• Journal of Food Hygiene and Safety
• /
• v.24 no.2
• /
• pp.148-153
• /
• 2009

In this study, we have developed a fluorescence chromatographic assay for the quantification of total cholesterol in serum, which is a well-known risk predictor for cardiovascular diseases. The new assay system consists of a chromatographic strip in a cartridge, enzyme buffer containing cholesterol esterase, cholesterol oxidase, horseradish peroxidase, and color developer AEC, and a laser fluorescence scanner. The correlation coefficient (r) between cholesterol concentration and relative fluorescence units was 0.968 in the new assay, showing a reliable linearity through the tested range of cholesterol. Recovery test and comparability with a Hitachi 747 instrument showed 106.5-94% and r = 0.939 (p<0.001), respectively. The new assay system for cholesterol was developed as a pre-POCT platform conducted in clinics since it is fast (8 min) and uses a small volume of sample ($5\;{\mu}l$), and it may be applied for on-site diagnostics to replace expensive automated biochemical analyzer.

• Journal of Microbiology and Biotechnology
• /
• v.23 no.4
• /
• pp.527-533
• /
• 2013

Polyclonal antibodies labeled with a tracer have been commonly used as secondary antibodies in immunochemical assays to quantify the concentration of antibody-antigen complexes. The majority of these antibodies conjugated with a tracer are commercially available, with the exception of few untouched targets. This study focused on the production and application of mouse anti-quail IgY as an intermediate antibody to link between quail egg yolk IgY and goat anti-mouse IgG-HRP as primary and secondary antibodies, respectively. Subsequently, the produced mouse anti-quail IgY was labeled with horseradish peroxidase (HRP) and its efficiency on enzyme linked immunosorbent assay (ELISA) was compared with that of commercial rabbit anti-chicken IgY-HRP. As an intermediate antibody, mouse anti-quail IgY was successfully produced with good affinity and sensitivity (1:10,000) to the primary and secondary antibodies. Subsequently, mouse anti-quail IgY was effectively conjugated with HRP enzyme, resulting in a secondary antibody with good sensitivity (1:10,000) to quail anti-V. parahaemolyticus and V. vulnificus IgY. The detection limit was $10^5$ CFU/ml for both V. parahaemolyticus and V. vulnificus. The efficiency of the produced conjugate to detect quail IgY on ELISA was comparable to that of the commercial rabbit anti-chicken IgY-HRP, and hence the produced and labeled mouse anti-quail IgY-HRP can be used as a secondary antibody to detect any antibody produced in quail.

• The Korean Journal of Zoology
• /
• v.39 no.1
• /
• pp.26-35
• /
• 1996

The projection from the lateral reticular nucleus (LRN) to three subdivisions of the cerebellar vermal lobule VI was studied in the rat by utilizing the retrograde transport of wheatgerm agglutinin-conjugated horseradish peroxidase. Labelled neurons were located bilaterally throughout the LRN, but with ipsilateral predominance. There seemed to be a dorsal4o-ventral transition in the ipsilateral magnocellular neurons projecting to the cerebellar vermal lobules VIa-to-Vic. In the contralateral side, cells projecting to vermal lobule Via were observed at more rostral sections, whereas those projecting to lobule Vic were located at caudal sections. There were relatively few labelled neurons in parvocellular or subtrigeminal divisions at either side. Computer-aided three dimensional reconstruction of LRN projections to lobules Via/Vib or lobules Vib/Vic exhibited extensive oberlap within each combination of injection cases.

