Horseradish peroxidase (HRP) is generally used as a label enzyme in enzyme immunoassay (EIA).The procedure used for HRP detection in EIA is critical for sensitivity and precision.This paper describes a novel fluorimet...Horseradish peroxidase (HRP) is generally used as a label enzyme in enzyme immunoassay (EIA).The procedure used for HRP detection in EIA is critical for sensitivity and precision.This paper describes a novel fluorimetric assay for horseradish peroxidase (HRP) using sesamol as substrate.The principle of the assay is as follow:sesamol (3,4-methylenedioxy phenol) is reacted enzymatically in the presence of hydrogen peroxide to produce dimeric sesamol.The dimer is fluorescent and can be detected sensitively at ex.347 nm,em.427 nm.The measurable range of HRP was 1.0×10-18 to 1.0×10-15 mol/assay,with a detection limit of 1.0×10-18 mol/assay.The coefficient of variation (CV,n=8) was examined at each point on the standard curve,with a mean CV percentage of 3.8%.This assay system was applied to thyroid stimulating hormone (TSH) EIA using HRP as the label enzyme.展开更多
Equol is a metabolite of soybean isoflavone, daidzein, and many health benefits are expected. Endogenous equol in urine is S-equol and mostly exists as glucuronate or sulfate conjugate. In this study we preliminary es...Equol is a metabolite of soybean isoflavone, daidzein, and many health benefits are expected. Endogenous equol in urine is S-equol and mostly exists as glucuronate or sulfate conjugate. In this study we preliminary established the simple enzyme-linked immunosorbent assay (ELISA) without deconjugation, then developed the S-equol specific ELISA involves deconjugation showing high stereospecificity to S-equol without using stereospecific antibody. For the simple ELISA, we used a polyclonal antibody that targets the regions not influenced by inhibition by conjugation of glucuronate and sulfate and achieved the correlation coefficient;r = 0.975, but the value was 30 % lower than high performance liquid chromatography (HPLC). Developing upon this we invented the specific ELISA established from S-equol homogeneous combination for the standard and enzyme-labeled antigen to enhance stereospecificity. The correlation with HPLC was favorable: r = 0.986, y = 0.996x – 6. Compared to the previous method using (R,S)-equol combination, cross-reactivity with R-equol was reduced from 65 to 13 %, and that with daidzein from 0.31% to 0.08%, markedly increased in the specificity. This study is expected to be applied for both simple clinical researches, and stereospecific immunoassays in which specific antibody preparation is difficult.展开更多
It is known that the factors identified as contributing to the bactericidal activity of honeys are the high sugar concentration, hydrogen peroxide (H<sub>2</sub>O<sub>2</sub>) and low pH, and i...It is known that the factors identified as contributing to the bactericidal activity of honeys are the high sugar concentration, hydrogen peroxide (H<sub>2</sub>O<sub>2</sub>) and low pH, and its bactericidal components depend on honey plant and natural environment. Manuka honey has been studied a lot about bactericidal effect. However, since Japanese honeys are mainly used as food, detailed analyzes of bactericidal components and its actions have not been reported. Therefore, we analyzed bactericidal components contained in nine Japanese honeys using Lucigenin-CL-HPLC. As our results, four species components of H<sub>2</sub>O<sub>2</sub>, glucose, fructose and methylglyoxal were detected from nine Japanese honeys. The H<sub>2</sub>O<sub>2</sub> concentrations were 4.1 × 10<sup>-5</sup> - 1.8 × 10<sup>-4</sup> mol/L, the glucose concentrations were 1.4 - 2.8 mol/L, the fructose concentrations were 1.9 - 2.4 mol/L, the methylglyoxal concentrations were 4.0 × 10<sup>-3</sup> - 1.6 × 10<sup>-2</sup> mol/L. In this study, we confirmed that methylglyoxal is also contained in Japanese honeys, although it is a bactericidal component which is reported to be characteristic of manuka honey. It is considered that the further study of Japanese honeys is useful for more safe use, quality control, and clinical application.展开更多
文摘Extracts of 16 natural medicine powders {Galla chinensis,Malloti cortex,Cassiae semen,Sophorae radix,Myricae cortex,Crataegi fructus,Gambit,Mume fructus,Geranii herba,Phellodendri cortex,Coptidis rhizoma,Swertiae herba,and Cinnamomi cortex） were assayed for reactive oxygen concentrations using the peroxyoxalate chemiluminescent detection system.High luminescence intensity was observed in Galla chinensis,Geranii herba,Malloti cortex,Myricae cortex,and Cinnamomi cortex.Additional experiments identified the reactive oxygen species as hydrogen peroxide.Galla chinensis generated 2.4 × 10^（-4） mol/L hydrogen peroxide from a 1 mg/mL solution.In bacterial growth tests,Galla chinensis extract had antibacterial activity against Escherichia coli.Staphylococcus aureus,Bacteroides thetaiotaomicron,Campylobacter sputorum biovar sputorum.Streptococcus salivarius thermophilus,Lactobacillus casei,and Bifidobacterium longum infantis.This antibacterial activity was decreased by the addition of catalase.It revealed that hydrogen peroxide which Galla chinensis produced participated in antibacterial activity.
