• 약학회지
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• 제41권1호
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• pp.74-80
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• 1997

A polygonal antibody against recombinant hirudin was raised for the development of a ELISA in biological fluids. Recombinant hirudin was conjugated to maleimide activated carrie r protein, KLH and injected to a rabbit. The third booster collection of antiserum was used as primary antibody for the ELISA. The titer for the detection antibody was determined. The direct ELISA could determine the concentration of hirudin in the range of ~10ng/ml. Affinity pulified IgG was obtained and conjugated to horseradish peroxidase. Purified IgG and IgG-HRP could be used as capture and detection antibody, respectively. Although sandwich ELISA would not give the satisfactory results. it could apply for the detection of hirudin level in the range of ~20 ${\mu}$g/ml.

• Animal cells and systems
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• 제6권2호
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• pp.159-165
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• 2002

Projections from the prefrontal cortex to subdivisions of the dorsal raphe nucleus were investigated in the rat using retrograde and anterograde tracing methods. A retrograde tracer, gold-conjugated horseradish peroxidase (WGA-apo-HRP-gold), was injected into each subdivision of the dorsal raphe including lateral wing, dorsomedial, and ventromedial areas. The majority of retrogradely labeled cells were located in the prelimbic, infralim-bic, and dorsal peduncular areas of the medial prefrontal cortex. A few cells were also identified in the cingulate, various regions of the orbital, and agranular insular cortices. Secondly, an anterograde tracer, Phaseolus vulgaris leucoagglutinin (PHA-L), was injected into the medial prefrontal cortex involving the prelimbic or infralimbic areas. Axonal fibers with varicosities were identified in all subdivisions of the DR including the lateral wing, dorsomedial, and ventromedial areas. Projections were bilateral, with ipsilateral predominance. Axonal fibers were observed at the lateral border of medial longitudinal fasciculus or in the interfascicular region at the midline. The present findings demonstrate that both the midline and lateral wing regions of the dorsal raphe nucleus receive excitatory input from cognitive and emotional centers of the cerebral cortex.

• 약학회지
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• 제35권6호
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• pp.486-496
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• 1991

Spinal parasympathetic outflows originate in the sacral parasympathetic nuclei. The sacral parasympathetic nuclei receive inputs from the brainstem. Many areas in the medulla appear to influence sympathetic outflow of the spinal cord. Whether neurons in these areas of the medulla may project to the lumbosacral cord to affect the parasympathetic outflow has not been studied clearly. Thus, this study was intended to investigate origins of cells projecting from the medulla to the sacral parasympathetic nuclei of the spinal cord. In 3 cats, horseradish peroxidase (HRP) was injected into the lower lumbar spinal cord. HRP labeled neurons were found mainly in the following areas: nucleus retroambiguus, nucleus tractus solitarius, raphe complex and ventrolateral area of the rostral medulla. Most of these areas are known to be involved in regulation of sympathetic activity, and, thus, these results indicate that these areas are likely to affect the sacral parasympathetic outflow as they do for the sympathetic nerves.

• 전기화학회지
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• 제7권1호
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• pp.44-48
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• 2004

The two-component type enzyme amplified amperometric DNA assay is described to use an ambient $O_2$ of the substrate of the DNA labeling enzyme. Although the assay detects DNA only at > 0.5M concentration, a concentration $\~10^6$ fold higher than the sandwich-type enzyme amplified amperometric DNA assay, it can be run with an always available substrate. The assay utilizes screen-printed carbon electrodes (SPEs) which were pre-coated by a co-electrodeposited film of an electron conducting redox hydrogel and a 37-base long single-stranded DNA sequence. The DNA in the electron conducting film hybridizes and captures, when present, the 37-base long detection-DNA, which is labeled with bilirubin oxidase (BOD), an enzyme catalyzing the four-electron reduction of $O_2$ to water. Because the redox hydrogel electrically connects the BOD reaction centers to the electrode, completion of the sandwich converts the film from non-electrocatalytic to electrocatalytic for the reduction of $O_2$ to water when the electrode is poised at 200 mV vs. Ag/hgCl. The advantage or the assay over the earlier reported sandwich type enzyme amplified amperometric DNA assay, in which the amplifying enzyme was horseradish peroxidase, is that it utilizes ambient $O_2$ instead of the less stable and naturally unavailable $H_2O_2$.

