Horseradish Peroxidase System Research Articles

Montelukast treatment (cysteinyl leukotriene receptor antagonist) in a model of food allergy: modifications in lymphatic cell population from rectal mucosa

The aim is to determine immunopathological modifications in rectal mucosa from rabbits after local challenge in ovalbumin (OVA) sensitized animals previously treated with montelukast. thirty two rabbits divided into four groups: G1: normal; G2: subcutaneously OVA sensitized; G3: sensitized, locally OVA challenged and sampled 4 hours after challenge; and G4: sensitized, locally OVA challenged and treated 4 hours before challenge with montelukast (0.15 mg/kg). Specific anti-OVA IgE levels were evaluated by passive cutaneous anaphylaxis test (PCA). In each group 200 high microscopical power fields (HPF) were counted. Results were expressed as arithmetic mean and SE. Anti -CD4, CD5, micro chain monoclonal antibodies were used. Avidin biotin horseradish peroxidase system was used. CD 4: G1: 8.3 +/- 0.06; G2: 13.4 +/- 0.08, G3: 8.25 +/- 0.06, G4: 11.8 +/- 0.02. CD 5: G1: 7.3 +/- 0.05; G2: 9.4 +/- 0.05, G3: 11.3 +/- 0.06, G4: 8.1 +/- 0.06. mu chain: G1: 10.4 +/- 0.06; G2: 3.8 +/- 0.02, G3: 6.0 +/- 0.10, G4: 2.2 +/- 0.10. In all cases, experimental groups (G3 vs. G4) presented statistical significant differences (p < 0.05). CD4+, CD5+ cells and mu chain+ decrease in experimental group (G4), probably due to lymphocyte migration inhibition to challenged mucosa. mu chain+ cell decrease could be based on B cell activation and expression of different surface immunoglobulins. Cells expressing mu chain decreased in G2 and G3 likely due to activation of B cells and subsequent expression of other immunoglobulin chains in cell surface. We conclude that obtained data are important to elucidate immunopathology of local anaphylactic reaction in rectal mucosa from systemic sensitized animals after treatment with montelukast.

Fabrication of reagentless glucose biosensors: A comparison of mono-enzyme GOD and bienzyme GOD–HRP systems

Glucose oxidase (GOD) and horseradish peroxidase (HRP), entrapped alone and simultaneously in silica sol–gel (SG) network on gold nanoparticles (GNPs) modified indium tin oxide (ITO) electrode, were used to fabricate mono-enzyme GOD and bienzyme GOD–HRP glucose biosensors. The optimal conditions for the construction and the analytical performances of the biosensors were studied and compared. The optimal GOD concentration in mixed solution for the mono-enzyme and bienzyme glucose biosensors was about 250 U/mL, and the optimal GOD/HRP ratio was approximately 2 for the bienzyme glucose biosensors. Amperometric response of glucose was evaluated by holding the biosensors at 0.1 V (versus SCE). The linear ranges of detection for glucose were between 0.05–4.0 mmol/L and 0.02–3.2 mmol/L, respectively. The sensitivity of the bienzyme glucose biosensor was approximately 2.4-fold higher than that of mono-enzyme glucose biosensor. Both glucose biosensors showed rapid response, high selectivity and long-term stability.

Determination of Nitric Oxide Using Horseradish Peroxidase by UV Second-order Derivative Spectrometry

Based on the reaction of horseradish peroxidase (HRP) with nitric oxide (NO), a novel detection method of NO has been developed. The method uses second-order derivative spectrometry in an anaerobic phosphate buffer solution. The effects of pH and HRP concentration on the determination of NO in HRP system were investigated, and the conditions for the measurements were optimized. Some possible coexisting substances, such as nitrite, nitrate and hydrogen peroxide, were tested. The linear regression equation of standard curve was found to be h = 8.89 x 10(-2)c(NO)-1.56 x 10(-3) with the relevant coefficient of 0.9966 (n = 5) in the NO concentration range of 0.085-1.3 microM. The relative standard deviations were less than 3%. Based on the standard deviation of 5 blank measurements and a signal-to-noise ratio of 3, the detection limit for NO was 0.032 microM. The proposed method was successfully applied to the determination of NO levels in serum samples.

