Immunochromatographic Assay Based on Polydopamine-Decorated Iridium Oxide Nanoparticles for the Rapid Detection of Salbutamol in Food Samples.

Salbutamol (SAL), a ß-2 adrenoreceptor agonist, is an unpopular addition to livestock and poultry, causing several side effects to human health. Thus, it is very important to develop a simple and rapid analytical method to screen SAL in the field of food safety. Here, we present an immunochromatographic assay (ICA) method for sensitively detecting SAL with polydopamine-decorated iridium oxide nanoparticles (IrO2@PDA NPs) as a signal tag. The IrO2@PDA with excellent hydrophilicity, biocompatibility, and stability was synthesized by oxidating self-polymerization of dopamine hydrochloride (DAH) on the surface of IrO2 NPs and used to label monoclonal antibodies (mAbs) through simple physical adsorption. Compared with IrO2 NPs, the IrO2@PDA also possessed superior optical properties and higher affinity with mAbs. With the proposed method, the limit of detection for SAL was 0.002 ng/mL, which was improved at least 24-fold and 180-fold compared with the IrO2 NPs-based ICA and conventional gold nanoparticles-based ICA, respectively. Furthermore, the SAL residuals in pork, pork liver, and beef were successfully detected by the developed biosensor and the recoveries ranged from 85.56% to 115.56%. Briefly, this work indicated that the powerful IrO2@PDA-based ICA can significantly improve detection sensitivity and has huge potential for accurate and sensitive detection of harmful small molecules analytes in food safety fields.

Label-Free Impedimetric Immunosensors Modulated by Protein A/Bovine Serum Albumin Layer for Ultrasensitive Detection of Salbutamol.

The sensing properties of immunosensors are determined not only by the amount of immobilized antibodies but also by the number of effective antigen-binding sites of the immobilized antibody. Protein A (PA) exhibits a high degree of affinity with the Fc part of IgG antibody to feasibly produce oriented antibody immobilization. This work proposes a simple method to control the PA surface density on gold nanostructure (AuNS)-deposited screen-printed carbon electrodes (SPCEs) by mixing concentration-varied PA and bovine serum albumin (BSA), and to explore the effect of PA density on the affinity attachment of anti-salbutamol (SAL) antibodies by electrochemical impedance spectroscopy. A concentration of 100 µg/mL PA and 100 µg/mL BSA can obtain a saturated coverage on the 3-mercaptoproponic acid (MPA)/AuNS/SPCEs and exhibit a 50% PA density to adsorb the amount of anti-SAL, more than other concentration-varied PA/BSA-modified electrodes. Compared with the randomly immobilized anti-SAL/MPA/AuNS/SPCEs and the anti-SAL/PA(100 µg/mL):BSA(0 µg/mL)/MPA/AuNS/SPCE, the anti-SAL/PA(100 µg/mL): BSA(100 µg/mL)/MPA/AuNS/SPCE-based immunosensors have better sensing properties for SAL detection, with an extremely low detection limit of 0.2 fg/mL and high reproducibility (<2.5% relative standard deviation). The mixture of PA(100 µg/mL):BSA(100 µg/mL) for the modification of AuNS/SPCEs has great promise for forming an optimal protein layer for the oriented adsorption of IgG antibodies to construct ultrasensitive SAL immunosensors.

Serious adverse reaction to timolol eye drops in a 7-year-old boy with glaucoma and asthma

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Dual-signal amplified electrochemiluminescence immunoassay for salbutamol based on quantum dots and gold nanoparticle-labeled horseradish peroxidase.