• Journal of Food Hygiene and Safety
• /
• v.32 no.6
• /
• pp.447-454
• /
• 2017

A new amperometric biosensor has been developed for the detection of hydrogen peroxide ($H_2O_2$). The sensor was fabricated through the one-step deposition of a biocomposite layer onto a glassy carbon electrode at neutral pH. The biocomposite, as a $H_2O_2$ sensing element, was prepared by the electrochemical deposition of a homogeneous mixture of graphene oxide, aniline, and horseradish peroxidase. The experimental results clearly demonstrated of that the sensor possessed high electrocatalytic activity and responded to $H_2O_2$ with a stable and rapid manners. Scanning electron microscopy, cyclic voltammetry, and amperometry were performed to optimize the characteristics of the sensor and to evaluate its sensing chemistry. The sensor exhibited a linear response to $H_2O_2$ in the range of 10 to $500{\mu}M$ concentrations, and its detection limit was calculated to be $1.3{\mu}M$. The proposed sensing-chemistry strategy and the sensor format were simple, cost-effective, and feasible for analysis of "food-grade $H_2O_2$" in food samples.

• Journal of Microbiology and Biotechnology
• /
• v.17 no.4
• /
• pp.600-603
• /
• 2007

The effect of a water-miscible ionic liquid, 1-butyl-3-methylimidazolium tetrafluoroborate $([BMIM][BF_4])$, on the horseradish peroxidase (HRP)-catalyzed oxidation of 2-methoxyphenol (guaiacol) with hydrogen peroxide $(H_2O_2)$ was investigated. HRP maintains its high activity in the aqueous mixtures containing various concentrations of the ionic liquid and even in 90% (v/v) ionic liquid. In order to minimize the effect of solution viscosity on the kinetic constants of HRP catalysis, the enzymatic reactions in the subsequent kinetic study were performed in water-ionic liquid mixtures containing 25% (v/v) ionic liquid at maximum. As the concentration of $[BMIM][BF_4]$ increased for the oxidation of guaiacol by HRP, the $K_m$ value increased with a slight decrease in the $K_{cat}$ value: The $K_m$ value increased from 2.8 mM in 100% (v/v) water to 22.5mM in 25% (v/v) ionic liquid, indicating that ionic liquid significantly weakens the binding affinity of guaiacol to HRP.

• Journal of Sensor Science and Technology
• /
• v.20 no.1
• /
• pp.35-39
• /
• 2011

A disposable electrochemical immunosensor system has been developed for the detection of herbicide in aqueous samples. Disposable screen printed carbon electrodes(SPCE) were used as basic electrodes and an enzyme, horseradish peroxidase (HRP), and anti-herbicide antibodies was immobilised on to the working electrode of SPCE by using avidin-biotin coupling reactions. An herbicide-glucose oxidase conjugates have been used for the competitive immunoreaction with sample herbicides. The enzymatic reaction between the conjugated glucose oxidase and glucose added generates hydrogen peroxide, which was reduced by the peroxidase immobilised. The latter process caused an electrical current change, due to direct re-reduction of peroxidase by a direct electron transfer mechanism, which was measured to determine the herbicides in the sample. The optimal operational condition was found to be: $20\;{\mu}gl-1$ deglycosylated avidin loading to the working electrode and working potential +50 mV vs. Ag/AgCl. The total assay time was 15 min after sample addition. The detection limits for herbicides, atrazine and simazine, were found to be 3 ppb and 10 ppb, respectively.

• Molecules and Cells
• /
• v.21 no.1
• /
• pp.161-165
• /
• 2006

Dichlorodihydrofluorescein ($DCFH_2$) is a widely used probe for intracellular $H_2O_2$. However, $H_2O_2$ can oxidize $DCFH_2$ only in the presence of a catalyst, whose identity in cells has not been clearly defined. We compared the peroxidase activity of Cu,Zn-superoxide dismutase (CuZnSOD), cytochrome c, horseradish peroxidase (HRP), $Cu^{2+}$, and $Fe^{3+}$ under various conditions to identify an intracellular catalyst. Enormous increase by bicarbonate in the rate of $DCFH_2$ oxidation distinguished CuZnSOD from cytochrome c and HRP. Cyanide inhibited the reaction catalyzed by CuZnSOD but accelerated that by $Cu^{2+}$ and $Fe^{3+}$. Oxidation of $DCFH_2$ by $H_2O_2$ in the presence of a cell lysate was also enhanced by bicarbonate and inhibited by cyanide. Confocal microscopy of $H_2O_2$-treated cells showed enhanced DCF fluorescence in the presence of bicarbonate and attenuated fluorescence for the cells pre-incubated with KCN. Moreover, DCF fluorescence was intensified in CuZnSOD-transfected HaCaT and RAW 264.7 cells. We propose that CuZnSOD is a potential intracellular catalyst for the $H_2O_2$-dependent oxidation of $DCFH_2$.