Pleiotrophin Regulates Lung Epithelial Cell Proliferation and Differentiation during Fetal Lung Development via β-Catenin and Dlk1

The role of pleiotrophin in fetal lung development was investigated. We found that pleiotrophin and its receptor, protein-tyrosine phosphatase receptor beta/zeta, were highly expressed in mesenchymal and epithelial cells of the fetal lungs, respectively. Using isolated fetal alveolar epithelial type II cells, we demonstrated that pleiotrophin promoted fetal type II cell proliferation and arrested type II cell trans-differentiation into alveolar epithelial type I cells. Pleiotrophin also increased wound healing of injured type II cell monolayer. Knockdown of pleiotrophin influenced lung branching morphogenesis in a fetal lung organ culture model. Pleiotrophin increased the tyrosine phosphorylation of beta-catenin, promoted beta-catenin translocation into the nucleus, and activated T cell factor/lymphoid enhancer factor transcription factors. Dlk1, a membrane ligand that initiates the Notch signaling pathway, was identified as a downstream target of the pleiotrophin/beta-catenin pathway by endogenous dlk1 expression, promoter assay, and chromatin immunoprecipitation. These results provide evidence that pleiotrophin regulates fetal type II cell proliferation and differentiation via integration of multiple signaling pathways including pleiotrophin, beta-catenin, and Notch pathways.

Micro-plate magnetic chemiluminescence immunoassay and its applications in carcinoembryonic antigen analysis

A micro-plate magnetic chemiluminescence immunoassay was developed for rapid and high throughput detection of carcinoembryonic antigens (CEA) in human sera. This method was based on a sandwich immunoreaction of fluorescein isothiocyanate (FITC)-labeled anti-CEA antibodies, CEA antigens, and horseradish peroxidase (HRP)-conjugated anti-CEA antibodies in micro-plate. The immunomagnetic particles coated with anti-FITC antibodies were used as the solid phase for the immunoassay. The separation procedure was carried out by a magnetic plate adaptor and the luminol-hydrogen peroxide (H2O2)-HRP system was employed for the chemiluminescence detection. The proposed method combined the advantages of the micro-plate reactor and magnetic particle separation technology with the linear range of 5–250 ng·mL−1. The detection limit of CEA was 0.61 ng·mL−1. The coefficient of the variation was less than 7% and 13% for intra-assay and inter-assay precision, respectively. Compared with the commercial micro-plate chemiluminescent kit, the proposed method showed a good correlation.

Investigation of voltammetric enzyme-linked immunoassay system based on N-heterocyclic substrate of 2,3-diaminopyridine

A new voltammetric enzyme-linked immunoassay system using the electrochemical substrate 2,3-diaminopyridine (DAP) and horseradish peroxidase (HRP) system has been developed. DAP is oxidized with H 2O 2 catalyzed by HRP, and the resulting electroactive product produces a sensitive voltammetric peak at potential of −0.72 V ( vs. saturated calomel electrode (SCE)) in Britton–Robinson (BR) buffer solutions. The enzyme-catalyzed reaction conditions and voltammetric detection conditions have been investigated in detail. Under the selected optimum conditions, the linear range for detection of free HRP is from 6.0 × 10 −11 to 1.0 × 10 −8 g mL −1 with a detection limit of 1.0 × 10 −12 g mL −1. The new voltammetric detection system has been successfully applied for the assay of prostate specific antigen (PSA) in human serum ranging from 0.4 to 100 ng mL −1 with a detection limit of 0.1 ng mL −1, which is five times lower than that of traditional o-phenylenediamine (OPD) spectrophotometric enzyme-linked immunosorbent assay (ELISA) method. The proposed N-heterocyclic electrochemical detection system of DAP–H 2O 2–HRP has provided a new and much improved immunoassay method.

