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As an alternative to radioimmunoassays, a simple, highly sensitive and quick enzymeimmunoassay (EIA) for determination of cortisol in blood plasma of yaks on microtiterplates using second antibody coating technique and cortisol-horseradish peroxidase as a label has been developed. The wells of the microtiterplate were coated with affinity-purified goat IgG (antirabbit IgG) that binds the hormone specific antibody. The EIA was carried out directly in 20 microl of heat treated plasma after 1:5 dilution with PBS. The cortisol standard curve, with doses ranged from 0.4 to 100 pg/well. The sensitivity of the assay was 20 pg/ml. Cortisol standard curve in buffer showed parallelism with serially diluted yak plasma containing high endogenous cortisol. Intra- and inter-assay coefficients of variation (CV) determined using pooled plasma was found 6.58 and 11.35%, respectively. Recovery of known concentrations of added cortisol in charcoal stripped plasma was linear (r = 0.98). For biological validation of cortisol enzymeimmunoassay, the blood samples were collected from yak cows at -48 and -24h before and 0, 12, 24, 36, 48, 60, 72, 84 and 96 h after dexamethasone administration. The plasma cortisol before dexamethasone administration was significantly (P < 0.05) higher than after dexamethasone administration. The developed EIA was further validated biologically by estimating cortisol in peri-parturient cows beginning day 10 prior to calving till day 10 post-calving; the concentrations were along with the expected lines as reported in bovine. In conclusion, the EIA developed in this study is simple, highly sensitive, valid and sufficiently reliable method for estimation of cortisol directly in bovine plasma.
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PMID:Application of sensitive enzymeimmunoassay for determination of cortisol in blood plasma of yaks (Poephagus grunniens L.). 1765 48

In order to study the effects of phosphorothioated antisense oligodeoxynucleotides (ASODN) on the expression of VEGF in human lymphoma cell line Namalwa cells, human lymphoma cell line Namalwa cells were incubated with VEGF ASODN (the final concentrations of VEGF ASODN were 5, 10, 20 micromol/L respectively), or scrambled sequence for 24 or 48 hours. The expressions of VEGF mRNA and VEGF protein were detected by reverse transcriptase-polymerase chain reaction and streptavidin peroxidase (SP) immunohistochemistry respectively. The results showed that the expression levels of VEGF mRNA in Namalwa cells treated with three concentration levels (5, 10, 20 micromol/L of ASODN) were 1.38, 0.96 and 0.57 respectively. Those in PBS-treated cells and scrambled sequence treated cells were 1.79 and 1.84. When treated with 20 micromol/L VEGF ASODN for 48 hours, VEGF protein of Namalwa cells decreased greatly. Meanwhile, there was no obvious change in the scrambled sequence treated group. It is concluded that VEGF ASODN can suppress the VEGF expression in Namalwa cells in vitro.
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PMID:Effect of antisense oligodeoxynucleotides on the expression of vascular endothelial growth factor in Namalwa cell in vitro. 1770 18

Staphylococcal enterotoxin B (SEB) is one of many toxins produced by the Gram-positive bacterium Staphylococcal aureus. While SEB is known as the causative agent of certain food poisonings it is also considered abiological select agent. Thus, rapid and accurate identification of SEB during either surveillance or in response to a biothreat is critical to the mitigation of the suspect agent. This report presents a new conductometric immune-biosensor for the detection of SEB based on immobilization of horseradish peroxidase (HRP)-labeled SEB antibody (HRP-anti-SEB) onto nanogold/chitosan-multiwalled carbon nanotube (Au/CTS-MWNT)-functionalized biorecognition interface. The formation of the antibody-antigen complex by a simple one-step immunoreaction between the immobilized HRP-anti-SEB and SEB in sample solution introduced a barrier of electrical communication between the immobilized HRP and the base surface, thus local conductivity variations could be evaluated by the bio-electrocatalytic reaction of HRP in 0.02 M PBS (pH 6.8) containing 0.15 mM H(2)O(2), 0.06 M KI and 0.1 M NaCl. Under optimal conditions, the proposed immune-biosensor exhibited a good conductometric response relative to SEB concentration in a linear range from 0.5 to 83.5 ng/ml with a correlation coefficient of 0.998. The developed immune-biosensor showed an acceptable accuracy, reproducibility and stability. Milk samples spiked with various concentrations of SEB gave an average of 116% recovery of the toxin.
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PMID:Conductometric immunosensors for the detection of staphylococcal enterotoxin B based bio-electrocalytic reaction on micro-comb electrodes. 1794 20