This study describes a novel electrochemical immunosensor to amplify the electrochemiluminescence (ECL) signal for the ultrasensitive detection of salbutamol (SAL) using quantum dots (QDs) and gold nanoparticle (AuNP) conjugated horseradish peroxidase (HRP). The electrochemical detection was based on the HRP catalyzed consumption of self-produced H2O2, which has been extensively used as a co-reactant of QDs, by o-phenylenediamine (OPD). The enzymatic reaction rate is proportional to the amount of HRP bound to the electrode. In the presence of a SAL standard solution, the immobilized SAL coating antigens competed with the SAL solution for the Ab-AuNPs-HRP complexes. With an increase in the SAL concentration, the amount of immobilized HRP decreases, which leads to an increase in the ECL intensity. Under optimized conditions, the ECL intensity changes linearly with the logarithm of the SAL concentration in the range of 0.05-500 ng mL(-1) with a detection limit of 0.017 ng mL(-1) (S/N = 3). The ECL immunosensor possesses high sensitivity, satisfactory reproducibility and selectivity, and may provide a feasible route for practical application.

Highly selective and sensitive detection of ß-agonists using a surface plasmon resonance sensor based on an alkanethiol monolayer functionalized on a Au surface.

Immunosensor surfaces for surface plasmon resonance (SPR) have been constructed using a functionalized succinimidyl propanethiol monolayer as a linker to immobilize ß-agonist protein conjugates on a Au surface. Because ß-agonist is a small molecule, an indirect competitive inhibition immunoassay was used for detection. The lowest detection limits for ractopamine and salbutamol were 10 ppt (10 pg mL(-1)) and 5 ppt (5 pg mL(-1)), respectively. The fabricated immunosensor surface can be used again for detection after regeneration in 0.1 M sodium hydroxide. It was found that the same sensor surface could be reused for performing over 100 rapid immunoreactions. Moreover, one immunosensing-regeneration cycle requires only 600 s. The fabricated immunosensor surfaces were characterized using SPR and scanning tunneling microscopy observation. In the kinetic study of the indirect competitive immunosensing inhibition, the affinity constant (K1) of salbutamol antibody was smaller than the K1 of ractopamine antibody. Compared to a previous study of clenbuterol detection, it was concluded that the high K1 was coupled with low sensitivity. In the selectivity study, both immunosensor surfaces provided >90% of confidence level for the specific detection of ß-agonist compounds. The fabrication of highly selective and sensitive sensor surfaces for detecting ß-agonist compounds was confirmed.

[Preparation of salbutamol polyclonal antibodies and development of indirect competitive enzyme-linked immunoassay].

OBJECTIVE: To prepare the antibodies against salbutamol (SAL) with high sensitivity and to develop an indirect competitive enzyme-linked immunoassay (ic-ELISA) for fast detection of SAL. METHODS: The New Zealand white rabbits were immunized with SAL in a small dose and long period mode. The method of ic-ELISA was optimized and adopted for the detection of a series of SAL samples, then the standard curve of SAL was established. The precision and the recoveries of the method were determined. RESULTS: The antibodies with high sensitivity towards SAL were prepared with a IC50 of 12.21 ng/ml. The ic-ELISA method for SAL measurement was established, the recoveries of measurement was between 95%-105% and the CV was <3%. CONCLUSION: The antibodies against salbutamol have been prepared and an indirect competitive enzyme-linked immunoassay for fast and specific detection of SAL has been developed.

Effect of inhaled inactivated Mycobacterium phlei in children with moderate asthma.

Bacillus Calmette-Guérin and other mycobacterial vaccines are important therapeutic methods in a series of chronic inflammatory disorders characterized by Th1/Th2 imbalance in which Th2 type cells and cytokines often increase. However, few studies have investigated whether it can reduce or prevent the symptoms and attacks in children with asthma. This study evaluated the effect of inactivated Mycobacterium phlei inhaled by an atomizing device placed on asthmatic children. In this randomized, single-center, Seretide-controlled study, children aged 4-12 years with newly diagnosed, moderate, persistent asthma were treated with either inhaled inactivated M. phlei or inhaled Seretide patch. The efficacy of inhaled inactivated M. phlei was related with the alleviation of asthma symptoms, improvement of lung function and reduction of bronchial hyper-responsiveness and total serum IgE, which was similar with Seretide. These findings may have important clinical value in confirming inhaled inactivated M. phlei as a new therapeutic method in moderately asthmatic children.