Detection of Salmonella typhimurium using an electrochemical immunosensor

An electrochemical immunosensor based on screen-printed gold working electrode with onboard carbon counter and silver–silver chloride pseudo-reference electrode for Salmonella typhimurium detection is described in this paper. Monoclonal anti-S. typhimurium antibody was immobilized using physical and covalent immobilization via amine coupling of carboxymethyldextran on the surface of the gold working electrode. A direct sandwich enzyme-linked immunosorbent assays (ELISA) format was then developed and optimized using a polyclonal anti-Salmonella antibodies conjugated to horseradish peroxidase (HRP) as the enzyme label. 3,3′,5,5′-Tetramethylbenzidine dihydrochloride (TMB)/H2O2 was used as the enzyme mediator/substrate system. Electrochemical detection was conducted using chronoamperometry at −200mV vs. onboard screen-printed Ag–AgCl pseudo-reference electrode. The applied potential was selected through the study of the electrochemical behaviour of bare gold electrode with TMB–H2O2–IgG–HRP system. S. typhimurium detection of 5×103cellsml−1 and ∼20cellsml−1 was achieved respectively for physical and covalent antibody immobilization. The developed sensor was then compared to a commercial ELISA kit and a chromogenic agar plating method for meat samples analysis. The sensor format shows a promising technology for simple and sensitive detection system for Salmonella contamination. Rapid detection of Salmonella is a key to the prevention and identification of problems related to health and safety.

Chemiluminometric Determination of the Pesticide Pirimicarb by a Flow Injection Analysis Assembly

A flow injection (FI) method is described for the determination of pirimicarb. It was found that an enhanced chemiluminescence (CL) signal is obtained when employing the luminol–H2O2–horseradish peroxidase (HRP) system. Under the optimum experimental conditions, the enhanced CL intensity was linear with the concentration 4.25–30.75 ng mL−1 (r = 0.997, n = 8) with a relative standard deviation of 0.99%, containing 12.75 ng mL−1 (n = 8). The limit of detection of the investigated compound was 0.12 ng mL−1. The method shows a moderate selectivity against other pesticides (Amitrole, Atrazine, 2,4,5-T, Dichlorprop, and Metamidophos).The proposed method was sensitive, simple, rapid, and successfully applied to the determination of pirimicarb when it is applied in freshwater; the mean recoveries were 98.3–118.5%.

Capillary electrophoresis enzyme immunoassay for alpha‐fetoprotein and thyroxine in human serum with electrochemical detection

A novel CE-based enzyme immunoassay (CE-EIA) method was developed in o-aminophenol (OAP)-H(2)O(2)-horseradish peroxidase (HRP) system and applied to benign liver disease and hyperthyroidism research in the clinical practical field. In the presented method, after the enzyme immunoreaction, the HRP-labeled antibody or HRP-labeled antigen catalyzed the enzyme substrate OAP and H(2)O(2). The product of the enzymatic catalysis reaction 2-aminophenoxazine-3-one (AP) was determined using electrochemical detection on a Pt electrode at the outlet of the reaction capillary. Factors influencing the performance, including running buffer concentration, separation, and detection voltage, were investigated to the optimum conditions. Noncompetitive and competitive models were utilized to detect alpha-fetoprotein (AFP) and thyroxine (T(4)) in human sera, respectively. The linear ranges and the detection limits (S/N = 3) were from 1.5 to 66.6 ng/mL and 0.48 ng/mL for AFP, and from 1.7 to 260.0 ng/mL and 1.0 ng/mL for T(4). The results of this method were linear proportional to those of spectrophotometric ELISA method, giving a good prospect for a new clinical diagnostic instrument.

Quantum dots–bienzyme hybrid system for the sensitive determination of glucose

In this paper, we attempt to develop a sensitive detection method for glucose with the combination of the unique optical property of quantum dots and the specificity of enzymatic reactions. With glucose and hydroquinone as substrates, benzoquinone that intensively quenches the photoluminescence of quantum dots can be produced via the catalysis of bienzyme (glucose oxidase and horseradish peroxidase) system. A relatively low detection limit of 1.0×10−8mol/L can be achieved. Two linear ranges from 1.0×10−6 to 1.5×10−4M and from 1.5×10−4 to 1.0×10−3M were obtained. For the detection of 1.0×10−4M glucose, six replicative measurements showed the reproducibility (R.S.D.) of 4.43%. The quantum dots–enzymes system possesses advantages of simple procedure without modification of quantum dots or immobilization of enzymes, low cost, high sensitivity and short detection times within several minutes.

