• Biomolecules & Therapeutics
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• 제23권5호
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• pp.486-492
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• 2015

Drug metabolism mostly occurs in the liver. Cytochrome P450 (CYP) is a drug-metabolizing enzyme that is responsible for many important drug metabolism reactions. Recently, the US FDA and EU EMA have suggested that CYP enzyme induction can be measured by both enzymatic activity and mRNA expression. However, these experiments are time-consuming and their interassay variability can lead to misinterpretations of the results. To resolve these problems and establish a more powerful method to measure CYP induction, we determined CYP induction by using luminescent assay. Luminescent CYP assays link CYP enzyme activity to firefly luciferase luminescence technology. In this study, we measured the induction of CYP isozymes (1A2, 2B6, 2C9, and 3A4) in cryopreserved human hepatocytes (HMC424, 478, and 493) using a luminometer. We then examined the potential induction abilities (unknown so far) of mesalazine, a drug for colitis, and mosapride citrate, which is used as an antispasmodic drug. The results showed that mesalazine promotes CYP2B6 and 3A4 activities, while mosapride citrate promotes CYP1A2, 2B6, and 3A4 activities. Luminescent CYP assays offer rapid and safe advantages over LC-MS/MS and qRT-PCR methods. Furthermore, luminescent CYP assays decrease the interference between the optical properties of the test compound and the CYP substrates. Therefore, luminescent CYP assays are less labor intensive, rapid, and can be used as robust tools for high-throughput CYP screening during early drug discovery.

• 한국진공학회:학술대회논문집
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• 한국진공학회 2016년도 제50회 동계 정기학술대회 초록집
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• pp.360.1-360.1
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• 2016

Hydroxyapatite (Ca10(PO4)6(OH)2, HAP) particles have attracted a great deal of attention in biomedical fields due to their good biocompatibility, bioactivity and fairly broad applications as drug delivery, dental implant, bone cement, and etc. Thus, many researchers have made an effort to add new functionalities such as luminescence, drug delivery, and bone regeneration properties up to HAP powders by controlling their nanostructure as well as composition. In this research, the mesoporous strontium substituted HAP (Sr-HAP) microspheres were synthesized using a hydrothermal method. In this synthesis, aspartic acid monomers were utilized to form microsphere by controlling surface energy of HAP particles and Sr ions were substituted into Ca ion sites, which induced luminescence property in HAP powders. Moreover, the change in the amount of Sr substitution was found to influence the particle size, morphology, and concurrently surface area, which led to changing drug loading as well as drug release property. The amount of Sr influences the morphology, luminescent properties, particle size, surface area cell viability and drug loading property, which are investigated by SEM, TEM, XRD, FTIR, BET, XPS and in vitro test such as MTT assay and drug release test. In particular, the multifunctional Sr-HAP with molar ratios of 0.25 (Sr/(Ca+Sr)) possessed the strongest luminescent property as well as the superior drug loading and sustained release properties that were correspondent with large surface area and pore size. Our study indicates that the fabricated multifunctional Sr-HAP microspheres are quite useful for bone regeneration and drug delivery.

• Journal of Microbiology and Biotechnology
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• 제26권3호
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• pp.588-595
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• 2016

IFN-γ release assays (IGRAs) have been developed as viable alternative diagnostic tools for detecting latent tuberculosis infection (LTBI). A customized homogeneous sandwich luminescent oxygen channeling immunoassay (LOCI) was used to quantify IFN-γ levels in IGRAs. Samples were collected from healthy volunteers (n = 40) who were T-Spot-negative and T-Spot-positive patients (n = 32) at rest. Then the amount of IFN-γ in the supernatant of IGRAs was measured by LOCI. The results demonstrated a low background, and high sensitivity, specificity, accuracy, and reproducibility, and a short assay time (only 30 min) with LOCI for IFN-γ. The recovery range was 81.63-102.06%, the coefficients of variation were below 5%, and the limit of detection was 19.0 mIU/ml. Excellent agreement between LOCI IFN-γ and the T-SPOT.TB test was obtained (97.2% agreement, κ = 0.94). The LOCI IFN-γ concentrations were significantly higher in T-Spot-positive patients than in the healthy group (p < 0.001). Moreover, as observed for the comparative LOCI IFN-γ assay, IFN-γ concentrations were related to the numbers of T-SPOT.TB spots. We have established an in vitro blood test for LTBI diagnosis, defined as LOCI IFN-γ. A high level of agreement between the LOCI IFN-γ method and T-SPOT.TB assay was observed in clinical studies that showed the LOCI IFN-γ method could determine LTBI. This study shows acceptable performance characteristics of the LOCI IFN-γ assay to diagnose LTBI.