• BMB Reports
• /
• v.33 no.4
• /
• pp.312-316
• /
• 2000

Effects of various nitrogenous compounds on the peroxidative activity of Korean radish (Rophanus sativus L.) isoperoxidase $A_1$ were examined by using anilino substrates, such as dianisidine and phenylenediamine. We also used phenolic substrates such as guaiacol, chlorogenic acid, caffeic acid, ferulic acid and esculetin. The peroxidation of dianisidine was stimulated by adenine and imidazole as much as 5 fold and 11 fold, respectively at pH 8. Moreover, about 4.8 fold and 8 fold stimulation of phenylenediamine peroxidation occurred by adenine and imidazole, respectively at pH 8. The stimulation by adenine and imidazole did not occur at the acidic pH range. The peroxidations of phenolic substrates, such as guaiacol, chlorogenic acid, caffeic acid, ferulic acid and esculetin, were not boosted greatly by any of the nitrogenous compounds tested. Notably, ammonium salt, which has been known for the excellent booster of horseradish peroxidase, did not affect the peroxidation of the Korean radish isoperoxidase $A_1$. The kinetic studies of dianisidine peroxidation with imidazole, as a model of boosting reaction, showed that neither the affinity of imidazole against dianisidine, nor the activation energy of dianisidine peroxidation changed during the activity boosting of isoperoxidase $A_1$.

• Journal of Microbiology and Biotechnology
• /
• v.13 no.5
• /
• pp.773-777
• /
• 2003

The color of Congo red hinders the spectrometric measurements of a concentration of hydrogen peroxide and enzyme activity (Horseradish peroxidase; HRP) during enzymatic decoloring of Congo red. In this study, a method was developed to measure peroxidase activity and hydrogen peroxide concentration in the presence of Congo red. The oxidation product of HRP/hydrogen peroxide and ABTS(2,2'-azino-bis-(3-ethylbenzotriazoline-6-sulfonic acid)) formed a dark green color. The spectrum of this product showed absorption bands at 420 nm and 734 nm. When compared with the Congo red spectrum, the absorption at 734 nm of this product did not overlap with Congo red, thus making the hydrogen peroxide measurement possible even in the presence of Congo red. Kinetic study of decoloring of Congo red performed by this method showed that the decoloring reaction followed the Michaelis-Menten kinetics. Pulse feeding of hydrogen peroxide, upon depletion, significantly increased the decoloring of Congo red. This result shows that this newly developed technique can monitor, predict, and improve the enzymatic decoloring process.

• Korean Journal of Environmental Agriculture
• /
• v.3 no.2
• /
• pp.37-42
• /
• 1984

The effect on the activities of peroxidase and lipoxygenase by chemicals was determined. Peroxidase activities of horseradish and apple were inhibited strongly by maneb and comparativelyby mancozeb, zineb. The inhibitory rate of enzyme activity was ranged in $12.5{\sim}34.5%$ by maneb, $11.0{\sim}30.5%$ by mancozeb, and $9.7{\sim}27.5%$ by zineb in the level of $10{\sim}30\;ppm$ at the final concentration. No signification was in the peroxidase activity of apple during ripening between control and plot, treated with 500 ppm mancozeb. On the activity of purified soybean lipoygenase, zineb inhibited it more strongly than carbofuran or phosphamidon, but maneb did not have inhibitory effect on that. The inhibition ranges of $14.0{\sim}40.0%$ by zineb, $6.5{\sim}20.0%$ by carbofuran and $4.5{\sim}13.0%$ by phosphamidon were shown in the final pesticide concentration of $10{\sim}30$ ppm. But in $100{\sim}200$ ppm, lipoxygenase activity was almost inhibited by carbofuran and phosphamidon.