Differential Expression of Novel Tyrosine Kinase Substrates during Breast Cancer Development

To identify novel tyrosine kinase substrates that have never been implicated in cancer, we studied the phosphoproteomic changes in the MCF10AT model of breast cancer progression using a combination of phosphotyrosyl affinity enrichment, iTRAQ technology, and LC-MS/MS. Using complementary MALDI- and ESI-based mass spectrometry, 57 unique proteins comprising tyrosine kinases, phosphatases, and other signaling proteins were detected to undergo differential phosphorylation during disease progression. Seven of these proteins (SPAG9, Toll-interacting protein (TOLLIP), WBP2, NSFL1C, SLC4A7, CYFIP1, and RPS2) were validated to be novel tyrosine kinase substrates. SPAG9, TOLLIP, WBP2, and NSFL1C were further proven to be authentic targets of epidermal growth factor signaling and Iressa (gefitinib). A closer examination revealed that the expression of SLC4A7, a bicarbonate transporter, was down-regulated in 64% of the 25 matched normal and tumor clinical samples. The expression of TOLLIP in clinical breast cancers was heterogeneous with 25% showing higher expression in tumor compared with normal tissues and 35% showing the reverse trend. Preliminary studies on SPAG9, on the other hand, did not show differential expression between normal and diseased states. This is the first time SLC4A7 and TOLLIP have been discovered as novel tyrosine kinase substrates that are also associated with human cancer development. Future molecular and functional studies will provide novel insights into the roles of TOLLIP and SLC4A7 in the molecular etiology of breast cancer.

CE‐based simultaneous liquid‐phase noncompetitive enzyme immunoassay for three tumor markers in human serum using electrochemical detection

A method for indirectly detecting horseradish peroxidase (HRP) was described by CE with electrochemical detection. Details of selection for optimum conditions were presented. The detection limit of free HRP was 1.09 x 10(-12) M or 0.94 zmol (S/N = 3). A novel CE-based liquid-phase binding noncompetitive enzyme immunoassay (CE-EIA) was developed. In this method, after the noncompetitive immunoreaction in liquid phase, the free enzyme (HRP)-labeled antibody (Ab*) and the bound enzyme-labeled complex (Ag-Ab*) were separated and then the system of HRP catalyzing H(2)O(2)/o-aminophenol (OAP) reaction was adopted. Prostate specific antigen (PSA), carcinoembryonic antigen (CEA), and human chorionic gonadotropin (HCG) in human serum samples were detected without any sample preparation, with the detection limits (S/N = 3) of 0.22, 0.17 and 0.30 ng/mL, respectively. This technique has been successfully applied to detect simultaneously PSA, CEA, and HCG in 12 min, upon adding these three antigens into human serum to simulate patient serum. It proves that the CE-EIA technique proposed could be developed into a sensitive and new method for simultaneous clinical assay of multianalytes.

Functional LCAT deficiency in human apolipoprotein A-I transgenic, SR-BI knockout mice

Reduction of plasma LCAT activity has been observed in several conditions in which the size of HDL particles is increased; however, the mechanism of this reduction remains elusive. We investigated the plasma activity, mass, and in vivo catabolism of LCAT and its association with HDL particles in human apolipoprotein A-I transgenic, scavenger receptor class B type I knockout (hA-ITg SR-BI-/-) mice. Compared with hA-ITg mice, hA-ITg SR-BI-/- mice had a 4-fold higher total plasma cholesterol concentration, which occurred predominantly in 13-18 nm diameter HDL particles, a significant reduction in plasma esterified cholesterol-total cholesterol (EC/TC) ratio, and significantly lower plasma LCAT activity, suggesting a decrease in LCAT protein. However, LCAT protein in plasma, hepatic mRNA for LCAT, and in vivo turnover of 35S-radiolabeled LCAT were similar in both genotypes of mice. HDL from hA-ITg SR-BI-/- mice was enriched in sphingomyelin (SM), relative to phosphatidylcholine, and had less associated [35S]LCAT radiolabel and endogenous LCAT activity compared with HDL from hA-ITg mice. We conclude that the decreased EC/TC ratio in the plasma of hA-ITg SR-BI-/- mice is attributed to a reduction in LCAT reactivity with SM-enriched HDL particles.