Highly sensitive near-simultaneous assay of multiple "lean meat agent" residues in swine urine using a disposable electrochemiluminescent immunosensors array.

Nowadays, ß(2)-agonists are abused illegally as "lean meat agents" for food-producing animals, and cause increasing food-safety accidents in some countries. Due to their hazard to the human health, "lean meat agents" are banned in most countries and required to be routinely monitored. We herein report a disposable electrochemiluminescent immunosensors array for near-simultaneous assay of multiple ß(2)-agonist residues in swine urine, by using ractopamine and salbutamol as the models. In this investigation, a screen-printed carbon electrodes array was assembled and acted as the substrate of the immunosensors array. Then the immunosensors array was constructed by site-selectively immobilizing the antigens of ractopamine and salbutamol on the working electrodes of array. After the competitive immuno-binding, with the aid of a homemade single-pore-four-throw switch, the electrochemiluminescent signals of the two ß(2)-agonists were sequentially detected using a non-array detector. The limits of detection for ractopamine and salbutamol were 8.5 and 17pg/mL, respectively, which were much lower than those of the most previous reports. Compared with other routine methods based on chromatography and ELISA, this method is more suitable for screening of multiple ß(2)-agonists in quantities of samples, owing to its merits of low cost, user-friendliness and high throughput, and shows great promise in food safety and agonist surveillance.

A high-affinity anti-salbutamol monoclonal antibody: key to a robust lateral-flow immunochromatographic assay.

Among the components that make up a lateral-flow immunochromatographic assay (ICA), antibody is the key. In this paper, salbutamol (SAL) as a model analyte was meticulously designed to prepare immunogen and coating antigen in distinctly different ways. Four hybridoma cell lines were prepared and identified. Among them, C9 had highest affinity, best dose-response behavior, lowest limit of detection, and highest specificity and was chosen to be labeled with colloidal gold as the detector reagent and applied on the conjugate pad. Goat anti-mouse antibody and SAL-BSA conjugate were sprayed on a nitrocellulose membrane as test line and control line, respectively. Under the optimized conditions, the ICA strip was constructed based on a binding inhibition format. Color intensity on the test line was visually distinguishable from that of the negative sample within 5 min, with the visual detection limit of 1 ngml(-1) in phosphate-buffered saline. Cross-reactions with other ß-agonists were not found (<1%). The results from ICA were in a good agreement with those obtained by enzyme-linked immunosorbent assay. The developed ICA has potential as a useful on-site screening tool for SAL in swine urine.

ß2-Adrenergic agonists bias TLR-2 and NOD2 activated dendritic cells towards inducing an IL-17 immune response.

This study tested the hypothesis that activation of ß2-adrenoceptors on DCs influences NOD2 signaling along with its cross-talk with Toll-like receptor-2 resulting in altered Th cell priming ability. Th17 cells are a newly discovered lineage of CD4(+) T cells involved in defense against extracellular bacteria and also implicated in autoimmune disorders. Initiation and polarization of the adaptive immune response is controlled by innate immune recognition mediated by DCs. Previous studies demonstrated that adrenergic receptors modulate cytokine production by DCs and affect their Th cell priming ability. We show that the ß2-adrenoceptor agonist salbutamol enhanced IL-6 production in murine bone marrow-derived DCs stimulated with the nucleotide-binding oligomerization domain 2 ligand muramyl dipeptide. However, when the Toll-like receptor-2 ligand Pam3CysSK4 was added, salbutamol inhibited IL-12 but did not alter IL-6 and IL-23 expression. Gene expression analysis showed that salbutamol inhibited the p40 subunit as well as IL-12p35, while IL-23p19 and IL-6 were stimulated. Therefore, ß2-adrenoceptors modulated cytokine production resulting in a Th17 cell priming cytokine pattern. Indeed, when antigen-pulsed DCs stimulated by muramyl dipeptide or Pam3CysSK4+muramyl dipeptide in the presence of salbutamol were used for in vivo immunization, the resulting Th17/Th1 cell ratio was increased as evaluated by IL-17 and IFN-γ production. In addition, intradermal injection of norepinephrine along with Pam3CysSK4+muramyl dipeptide increased the Th17 response to an immunogenic protein and this effect was reversed by a ß2-adrenoceptor antagonist. Thus, ß2-adrenoceptors may be involved in the regulation of defense against extracellular bacteria and the pathogenesis of inflammatory diseases.