• KSBB Journal
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• 제22권6호
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• pp.426-432
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• 2007

QDs을 기반으로 하는 OsFIA는 매우 빠르고 간단히 수행될 수 있다. 또한 이 분석법은 고체상의 담체나 결합/잔류시약의 분리 등과 같은 여러 과정을 필요로 하지 않으며, 적은 양의 시약으로도 분석이 가능하다. 본 분석법은 높은 감도로 항원을 측정할 수 있으며, 일상적인 분석에도 쉽게 도입될 수 있을 것이다. 선형 범위 내에서 측정 가능한 receptor의 최소농도는 0.05 nM (2.65 ng/mL) 정도이다. 또한, 일반적으로 상용화된 항체를 가치고 수행이 가능하다. 이 OsFIA 분석법은 기존의 실험적 sandwich immunoassay의 효과적인 대안으로 제시된다.

• Journal of Microbiology
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• 제38권3호
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• pp.150-155
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• 2000

A bioluminescent assay method for detecting the activity of phospholipase C(PLC; phosphatidyl choline cholinephosphohydrolase, EC 3.1.4.3) was developed using bioluminescent marine bacteria. Phospholipase C from Bacillus cereus and sn-1,2- dimyristoyl glycerol was further hydrolyzed with lipase from Candida ecylidracea. The hydrolyzed myristic acid was quantified using a dark mutant of Vibrio harveyi (designated as M-17). The in vivo light intensity of which was stimulated specifically up to one thousand fold in the presence of myristic acid. The rates of the hdrolysis of the DMPC substrate by the phospholipase measured by the luminescence method were linear with time and the were estalished to detect as little as 0.1 mUnit of phospholipase C and 5 nM of myristic acid production.

• 한국환경보건학회:학술대회논문집
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• 한국환경보건학회 2004년도 International Conference Global Environmental Problems and their Health Consequences
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• pp.146-150
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• 2004

Degradation efficiency of pentachlorophenol (PCP) by using direct UV photolysis and $TiO_2$ photocatalysis was evaluated with both chemical analyses and acute toxicity assessment employing luminescent bacteria Vibrio fischeri. PCP was chosen as a target compound in this study because of its wide application as fungicide, bactericide, insecticide and wood preservative in agriculture and many industries, in addition to its well-known environmental consequences. The acute toxicity to the microbe was reduced by >60% when applying UV alone, and was completely removed when treated with $UV-TiO_2$ combinations. Toxicity reduction pattern determined with the Microtox Assay generally corresponds with the chemistry data: However, it should be noted that toxicity was greater than expected by the chemistry data. Formation of TCBQ, a toxic byprodut, could not explain observed microbial toxicity. These observations are probably due to the presence of unidentified toxic PCP byproducts, which may include polychlorinated dibenzodioxins and polychlorinated dibenzofurans. When Microtox results were compared between different exposure time, i.e.,5 min and 15 min, an interesting pattern was noted with $UVA-\;TiO_2$ treatment. While no microbial toxicity was observed with 5 min exposure, an EC50 value of 45.4% was estimated with 15 min exposure, which was not observed in $UVB-\;TiO_2$ exposure. This result may suggest the presence of unidentified toxic degradation products generated in the later stage of treatment. Based on this study, $TiO_2$ photocatalyst, together with UVB photolysis could improve the removal of both PCP and its toxic derivatives in more efficient way. The Microtox Assay is promising and economical method for monitoring efficiency of wastewater treatment processes.

• Biomolecules & Therapeutics
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• 제31권1호
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• pp.127-138
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• 2023

Glycogen synthase kinase-3β (GSK-3β) is an important serine/threonine kinase that implicates in multiple cellular processes and links with the neurodegenerative diseases including Alzheimer's disease (AD). In this study, structure-based virtual screening was performed to search database for compounds targeting GSK-3β from Enamine's screening collection. Of the top-ranked compounds, 7 primary hits underwent a luminescent kinase assay and a cell assay using human neuroblastoma SH-SY5Y cells expressing Tau repeat domain (Tau_RD) with pro-aggregant mutation ΔK280. In the kinase assay for these 7 compounds, residual GSK-3β activities ranged from 36.1% to 90.0% were detected at the IC₅₀ of SB-216763. In the cell assay, only compounds VB-030 and VB-037 reduced Tau aggregation in SH-SY5Y cells expressing ΔK280 Tau_RD-DsRed folding reporter. In SH-SY5Y cells expressing ΔK280 Tau_RD, neither VB-030 nor VB-037 increased expression of GSK-3α Ser21 or GSK-3β Ser9. Among extracellular signal-regulated kinase (ERK), AKT serine/threonine kinase 1 (AKT), mitogen-activated protein kinase 14 (P38) and mitogenactivated protein kinase 8 (JNK) which modulate Tau phosphorylation, VB-037 attenuated active phosphorylation of P38 Thr180/ Tyr182, whereas VB-030 had no effect on the phosphorylation status of ERK, AKT, P38 or JNK. However, both VB-030 and VB-037 reduced endogenous Tau phosphorylation at Ser202, Thr231, Ser396 and Ser404 in neuronally differentiated SH-SY5Y expressing ΔK280 Tau_RD. In addition, VB-030 and VB-037 further improved neuronal survival and/or neurite length and branch in mouse hippocampal primary culture under Tau cytotoxicity. Overall, through inhibiting GSK-3β kinase activity and/or p-P38 (Thr180/Tyr182), both compounds may serve as promising candidates to reduce Tau aggregation/cytotoxicity for AD treatment.