Gold nanoparticles stabilized by chitosan (AuCS) were hybridized with exfoliated clay nanoplates through electrostatic interaction. The resulting clay-chitosan-gold nanoparticle nanocomposite (Clay/AuCS) was used to modify glassy carbon electrode (GCE). HRP, a model peroxidase, was entrapped between the Clay/AuCS film and another clay layer. UV-vis spectrum suggested HRP retained its native conformation in the modified film. Basal plane spacing of clay obtained by X-ray diffraction (XRD) indicated that there was an intercalation-exfoliation-restacking process among HRP, AuCS and clay during the modified film drying. The immobilized HRP showed a pair of quasi-reversible redox peaks at -0.195 V (vs. saturated Ag/AgCl electrode) in 0.1M PBS (pH 7.0), and the biosensor displayed a fast amperometric response to H(2)O(2) with a wide linear range of 39 microM to 3.1 mM. The detection limit was 9.0 microM based on the signal to noise ratio of 3. The kinetic parameters such as alpha (charge transfer coefficient), k(s) (electron transfer rate constant) and K(m) (Michaelis-Menten constant) were evaluated to be 0.53, 2.95+/-0.20s(-1) and 23.15 mM, respectively.
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PMID:Direct electrochemistry and electrocatalysis of horseradish peroxidase based on clay-chitosan-gold nanoparticle nanocomposite. 1805 82

A disposable amperometric immunosensing strip was fabricated for rapid detection of Escherichia coli O157:H7. The method uses an indirect sandwich enzyme-linked immunoassay with double antibodies. Screen-printed carbon electrodes (SPCEs) were framed by commercial silver and carbon inks. For electrochemical characterization the carbon electrodes were coupled with the first E. coli O157:H7-specific antibody, E. coli O157:H7 intact cells and the second E. coli O157:H7-specific antibody conjugated with horseradish peroxidase (HRP). Hydrogen peroxide and ferrocenedicarboxylic acid (FeDC) were used as the substrate for HRP and mediator, respectively, at a potential +300 mV vs. counter/reference electrode. The response current (RC) of the immunosensing strips could be amplified significantly by 13-nm diameter Au nanoparticles (AuNPs) attached to the working electrode. The results show that the combined effects of AuNPs and FeDC enhanced RC by 13.1-fold. The SPCE immunosensing strips were used to detect E. coli O157:H7 specifically. Concentrations of E. coli O157:H7 from 10(2) to 10(7)CFU/ml could be detected. The detection limit was approximately 6CFU/strip in PBS buffer and 50CFU/strip in milk. The SPCE modified with AuNPs and FeDC has the potential for further applications and provides the basis for incorporating the method into an integrated system for rapid pathogen detection.
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PMID:Disposable amperometric immunosensing strips fabricated by Au nanoparticles-modified screen-printed carbon electrodes for the detection of foodborne pathogen Escherichia coli O157:H7. 1842 27