Preparation of anti-salbutamol antibody based on a new designed immunogen and development of a heterologous indirect ELISA for detection of salbutamol residue.

To synthesize salbutamol immunogen and develop an enzyme immunoassay (ELISA), a new salbutamol immunogen was synthesized using 4-aminobenzoic acid as a linker to connect hapten with carrier protein. An enzyme immunoassay based on the antibody prepared was developed and applied to detect salbutamol residue spiked in swine liver. An unusual coating antigen, clenbuterol-ovalbumin (OVA) conjugate instead of salbutamol-OVA conjugate, was used in the immunoassay and the results were discussed based on the structures of related compounds. The antibodies showed high sensitivity in the heterologous assay when using clenbuterol-OVA as a coating antigen, with an IC50 value of 8.97 ng mL(-1) toward salbutamol. The antibodies prepared showed high cross-reactivity with clenbuterol (107%) and were promising for the simultaneous determination of salbutamol and clenbuterol residues in food and food products. Recovery rates from the salbutamol-spiked swine liver samples were in the range of 70%-99%, while the intra-assay and inter-assay coefficients of variation were <13.3% and <14.3%, respectively. In summary, the antibodies of salbutamol have been successfully prepared. Sensitive and stable analysis for the detection of salbutamol residues in swine liver was obtained based on the competitive ELISA methods developed in this study.

Development of immunochromatographic assay for the on-site detection of salbutamol.

Salbutamol, one of the beta-agonists, is misused as a growth promoter in meat producing animals. In-house synthesized colloidal gold was conjugated with the polyclonal anti-salbutamol antibodies. A rapid immunochromatographic assay was developed in a competitive format. The salbutamol-BSA conjugate and goat anti-rabbit IgG were immobilized on a nitrocellulose membrane as test and control lines, respectively. The color intensity of a purple test line was inversely proportional to the amount of salbutamol presenting in the samples. The sensitivity was estimated to be about 80 ng/mL of salbutamol in PBS. The method can be useful as an "on-site" screening procedure for detection of salbutamol.

Modulation of protective immunity against herpes simplex virus via mucosal genetic co-transfer of DNA vaccine with beta2-adrenergic agonist.

Cholera toxin, which has been frequently used as mucosal adjuvant, leads to an irreversible activation of adenylyl cyclase, thereby accumulating cAMP in target cells. Here, it was assumed that beta(2)-adrenergic agonist salbutamol may have modulatory functions of immunity induced by DNA vaccine, since beta(2)-adrenergic agonists induce a temporary cAMP accumulation. To test this assumption, the present study evaluated the modulatory functions of salbutamol co-administered with DNA vaccine expressing gB of herpes simplex virus (HSV) via intranasal (i.n.) route. We found that the i.n. co-administration of salbutamol enhanced gB-specific IgG and IgA responses in both systemic and mucosal tissues, but optimal dosages of co-administered salbutamol were required to induce maximal immune responses. Moreover, the mucosal co-delivery of salbutamol with HSV DNA vaccine induced Th2-biased immunity against HSV antigen, as evidenced by IgG isotypes and Th1/Th2-type cytokine production. The enhanced immune responses caused by co-administration of salbutamol provided effective and rapid responses to HSV mucosal challenge, thereby conferring prolonged survival and reduced inflammation against viral infection. Therefore, these results suggest that salbutamol may be an attractive adjuvant for mucosal genetic transfer of DNA vaccine.