Lateral Spread of an Amplification Signal Using an Enzymatic System on a Conductive Surface

In this letter, we report a novel signal amplification phenomenon that rapidly and dramatically increases both the magnitude and the lateral extent of the original signal. This phenomenon utilizes an enzyme immobilized on a conductive surface to generate amplified signals at locations remote from the original site of enzyme activity. The result is demonstrated on a microfluidic platform using the established precipitating enzyme-substrate system of horseradish peroxidase (HRP) and 3,3',5,5'-tetramethylbenzidine (TMB) on a surface plasmon resonance (SPR) imaging system.

Magnetic Field Effects and Radical Pair Mechanisms in Enzymes: A Reappraisal of the Horseradish Peroxidase System

Reinvestigation by stopped-flow spectrophotometry of the previously observed influence of a static magnetic field on the horseradish peroxidase (HRP)-catalyzed reduction of hydrogen peroxide by Taraban et al. (J. Am. Chem. Soc. 1997, 119, 5768) did not reproduce the originally observed effects. No magnetic field effect was observed for static fields of up to 75 mT. Field-induced changes in both k1 and k2 reported in the original work were found to produce equal and opposite effects on the shape of the observed kinetic decay of the 418 nm spectroscopic signal as a result of the difference in the relative absorbances of Native HRP and Compound II.

Immunopathological modifications in the rectal mucosa from an animal model of food allergy

The aim is to determine immunopathological modifications in rectal mucosa from rabbit after local challenge in sensitized animals with ovalbumin (OVA). Thirty rabbits divided into three groups: G1: normal, G2: subcutaneously OVA sensitized, G3: sensitized, locally OVA challenged and sampled 4 hours after challenge. Specific anti-OVA IgE levels were evaluated by passive cutaneous anaphylaxis test (PCA). In each group 200 high microscopical power fields (HPF) were counted. Results were expressed as arithmetic mean and SE. Statistical analysis was made using Student t test. Anti-CD4, CD5, micro chain, CD25 and RLA II monoclonal antibodies were used. Avidin biotin horseradish peroxidase system was used. CD 4: G1: 8.3 +/- 0.06; G2: 13.4 +/- 0.08 and G3: 8.25 +/- 0.06. CD 5: G1: 7.3 +/- 0.05; G2: 9.4 +/- 0.05 and G3: 11.3 +/- 0.06. CD 25: G1: 13 +/- 0.08; G2: 15.1 +/- 0.13 and G3: 25.5 +/- 0.15. mu chain: G1: 10.4 +/- 0.06; G2: 3.8 +/- 0.02 and G3: 6.0 +/- 0.10. RLA II (DR): G1: 11.6 +/- 0.05; G2: 19.2 +/- 0.09 and G3: 19.1 +/- 0.11. In all cases, experimental groups (G2 and G3) presented statistical significant differences vs. control group (G1) (p < 0.001). Interleukin-2 receptor (CD25+ cells) increase in experimental groups. Cells expressing micro chain decreased in G2 and G3 likely due to activation of B cells and subsequent expression of other immunoglobulin chains in cell surface. RLA II expression is higher in G2 and G3. This receptor is considered an activation marker expressed by macrophages, T and B cells. We conclude that obtained data are important to elucidate immunopathology of local anaphylactic reaction in rectal mucosa from systemic sensitized animals.

Oxidative activation of linezolid to a reactive species: Implications for linezolid adverse drug reactions

Background Linezolid is a novel oxazolidone antimicrobial agent which is effective against gram positive bacteria. It is extensively metabolized. Serious adverse drug reactions to linezolid occur relatively frequently and can be life threatening. The mechanism of these and the potential role of metabolites is not known. Methods Linezolid was incubated in increasing concentrations and over increasing incubation times with Jurkat e6.1 lymphoblasts in the presence and absence of a murine microsomal activating system or a horseradish-peroxidase (HRP) activating system. Viability was determined after incubation using a tetrazolium-based assay. Results No toxicity was demonstrated with linezolid over a concentration range from 0 to 1200 μM in the absence of murine microsomes. In contrast, concentration-dependent toxicity was demonstrated when cells were incubated in the presence of the HRP system (100±5% viability at 0μM, 20± 3% viability at 625 μM, p<0.05). At concentrations greater than 1000 μM, cell viability increased back towards baseline (80±5% at 1250 μM, p<0.05). Conclusion Oxidative activation of linezolid appears to produce metabolite(s) toxic to lymphoblasts not dependent on CYP450-dependent metabolism. At high concentrations linezolid may act as an anti-oxidant. This suggests that reactive linezolid metabolites may play a role in linezolid-related adverse drug reactions. Clinical Pharmacology & Therapeutics (2005) 77, P69–P69; doi: 10.1016/j.clpt.2004.12.157