Horseradish peroxidase (HRP) was successfully immobilized on vertically oriented TiO(2) nanotube arrays (NTAs), which was prepared by a seeded-growth mechanism. The nanotubular structure of TiO(2) was characterized by scanning electron microscope (SEM). After encapsulated HRP on TiO(2) nanotube arrays, the direct electron transfer of HRP was observed. Owing to the redox reaction of electroactive center of HRP, the HRP/TiO(2) NTAs modified electrode exhibited a pair of quasi-reversible peaks with the peak-to-peak separation of 70 mV and the formal potential of -0.122 V (vs. SCE) in 0.2mol L(-1) phosphate buffer solution (PBS, pH 7.0). The number of transference electron was 0.84 and the direct electron transfer (ET) constant (k(s)) was 3.82 s(-1). The HRP/TiO(2) NTAs modified electrode displayed an excellent electrocatalytic performance for H(2)O(2) and the formal Michaelis-Menten constant (K(m)(app)) was 1.9 mmol L(-1). The response currents had a good linear relation with the concentration of H(2)O(2) from 5.0 x 10(-7) mol L(-1) to 1.0 x 10(-5) mol L(-1) and 5.0 x 10(-5) mol L(-1) to 1.0 x10(-3) mol L(-1), respectively.
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PMID:Direct electrochemistry of horseradish peroxidase on TiO(2) nanotube arrays via seeded-growth synthesis. 1848 91

The excitatory amino acid glutamate plays an important role in the development of neuronal sensitization and the ionotropic N-methyl-d-aspartate receptor (NMDAR) is one of the major receptors involved. The objective of this study was to use a cat model of gastroesophageal reflux disease (GERD) to investigate the expression of the NR1 and NR2A subunits of NMDAR in the vagal and spinal afferent fibers innervating the esophagus. Two groups of cats (Acid-7D and PBS-7D) received 0.1 N HCl (pH 1.2) or 0.1 M PBS (pH 7.4) infusion in the esophagus (1 ml/min for 30 min/day for 7 days), respectively. NR1 splice variants (both NH(2) and COOH terminals) and NR2A in the thoracic dorsal root ganglia (DRGs), nodose ganglia (NGs), and esophagus were evaluated by RT-PCR, Western blot, and immunohistochemistry. Acid produced marked inflammation and a significant increase in eosinophil peroxidase and myeloperoxidase contents compared with PBS-infused esophagus. The NR1-4 splice variant gene exhibited a significant upregulation in DRGs and esophagus after acid infusion. In DRGs, NGs, and esophagus, acid infusion resulted in significant upregulation of NR1 and downregulation of NR2A subunit gene expression. A significant increase in NR1 polypeptide expression was observed in DRGs and NGs from Acid-7D compared with control. In conclusion, long-term acid infusion in the cat esophagus resulted in ulcerative esophagitis and differential expressions of NR1 and NR2A subunits. It is possible that these changes may in part contribute to esophageal hypersensitivity observed in reflux esophagitis.
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PMID:Alterations in N-methyl-D-aspartate receptor subunits in primary sensory neurons following acid-induced esophagitis in cats. 1897 10

Pulmonary LPS exposure plays a key role in exacerbation of lung diseases such as chronic obstructive pulmonary disease and asthma. However, little is known about the effects of repeated LPS exposure in the lung microenvironment. We have developed a novel murine model of pulmonary LPS tolerance induced by intratracheal (i.t.) administration of LPS. First, we show that pulmonary LPS exposure does not induce whole-body refractoriness to systemic LPS, because i.t. administration followed by i.p. administration did not decrease plasma TNF-alpha. However, a local refractory state can be induced with two i.t. LPS exposures. Pulmonary LPS tolerance was induced by i.t. administration of 100 ng LPS at time 0 and 48 h. Nontolerant mice received PBS at time 0 and LPS at 48 h. Bronchoalveolar lavage levels of TNF-alpha were significantly attenuated in tolerant mice vs nontolerant mice (1597 pg/ml vs 7261 pg/ml). TNF-alpha mRNA was significantly reduced in bronchoalveolar lavage cells (5-fold) and lung tissue (10-fold). No reduction was seen in neutrophil numbers in the bronchoalveolar lavage fluid, myeloperoxidase activity, or expression of neutrophil chemoattractants CXCL1 and CXCL2, reflecting the specificity of the response. The reduction in TNF-alpha was accompanied by a significant increase in soluble receptors, TNF-SRI (159 pg/ml vs 206 pg/ml) and TNF-SRII (1366 pg/m vs 2695 pg/ml). In conclusion, pulmonary LPS tolerance results in a specific reduction in TNF-alpha expression, while the neutrophilic response is unaffected. This response may be a mechanism to limit tissue damage by reducing TNF-alpha levels, while still maintaining the antimicrobial capacity of the lung.
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PMID:Acute pulmonary lipopolysaccharide tolerance decreases TNF-alpha without reducing neutrophil recruitment. 1905 Feb 57

