM AUTHOR]

Nanobody (Nb), a new type of biorecognition element generally from Camelidae, has the characteristics of small molecular weight, high stability, great solubility and high expression level in E. coli. In this study, with 19-nortestosterone (19-NT), an anabolic androgenic steroid as target drug, three specific Nbs against 19-NT were selected from camel immune library by phage display technology. The obtained Nbs showed excellent thermostability and organic solvent tolerance. The nanobody Nb2F7 with the best performance was used to develop a sensitive indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) for 19-NT detection. Under optimized conditions, the standard curve of ic-ELISA was fitted with a half-maximal inhibitory concentration (IC50) of 1.03 ng/mL and a detection limit (LOD) of 0.10 ng/mL for 19-NT. Meanwhile, the developed assay had low cross- reactivity with analogs and the recoveries of 19-NT ranged from 82.61% to 99.24% in spiked samples. The correlation coefficient between ic-ELISA and the ultra-performance liquid chromatography/mass spectrometry (UPLC-MS/MS) method was 0.9975, which indicated that the nanobody-based ic-ELISA could be a useful tool for a rapid analysis of 19-NT in animal urine samples. [ABSTRACT FROM AUTHOR]

A series of haptens were rationally designed for producing monoclonal antibodies specific for EC and a simple fluorescence immunoassay platform was developed for the sensitive determination of EC based on alkaline phosphatase (ALP)-triggered Cu+ quenching of CdSe quantum dots (QDs). It was noted that Cd as a fluorescence substrate in CdSe QDs can be selectively substituted by Cu+ that resulted in a more significant fluorescence quenching in comparison with Cu2+. Meanwhile, because ALP catalyzed ascorbic acid phosphate and then assisted the transformation of Cu2+ to Cu+, the change in fluorescence intensity was found to be proportional to ALP concentration. After simple magnetic separation, the sensitivity and linear range of the established assay were improved approximately 53-fold and an order of magnitude, respectively, when compared with the conventional ELISA. The proposed platform was able to both amplify the signal and eliminate matrix interferences, making it a promising to determine EC as well as other contaminants in complex food matrix in a highly sensitive and simple manner. [ABSTRACT FROM AUTHOR]

Histamine (HA) is an important food contaminant generated during food fermentation or spoilage. However, an immunoassay for direct (derivatization free) determination of HA has rarely been reported due to its small size to induce the desired antibodies by its current hapten-protein conjugates. In this work, despite violating the classical hapten design criteria which recommend introducing a linear aliphatic (phenyl free) linker into the immunizing hapten, a novel haptens, HA-245 designed and synthesized with a phenyl-contained linker, exhibited significantly enhanced immunological properties. Thus, a quality-improved monoclonal antibody (Mab) against HA was elicited by its hapten-carrier conjugates. Then, as the linear aliphatic linker contained haptens, Hapten B was used as linker-heterologous coating haptens to eliminate the recognition of linker antibodies. Indirect competitive ELISA (ic-ELISA) was developed with a 50% inhibition concentration (IC50) of 0.21 mg/L and a limit of detection (LOD) of 0.06 mg/L in buffer solution. The average recoveries of HA from spiked food samples for this ic-ELISA ranged from 84.1% and 108.5%, and the analysis results agreed well with those of referenced LC-MS/MS. This investigation not only realized derivatization-free immunoassay for HA, but also provided a valuable guidance for hapten design and development of immunoassay for small molecules. [ABSTRACT FROM AUTHOR]

Nanobodies are one-tenth the size of conventional antibodies and are naturally obtained from the atypical heavy-chain-only antibodies present in camelids. Their small size, high solubility, high stability, and strong resilience to organic solvents facilitate their use as novel analytical reagents in immunochemistry. In this study, specific nanobodies against pesticide carbofuran were isolated and characterized from an immunized library via phage display platform. We further established an indirect competitive enzyme-linked immunosorbent assay (ELISA) using nanobody Nb316 to detect carbofuran in vegetable and fruit samples. The results showed a half-maximal inhibitory concentration (IC50) of 7.27 ng/mL and a detection limit of 0.65 ng/mL. A simplified sample pretreatment procedure omitting the evaporation of organic solvent was used. The averaged recovery rate of spiked samples ranged between 82.3% and 103.9%, which correlated with that of standard UPLC–MS/MS method. In conclusion, a nanobody with high specificity for carbofuran was characterized, and a nanobody-based sensitive immunoassay for simple and rapid detection of carbofuran in real samples was validated. [ABSTRACT FROM AUTHOR]