Antibody production: Low dose immunogen vs. low incorporation hapten using salmeterol as a model.

Haptens are low molecular weight compounds that are non-immunogenic and so must be conjugated to carrier molecules to elicit an immune response. Doses of 50-1000 microg protein conjugate have been suggested for immunisation of rabbits with hapten-protein immunogens. Although larger doses may give a faster response, lower doses may result in higher affinity antibodies. The amount of hapten presented to the host's immune system can be controlled by varying either the amount of immunogen administered or the quantity coupled to a fixed amount of protein. This study compares the two approaches for the production of antibodies to the beta-agonist salmeterol as a model. A range of salmeterol-HSA conjugates was prepared, with varying molar ratios of hapten:protein (80:1, 15:1, 7.5:1, 4:1), for use as immunogens. The 80:1 immunogen was administered to different animals at four concentrations (0.5, 0.2, 0.1 and 0.05 mgdose(-1)) while the remaining three immunogens were administered at 0.5 mgdose(-1). The effects of immunogen dose and hapten incorporation on the titre and affinity of the antibodies produced to salmeterol were investigated. It was found that both approaches resulted in the production of more sensitive antibodies, although reducing the degree of hapten incorporation brought about a larger reduction in antibody titre. Reducing the degree of hapten incorporation also produced a more consistent pattern of results regarding antibody sensitivity (after the sixth immunisation in this study) which may make it easier to predict the most suitable time for antibody harvesting.

Modulation of protective immunity against herpes simplex virus via mucosal genetic co-transfer of DNA vaccine with beta2-adrenergic agonist

Cholera toxin, which has been frequently used as mucosal adjuvant, leads to an irreversible activation of adenylyl cyclase, thereby accumulating cAMP in target cells. Here, it was assumed that beta2-adrenergic agonist salbutamol may have modulatory functions of immunity induced by DNA vaccine, since beta2-adrenergic agonists induce a temporary cAMP accumulation. To test this assumption, the present study evaluated the modulatory functions of salbutamol co-administered with DNA vaccine expressing gB of herpes simplex virus (HSV) via intranasal (i.n.) route. We found that the i.n. co-administration of salbutamol enhanced gB-specific IgG and IgA responses in both systemic and mucosal tissues, but optimal dosages of co-administered salbutamol were required to induce maximal immune responses. Moreover, the mucosal co-delivery of salbutamol with HSV DNA vaccine induced Th2-biased immunity against HSV antigen, as evidenced by IgG isotypes and Th1/Th2-type cytokine production. The enhanced immune responses caused by co-administration of salbutamol provided effective and rapid responses to HSV mucosal challenge, thereby conferring prolonged survival and reduced inflammation against viral infection. Therefore, these results suggest that salbutamol may be an attractive adjuvant for mucosal genetic transfer of DNA vaccine.

Adrenergic modulation of dendritic cell cancer vaccine in a mouse model: role of dendritic cell maturation.

The aim of the present study was to evaluate the role of beta2-adrenergic receptors (beta2-ARs) in the outcome of a dendritic cell (DC)-based cancer vaccine in the murine E.G7-ovalbumin (OVA) model. We found that unlike the beta2-AR expressed on antigen loaded DCs, beta2-ARs expressed in the site of DCs inoculation may influence the efficacy of the antitumor response. Intradermal injection of Staphylococcus aureus peptidoglycan along with the beta2-AR-specific antagonist ICI 118,551 increased the local innate cytokine response in tumor-bearing mice. When the adoptive transfer of immature DCs loaded with OVA followed this skin preconditioning, the antitumor response was increased and tumor growth was significantly reduced. Surprisingly, when OVA-loaded mature DCs were used, the effect of the skin preconditioning was the opposite and tumor growth was similar to that observed in control, nonimmunized mice. The extent of the antitumor response on transfer of immature or mature DCs was mediated by a different migration in the draining lymph nodes and by a distinct recruitment of endogenous DCs that resulted in a modulation of the OVA-specific cytotoxic T lymphocyte response. The unexpected tolerogenic effect exerted by mature DCs on skin preconditioning was apparently mediated by the expression of a distinctive pattern of cytokines and of the suppressive enzyme indoleamine 2, 3-dioxygenase in draining lymph nodes. In conclusion, we found that beta2-ARs inhibition along with toll-like receptor2 activation at the site of cancer vaccination may either enhance the resulting antitumor response or be tolerogenic in dependence of the maturation state of the transferred DCs.