• Animal cells and systems
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• 제7권1호
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• pp.49-55
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• 2003

The fiber projection from the prefrontal cortex to the locus coeruleus (LC) in the periventricular region was analyzed in rat using anterograde and retrograde tracing methods. Following injection of an anterograde tracer, Phaseolus vulgaris leucoagglutinin (PHA-L), into prelimbic and infralimbic regions of the medial prefrontal cortex, labeled axonal fibers with varicosities were observed bilaterally within the LC, with ipsilateral predominance. Terminal labeling was also observed in the region medial to the nucleus at rostral to middle levels of the LC, whereas axonal labeling in the caudal LC was minimal. Anterogradely-labeled axonal fibers were not found in the subcoerulear region. A retrograde tracer, gold-conjugated and inactivated wheatgerm-agglutinin horseradish-peroxidase (WGA-apo-HRP-gold), was injected into several rostro-caudal levels of the LC. Majority of retrogradely-labeled cells were observed in the prelimbic or infralimbic regions of the medial prefrontal cortex when the injections were made into rostral to middle levels of the LC. Only a few cells were observed in cingulate, dorsal peduncular, orbital, or insular cortices. The present findings suggest that the nucleus LC receives restricted, excitatory inputs from cognitive, emotional, and autonomic centers of the cerebral cortex and might secondarily have influences on widespread brain regions via its diversified monoaminergic innervation.

• 식물병연구
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• 제25권3호
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• pp.131-135
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• 2019

Cylindrocarpon destructans causes ginseng root rot and produces radicicol that has an antifungal effect. In this study, we developed a method to detect this fungus using enzyme-linked immunosorbent assay (ELISA). Secreted proteins of C. destructans were used as antigens to obtain C. destructans-specific IgG from mouse. Out of 318 monoclonal antibodies generated from mouse, two antibodies (Cd7-2-2 and Cd7-2-10) showed highest specificity and sensitivity. Indirect ELISA using both antigens successfully detected C. destructans in soils, but direct ELISA using IgG conjugated with horseradish peroxidase failed to detect antigens in soils. The indirect ELISA developed here can efficiently detect the fungus and help manage ginseng root rot disease in fields.

• 센서학회지
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• 제32권1호
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• pp.16-21
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• 2023

This study reports the potential of using commercial copy papers as substrates for simple sensitive glucose detection. Typical paper-based devices use filter papers as porous substrates that can contain reagents; however, this is the first study to report the use of copy papers for the purpose of enhancing enzymatic colorimetric detection. Glucose detection using glucose oxidase, horseradish peroxidase and potassium iodide was performed on a copy paper, cellulose-based filter paper, and polyethylene film. The results indicated that the copy paper exhibited a stronger coloration than the other substrates. Reagents required for detection were dried on the copy paper, and a 3D-printed holder was designed to provide an environment for consistent imaging, making it a convenient cost-effective option for point-of-care testing using a mobile phone camera. The simple paper-based glucose sensor exhibited a linear range of 0.1-20 mM, limit of quantification of 0.477 mM, and limit of detection of 0.143 mM.

• KSBB Journal
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• 제11권6호
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• pp.681-688
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• 1996