• Biomolecules & Therapeutics
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• 제21권6호
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• pp.487-492
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• 2013

Cytochrome P450 4A11 (CYP4A11) is a fatty acid hydroxylase enzyme expressed in human liver. It catalyzes not only the hydroxylation of saturated and unsaturated fatty acids, but the conversion of arachidonic acid to 20-hydroxyeicosatetraenoic acid (20-HETE), a regulator of blood pressure. In this study, we performed a directed evolution analysis of CYP4A11 using the luminogenic assay system. A random mutant library of CYP4A11, in which mutations were made throughout the entire coding region, was screened with luciferase activity to detect the demethylation of luciferin-4A (2-[6-methoxyquinolin-2-yl]-4,5-dihydrothiazole-4-carboxylic acid) of CYP4A11 mutants in Escherichia coli. Consecutive rounds of random mutagenesis and screening yielded three improved CYP4A11 mutants, CP2600 (A24T/T263A), CP2601 (T263A), and CP2616 (A24T/T263A/V430E) with ~3-fold increase in whole cells and >10-fold increase in purified proteins on the luminescence assay. However, the steady state kinetic analysis for lauric acid hydroxylation showed the significant reductions in enzymatic activities in all three mutants. A mutant, CP2600, showed a 51% decrease in catalytic efficiency ($k_{cat}/K_m$) for lauric acid hydroxylation mainly due to an increase in $K_m$. CP2601 and CP2616 showed much greater reductions (>75%) in the catalytic efficiency due to both a decrease in $k_{cat}$ and an increase in Km. These decreased catalytic activities of CP2601 and CP2616 can be partially attributed to the changes in substrate affinities. These results suggest that the enzymatic activities of CYP4A11 mutants selected from directed evolution using a luminogenic P450 substrate may not demonstrate a direct correlation with the hydroxylation activities of lauric acid.

• 한국가축번식학회지
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• 제9권2호
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• pp.133-139
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• 1985

An immunoassay may be defined as an analytical procedure involving the competitive reaction between a limiting concentration of specific antibody and two populations of antigen, one of which is labelled or immobillized. The advent of immunoassay has revolutionised our knowledge of reproductive physiology and the practice of veterinary and clinical medicine. Radioimmunoassay (RIA) was the first of these methods to be developed, which meausred the analyte with good sensitivity, accuracy and precision (1,2). The essential components of RIA are:-(i) a limited concentration of antibodies, (ii) a reference preparation, and (iii) an antigen labelled with a radioisotope (usually tritium or iodine-125). Most procedures invelove isolating the antibody-bound fraction and measuring the amount of labelled antigen. Good facilities are available for scintilltion counting, data reduction nd statistical analysis. RIA is undergoing refinement through:-(i) the introduction of new techniques to separate the antibody-bound and free fractions which minimize the misclassification of labelled antigen into these compartments, and the amount of non-specfic binding. (3), (ii) the development of non-extration for the measurement of haptens (4), (iii) the determination of a, pp.rent free (i.e. non-protein bound) analytes (5), and (iv) the use of monoclonal antibodies(6). In 1968, Miles and Hales introduced in important new type of immunoassay which they termed immunora-diometric assay (IRMA) based on t도 use of isotopically labelled specific antibodies(7) in a move from limited to excess reagent systems. The concept of two-site IRMAs (with a capture antibody on a solid-phase, and a second labelled antibody to a different antigenic determinant of the analyte) has enabled the development of more sensitive and less-time consuming methods for the measurement of protein hormones ovar wide concentration of analyte (8). The increasing use of isotopic methos for diverse a, pp.ications has exposed several problems. For example, the radioactive half-life and radiolysis of the labelled reagent limits assay sensitivity and imposes a time limit on the usefulness of a kit. In addition, the potential health hazards associated with the use and disposal of radioactive cmpounds and the solvents and photofluors necessary for liquid scientillation counting are incompatable with the development of extra-laboratory tests. To date, the most practical alternative labels to radioisotopes, for the measurement of analytes in a concentration > 1 ng/ml, are erythrocytes, polystyrene particiles, gold sols, dyes and enzymes or cofactors with a visual or colorimetric end-point(9). Increased sensitivity to<1 pg/ml may be obtained with fluorescent and chemiluminescent labels, or enzymes with a fluorometric, chemiluminometric or bioluminometric end-point. The sensitivity of any immunoassay or immunometric assay depends on the affinity of the antibody-antigen reaction, the specific activity of the label, the precision with which the reagents are manipulated and the nonspecific background signal (10). The sensitivity of a limited reagent system for the measurement of haptens or proteins is mainly dependent upon the affinity of the antibodies and the smalleest amount of reagent that may be manipulated. Consequently, it is difficult in practice to improve on the sensitivity obtained with iodine-125 as the label. Conversely, with excess reagent systems for the measurement of proteins it is theoretically possible to increase assay sensitivity at least 1000 fold with alternative luminescent labels. To date, a 10-fold improvement has been achieved, and attempts are being made to reduce the influence of other variables on the specific signal from the immunoreaction.