Alveolar and airway sites of nitric oxide inflammation in treated asthma.

The goal of this study was to identify airway and alveolar site(s) of inflammation using exhaled nitric oxide (NO) as a marker in treated patients with asthma, including response to oral corticosteroids, and correlate these sites with expiratory airflow limitation. In 53 (24 male) patients with asthma, age 43 +/- 23 years (mean +/- SD) and all on inhaled corticosteroids, post 180 microg aerosolized albuterol, FEV(1) was 74 +/- 23% predicted and FEV(1)/FVC was 68 +/- 11%. Exhaled NO at 100 ml/second was 27 +/- 23 ppb (p < 0.001 compared with normal, 12 +/- 15 ppb). Bronchial NO maximal flux was 2.4 +/- 3.1 nl/second (p < 0.001 compared with normal, 0.85 +/- 0.55). Alveolar NO concentration was 7.0 +/- 7.4 ppb (p = 0.01 compared with the normal value, 3.2 +/- 2.0 ppb). There was no significant correlation between FEV(1) % predicted or lung elastic recoil and NO bronchial flux or alveolar concentration. However, there was a weak but significant correlation between NO bronchial flux and alveolar concentration (Spearman r = 0.50, p < 0.001). In 10 subjects with asthma on inhaled corticosteroids, 5 days of 30 mg prednisone resulted in isolated significant decreases in NO alveolar concentration, from 13 +/- 10 to 4 +/- 4 ppb (p = 0.002). Despite treatment, including inhaled corticosteroids, patients with asthma may have ongoing separate airway and alveolar sites of NO inflammation, the latter responsive to oral corticosteroids.

Effects of montelukast on surrogate inflammatory markers in corticosteroid-treated patients with asthma.

We evaluated whether montelukast conferred additive effects in patients with asthma receiving fluticasone/salmeterol (FP/SM) combination and FP alone. Twenty-two patients with mild to moderate asthma completed a double-blind, placebo-controlled study. After a 2-week run-in using FP 250 microg/SM 50 microg 1 puff twice daily, patients entered a randomized crossover period to receive additional montelukast 10 mg daily or placebo for 3 weeks each. For the first 2 weeks, they received FP/SM 1 puff BID, and then they received FP 250 microg 1 puff BID for the 3rd week. The primary outcome was adenosine monophosphate challenge threshold and recovery time; secondary outcomes included surrogate inflammatory markers and lung function. Compared with FP/SM run-in, adding montelukast to FP/SM was better (p < 0.05) than placebo for inflammatory markers but not for lung function. For adenosine monophosphate threshold, recovery, exhaled nitric oxide, and blood eosinophils, there were 1.4 (95% confidence interval, 1.1-1.8) geometric mean fold, 10 minutes (3-17 minutes), 2.1 parts per billion (0.2-3.9 parts per billion), and 88 (34-172) x 10(6)/L differences, respectively. The combination of FP plus montelukast was superior to FP/SM for inflammatory markers but was inferior for lung function. Thus, in patients taking FP/SM or FP, montelukast conferred complimentary effects on surrogate inflammatory markers, which were dissociated from lung function. Further studies are required to evaluate whether these effects of montelukast translate into clinical benefits.