文摘Horseradish peroxidase (HRP) is generally used as a label enzyme in enzyme immunoassay (EIA).The procedure used for HRP detection in EIA is critical for sensitivity and precision.This paper describes a novel fluorimetric assay for horseradish peroxidase (HRP) using sesamol as substrate.The principle of the assay is as follow:sesamol (3,4-methylenedioxy phenol) is reacted enzymatically in the presence of hydrogen peroxide to produce dimeric sesamol.The dimer is fluorescent and can be detected sensitively at ex.347 nm,em.427 nm.The measurable range of HRP was 1.0×10-18 to 1.0×10-15 mol/assay,with a detection limit of 1.0×10-18 mol/assay.The coefficient of variation (CV,n=8) was examined at each point on the standard curve,with a mean CV percentage of 3.8%.This assay system was applied to thyroid stimulating hormone (TSH) EIA using HRP as the label enzyme.
文摘Equol is a metabolite of soybean isoflavone, daidzein, and many health benefits are expected. Endogenous equol in urine is S-equol and mostly exists as glucuronate or sulfate conjugate. In this study we preliminary established the simple enzyme-linked immunosorbent assay (ELISA) without deconjugation, then developed the S-equol specific ELISA involves deconjugation showing high stereospecificity to S-equol without using stereospecific antibody. For the simple ELISA, we used a polyclonal antibody that targets the regions not influenced by inhibition by conjugation of glucuronate and sulfate and achieved the correlation coefficient;r = 0.975, but the value was 30 % lower than high performance liquid chromatography (HPLC). Developing upon this we invented the specific ELISA established from S-equol homogeneous combination for the standard and enzyme-labeled antigen to enhance stereospecificity. The correlation with HPLC was favorable: r = 0.986, y = 0.996x – 6. Compared to the previous method using (R,S)-equol combination, cross-reactivity with R-equol was reduced from 65 to 13 %, and that with daidzein from 0.31% to 0.08%, markedly increased in the specificity. This study is expected to be applied for both simple clinical researches, and stereospecific immunoassays in which specific antibody preparation is difficult.
文摘It is known that the factors identified as contributing to the bactericidal activity of honeys are the high sugar concentration, hydrogen peroxide (H<sub>2</sub>O<sub>2</sub>) and low pH, and its bactericidal components depend on honey plant and natural environment. Manuka honey has been studied a lot about bactericidal effect. However, since Japanese honeys are mainly used as food, detailed analyzes of bactericidal components and its actions have not been reported. Therefore, we analyzed bactericidal components contained in nine Japanese honeys using Lucigenin-CL-HPLC. As our results, four species components of H<sub>2</sub>O<sub>2</sub>, glucose, fructose and methylglyoxal were detected from nine Japanese honeys. The H<sub>2</sub>O<sub>2</sub> concentrations were 4.1 × 10<sup>-5</sup> - 1.8 × 10<sup>-4</sup> mol/L, the glucose concentrations were 1.4 - 2.8 mol/L, the fructose concentrations were 1.9 - 2.4 mol/L, the methylglyoxal concentrations were 4.0 × 10<sup>-3</sup> - 1.6 × 10<sup>-2</sup> mol/L. In this study, we confirmed that methylglyoxal is also contained in Japanese honeys, although it is a bactericidal component which is reported to be characteristic of manuka honey. It is considered that the further study of Japanese honeys is useful for more safe use, quality control, and clinical application.