페놀이 포함되어 있는 수용액에 HRP 및 $H_2O_2$를 첨가하여 페놀을 침전 및 제거시키는 방법에서, 페 놀의 종류와 페놀 및 $H_2O_2$의 농도가 HRP의 페놀제거 효율에 미치는 영향을 조사하였다. 여러 가지 페 놀성분은 최척 완충용액 (pH 5~7) 에서 HRP와 $H_2O_2$를 사용해서 90% 이상 제거할 수 있었다. $H_2O_2$ 의 농도가 10mM 이상일 때 HRP의 페놀제거 효율 이 급격히 감소하였으며 HzOz의 농도가 50mM 이 상얼 때 HRP의 페놀제거 효율은 거의 없었다. HRP 한 분자당 제거할 수 있는 페놀의 분자수 (turnover number)는 $H_2O_2$ 빛 페놀물질의 초기농 도가 각각 ImM일 때, p-ethoxyphenol의 경우 18047로 가장 켰으며 m-chlorophenol의 경우 1244로서 가장 척였다. 페놀과 반응 후($25^{\circ}C$, 24h) 반응물질로부터 분리된 HRP의 Soret 파장 (404nm)에셔의 흡광도는 원래효소의 48%로 감소 하였고 kcat 및 kcatjKm값은 각각 원래효소의 41 %와 51 %로 감소함으로서 페놀 제거시 HRP의 구 조변화 및 활성저하가 초래되었음을 알 수 있었다. 페놀과 함께 비 페놀계 방향성 물질인 벤젠, 에틸벤 젠, 툴루엔 (BET)을 포함하고 있는 용액 에 HRP 빛 $H_2O_2$를 첨가하면 BET의 제거율이 증가하였다.

• KSBB Journal
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• 제13권5호
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• pp.577-584
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• 1998

A sensitive membrane strip assay for plasma lipoprotein cholesterol that can be performed without handling reagents has been investigated. We previously developed an assay system with immobilized enzymes (cholesterol esterase and cholesterol oxidase) on the surfaces of nitrocellulose membrane(1). In such a case, the amount of enzymes present on the membrane was limited by its surface area and, thus, the detection capability was relatively poor (> 50 mg/dL cholesterol). To overcome this problem, we devised a new system with non-immobilized enzymes by placing them within interstitial spaces of a celullose membrane pad in a dry state. Upon contact with sample medium, the enzymes were immediately dissolved and participated in the reactions with cholesterol in a liquid phase. We constructed a user-friendly system consisting of four membrane pads fro sample application, cholesterol decomposition, color development as signal, and medium absorption to invoke a continuous flow (sequential location from the bottom). A sample containing lipoproteins was added into the application pad by capillary action and transferred to the next pad for decomposition. The decomposition pad (namely, enzyme pad) contained a detergent (sodium cholate) for the destruction of lipoprotein particles, the two enzymes for cholesterol decomposition, and a chromogen (3,3'-diaminobenzidine). As a consequence of the enzyme reactions, hydrogen peroxide was produced, and then reacted in the presence of the chromogen with horseradish peroxidase immobilized on the signal generation pad. Finally, a colorimetric signal directly proportional to the cholesterol concentration was produced. The detection limit determined from this system under optimal conditions was at least 2 times lower than of the enzyme-immobilized system.

• 한국식품위생안전성학회지
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• 제9권3호
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• pp.123-131
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• 1994

An enzyme-linked immunosorbent assay(ELISA) was developed for the detection of residual gentamicin(GM) in edible animal products. The immunogen(GM-KLH conjugate) and coating antigen(GM-BSA conjugate) were prepared by coupling GM sulfate to keyhole limpet hemocyanin(KLH) and bovine serum albumin(BSA) in the presence of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride, respectively. Polyclonal antibody to GM was produced in rabbits(New Zealand White, female) by using the immunogen and the antibody titer was measured by indirect ELISA. A competitive ELISA was developed using GM-bovine serum albumin conjugate as a coating antigen, GM(as standards or sample), polyclonal antibody to GM, secondary antibody conjugated with horseradish peroxidase as an enzyme, and H2O2 and o-phenylenediamine dihydrochloride as a substrate and a chromophore, respectively. The detection limit of GM was 10 ng/ml and the standard curve of GM(n=26) was linear up to 10 $\mu\textrm{g}$/ml in this competitive ELISA system. There were no cross-reactivities of the partially purified antibody between GM and the various antibiotice such as amikacin, benzyl-penicillin, chloramphenicol, erythromycin, furazlidone, kanamycin, neomycin, oleandomycin, streptomycin, sulfathiazole and thiamphenicol(CR50<0.05%)