• 한국식품위생안전성학회지
• /
• 제24권2호
• /
• pp.148-153
• /
• 2009

본 연구에서는 심혈관계 질환의 표지인자인 콜레스테롤의 농도를 새롭게 개발한 형광 크로마토그라피 방법에 의하여 측정 하고자 시도하였다. 개발된 측정 시스템은 크로마토그라피 스트립이 들어 있는 카트리지, 발색제 AEC, 콜레스테롤 옥사데이즈 (Cholesterol Oxidase, CO), 콜레스테롤 에스트라아제(Cholesterol Esterase, CE), 호스래 디쉬페록시다아제 (Horseradish peroxidase, HRP)를 포함하는 효소혼합 수용액, 그리고 형광 판독기로 구성되어 었다. 콜레스테롤 농도 변화에 따른 형광 세기 변화로 얻은 상관계수 r= 0.968로 측정 가능한 농도 범위에서 신뢰할 만한 직선성 관계를 제시하여 주었다. 회복률과 Hitachi 747 검사기기와 비교 테스트에서는 각각 106.5-94%와 상관관계 r = 0.939 (p<0.001)로 비교적 높은 유의성을 보여주었다. 따라서 본 연구에서 개발된 새로운 콜레스테롤 농도 측정법은 빠른 반응 시간 (8분)과 적은 시료량 ($5\;{\mu}l$)을 사용하기에 시료를 실험실로 운반할 필요없이 클리닉에서 측정 가능한 준현장검사 플랫폼형으로 개발되었으며 기존의 고가의 자동화 장비를 사용하는 생화학적 진단방식 대체용으로의 응용이 기대된다.

• Journal of Microbiology and Biotechnology
• /
• 제23권4호
• /
• 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.

• 한국동물학회지
• /
• 제39권1호
• /
• pp.26-35
• /
• 1996

쥐의 외측 망상핵으로부터 소뇌충부 제6엽 내의 각 소엽으로 신경 경로를 WGA-HRP를 이용한 역행수송법을 써서 조사하였다. 표지된 신경세포는 양측의 외측 망상핵에 모두 존재하였으나, 동축의 경우에 편중되어 있었다. 동측 또는 대측의 외측 망상핵의 큰 세포구획(magnocellular division)에서 국소순적으로 배열이 관찰되었는데, a소엽에서 c소엽으로의 투사가 동측의 큰 세포구획에서 등쪽에서 배쪽으로 분포양상을 보였으며, 대측의 큰세포구획에서는 다소 머리측에서 꼬리측 절편으로의 분포 양상을 보였다. 그 외 동측 또는 대측의 작은세포구획(parvocellular division) 및 삼차밑구획(subtrigeminal division)에서의 표지된 신경세포의 수는 극히 적었다. 한편 외측 망상핵으로부터 소뇌층부의 제6엽 a소엽/b소엽으로서의 투사에 관한 컴퓨터를 이용한 삼차원 재구성은 각 경우에 있어서 상당량의 투사의 중첩이 존재함을 보여주고 있다.

• 한국식품위생안전성학회지
• /
• 제32권6호
• /
• pp.447-454
• /
• 2017

본 연구의 목적은 식품 제조 중 표백 및 살균에 사용되는 과산화수소($H_2O_2$)의 잔류 농도 검출에 활용 될 수 있는 유리탄소전극 기반의 바이오센서의 개발이다. 미국 FDA 및 국내 식품의학안전처 등 식품 용 과산화수소(food grade $H_2O_2$)는 국내외적으로 35% $H_2O_2$ 수용액으로 규정한다. 연구에서 개발한 바이오센서는 감응 물질로 사용된 horseradish peroxidase를 graphene oxide와 aniline과 함께 biocomposite를 형성시킨 후 중성 pH에서 본 연구에서 새롭게 개발된 전기화학적 증착법을 수행하여 개발되었다. 센서구조 및 특성 평가를 위하여 SEM, 순환 전압 전류법 등을 수행하였으며 본 연구에서 개발된 바이오센서는 $10-500{\mu}M$ 농도의 $H_2O_2$에 대하여 직선상의 농도 의존적인 반응을 나타내었으며 최저 검출 한계는 $0.12{\mu}M$으로 산출되었다. 본 연구에서 개발된 센서의 전략적 가치는 향후 오징어포, 건어물 등 널리 유통되는 식품 중에 함유된 식품용 $H_2O_2$ 미량을 현장에서 쉽게 분석 할 수 있어서 비용-효과적 측면에서 그 가치가 우수하다는 것을 제시한다.

• Journal of Microbiology and Biotechnology
• /
• 제17권4호
• /
• 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.

• 센서학회지
• /
• 제20권1호
• /
• 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
• /
• 제21권1호
• /
• 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
• /
• 제33권4호
• /
• 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
• /
• 제13권5호
• /
• 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.

• 한국환경농학회지
• /
• 제3권2호
• /
• pp.37-42
• /
• 1984

화학물질(化學物質)에 의(依)한 서양고추냉이와 사과의 peroxidase 및 정제된 대두(大豆) lipoxygenase 역가저해(力價沮害)를 조사(調査)하였다. 서양고추냉이와 사과의 peroxidase 역가(力價)에 저해효과(沮害效果)를 주는 농약중(農藥中) maneb가 zineb의 효과(效果)보다 컸으며 10,20,30ppm의 처리농도(處理濃度)에서 maneb는 12.5,23.0,34.5%, zineb는 9.5, 17.5,27.5%의 역가저해(力價沮害)를 보였다. Maneb와 zineb의 혼합체(混合體)인 mancozeb 를 500 ppm의 농도(濃度)로 사과나무에 철포(撤布)했을때 사과의 peroxidase 역가변화(力價變化)는 무처리구(無處理區)와 별 차이(差異)가 없었다. 대두(大豆)의 lipoxygenase 역가(力價)에 미치는 영향(影響)은 zineb의 경우 가장 저해도(沮害度)가 컸으며 phosphamidon과 carbofuran 이 또한 저해효과(沮害效果)가 있었다. 그러나 maneb는 저해효과(沮害效果)가 없었다. 10, 20, 30 ppm의 처리농도하(處理濃度下)에서의 lipoxygenase 역가저해도(力價沮害度)는 zineb가 14.0, 27.0, 40.0%, carbofuran이 6.5, 12.5, 20.0% 그리고 phosphamidon이 4.5, 9.0, 13.0%였다. 또한 이들 세농약(農藥)은 $100{\sim}200\;ppm$에서 100% lipoxygenase 역가저해(力價沮害)를 보였다.