A simple sensor method was developed for aflatoxin M(1) analysis to be applied directly with milk by using antibody modified screen-printed carbon working electrode with carbon counter and silver-silver chloride pseudo-reference electrode. A competitive ELISA assay format was constructed on the surface of the working electrode using 3,3,5',5'-tetramethylbenzidine dihyrochloride (TMB)/H(2)O(2) electrochemical detection scheme with horseradish peroxidase (HRP) as the enzyme label. The performance of the assay and the sensor was optimised and characterised in pure buffer conditions before applying to milk samples. Extensive interference to the electroanalytical signal was observed upon the analysis of milk. Through a series of chemical fractionations of the milk, and testing the electrochemical properties of the fractions, the interference was attributed to whey proteins with focus towards alpha-lactalbumin. A simple pre-treatment technique of incorporating 18 mM calcium chloride, in the form of Dulbucco's PBS, in a 1:1 ratio to the milk sample or standards and also to the washing buffer stabilised the whey proteins in solution and eliminate the interfering signal. The resulting immunosensor was interference free and achieved a limit of detection of 39 ng l(-1) with a linear dynamic detection range up to 1000 ng l(-1). The developed immunosensor method was compared to a commercial ELISA kit and an in-house HPLC method. The immunsensor was comparable, in term of sensitivity, but vastly superior in term of portability and cost therefore a key instrument for the detection of aflatoxin M(1) at the source of the contamination.
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PMID:Development of an electrochemical immunosensor for aflatoxin M1 in milk with focus on matrix interference. 1916 7

Lung contusion is a common problem from blunt chest trauma caused by mechanical forces and by exposure to blast overpressure, often with fatal consequences. Lung contusion is also a risk factor for the development of pneumonia, severe clinical acute lung injury (ALI), and acute respiratory distress syndrome (ARDS). Infiltrating neutrophils are considered to be central mediators of lung injuries after blunt trauma. Recent studies have demonstrated that antioxidants reduced pulmonary inflammation in different models of lung damage. This study examined the effect of antioxidant N-acetylcysteine amide (NACA) on the progression of lung inflammation after exposure to a moderate level of blast overpressure (140 kPa). Rats were administered with NACA (i.p. 100 mg/kg) or placebo (PBS) 30, 60 min and 24 h after exposure. Nonblasted sham-injected animals served as controls. Neutrophil infiltration measured by myeloperoxidase (MPO) activity in the lung was significantly increased at 2 days after blast and returned to controls at 8 days. This increase corresponded with activation of integrin CD11b mRNA and lung inflammatory chemokine mRNA expression; macrophage inflammatory protein-1 (MIP-1), monocyte chemotactic peptide-1 (MCP-1), and cytokine-induced neutrophil chemoattractant-1 (CINC-1). At 8 days, all inflammatory mediators returned to control levels. In addition, expression of heme oxygenase-1 (HO-1) mRNA increased at 2 days after exposure. No changes were detected in the lung manganase superoxide dismutase (MnSOD) or glutathione reductase (GR) mRNA expression after blast. N-Acetylcysteine amide significantly reduced infiltration of neutrophils and CD11b mRNA activation in lungs, and completely blocked activation of MIP-1, MCP-1 and CINC-1 mRNA. The relatively higher inhibition of chemokine mRNAs compared with reduction in MPO activity and CD11b is in accordance with an antioxidant effect of NACA on reactive oxygen species (ROS) accumulation, rather than by an effect on neutrophil sequestration. The inhibition of HO-1 mRNA activation after blast was likely also related to the drug antioxidant effect.
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PMID:Attenuation of pulmonary inflammation after exposure to blast overpressure by N-acetylcysteine amide. 1917 37


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