有機溶媒／水系でのワサビペルオキシダーゼ触媒によるオレンジ２の脱色反応

Study was made to clarify the decoloration mechanism of Orange II in the presence of horseradish peroxidase (HRP)-hydrogen peroxide (H2O2) in organic solvents/water systems. The decoloration rate constant of Orange II decreased in the presence of organic solvents. The highest decoloration rate constant was noted for the methanol/water system. The decoloration reaction of Orange II was found to proceed even at high concentrations of methanol, while this was not the case for alcohols solvents. Reaction of the HRP system was examined by following the changes in HRP oxidation states using the methanol, ethanol and 2-propanol/water systems. An intermediate - Compound III - was formed in the HRP oxidation cycle of ethanol/water and 2-propanol/water systems but did not in the methanol/water system. HRP oxidation states and reaction cycle are importantly involved in the decoloration mechanism of Orange II.

Voltammetric Determination of Leucoindigo Adsorbed on Pretreated Carbon Paste Electrodes: Its Application in a Flow System

The determination of leucoindigo adsorbed on the surface of pretreated carbon paste electrodes has been performed by cyclic (CV) and alternating current voltammetry (ACV). The water-soluble compound called leucoindigo is obtained through the reduction of water insoluble indigo in an alkaline media and in presence of a dithionite salt (Na2S2O4) as a reducing agent. Cyclic voltammograms of leucoindigo show two reversible electrodic processes, in the aqueous 0.1 M Tris-HCl pH 7.2, at the formal potential of −0.4 V and +0.3 V (vs. Ag/AgCl/sat. KCl). In a batch protocol, leucoindigo was quantified by CV with a change of medium between accumulation and detection steps. Reversible voltammetric processes of leucoindigo were greatly enhanced by other voltammetric technique like alternating current voltammetry. Limit of detection in the nanomolar range was achieved for a 2 min accumulation time by ACV in a batch procedure. A flow system was also employed, with adsorptive voltammetric detection of leucoindigo, since this automates the methodology and decreases analysis time. Parameters related to the electrochemical technique are optimized and calibration plots obtained are reported. These data provide useful information about the suitability of using leucoindigo in the detection system of alkaline phosphatase (AP) and horseradish peroxidase (HRP) based affinity devices, in which indigo is generated by the enzymatic hydrolysis of the 3-indoxyl phosphate substrate.

Application of immobilized horseradish peroxidase for the removal of p-chlorophenol from aqueous solution

The development and application of an immobilized horseradish peroxidase (HRP) system with porous aminopropyl glass (APG) beads are reported. The optimal condition for the immobilization of HRP on APG with glutaraladhyde, which leads to a specific activity of 50.7 U/g APG, was identified. Upon immobilization, the operation pH range was broadened downward by one pH unit from 7.5–9.5 to 6.5–9.5 and optimal operation temperature was increased by at least 10 °C. Thermostability of HRP was also improved by approximately 20% upon immobilization. The application of such HRP immobilized APG for the removal of p-chlorophenol from water was investigated. An HPLC method for the quantification of p-chlorophenol was developed to substitute for spectrophotometric methods that are subject to interferences from proteins and other UV-absrobing compounds in the reaction mixtures. The polymerization of p-chlorophenol into insoluble precipitate was completed within 3 h after the initiation of reaction with the addition of hydrogen peroxide with a maximal removal efficiency of 25%, observed at pH 7.5. The addition of poly(ethylene glycol) significantly enhanced the removal efficiency presumably via the formation of a protective shield in the vicinity of the active site of HRP from the free radicals formed during polymerization and can thus reduce the amount of enzymes needed to achieve the desired removal efficiency. The reusability of the HRP immobilized APG was also demonstrated.