• Title/Summary/Keyword: Non-radioactive assay

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A Neutravidin-based Assay for Reverse Transcriptase Suitable for High Throughput Screening of Retroviral Activity

  • Brennan, Lyndall E.;Sune, Carlos;Klimkait, Thomas
    • BMB Reports
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    • v.35 no.3
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    • pp.262-266
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    • 2002
  • A non-isotopic neutravidin-based reverse transcriptase (RT) assay adapted for high throughput screening of HIV activity is described. Using a 96-well microtitre plate, HIV particles are lysed and the RT enzyme released into a reaction mixture containing poly(A) RNA, biotinylated oligo d(T) and fluorescein-labelled dUTP (FI-dUTP). With poly(A) as a template and oligo d(T) as primer, the viron RT incorporates FI-dUTP into an elongating DNA strand. The resulting product is captured on a neutravidin-coated 96-well plate and the unincorporated nucleotides removed by a series of washing steps. A simple ELISA is subsequently performed using a monoclonal antifluorescein antibody conjugated to alkaline phosphatase. Quantification of RT activity is facilitated by a colorimetric readout. The assay was validated in the context of a diagnostic HIV-1 phenotyping assay. Using supernatants from HIV-1 infected lymphocyte cultures the assay was shown to be as sensitive as a radioactive assay and the RT activity correlated well with levels of cell-asociated HIV-p24. Importantly, even minor reductions of RT activity by virus variants with reduced fitness could be distinguished.

Non-radio isotopic endpoint for local lymph node assay in Balb/c mice using ELISA based on Bromdeoxyuridine incorporation

  • Lee, Jong-Kwon;Park, Jae-Hyun;Kim, Hyung-Soo;Yun, So-Mi;Chung, Seung-Tae;Juno H. Eom;Park, Seung-Hee;Gi ho Seo;Oh, Hye-Young
    • Proceedings of the Korean Society of Toxicology Conference
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    • 2002.11b
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    • pp.185-185
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    • 2002
  • A murine local lymph node assay (LLNA) has been developed as an alternative test to guinea pig maximization test. The disadvantage of LLNA is the need for the use of radioactive material. In this study, we aimed to investigate the development of non-radio isotopic endpoint for local lymph node assay in Balb/c mice using Enzyme-linked immunosorbent assay (ELISA) based on Bromodeoxyuridine (BrdU) incorporation.(omitted)

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Development of a DDA+PGA-combined non-destructive active interrogation system in "Active-N"

  • Kazuyoshi Furutaka;Akira Ohzu;Yosuke Toh
    • Nuclear Engineering and Technology
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    • v.55 no.11
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    • pp.4002-4018
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    • 2023
  • An integrated neutron interrogation system has been developed for non-destructive assay of highly-radioactive special nuclear materials, to accumulate knowledge of the method through developing and using it. The system combines a differential die-away (DDA) measurement system for the quantification of nuclear materials and a prompt gamma-ray analysis (PGA) system for the detection of neutron poisons which disturb the DDA measurements; a common D-T neutron generator is used. A special care has been taken for the selection of materials to reduce the background gamma rays produced by the interrogation neutrons. A series of measurements were performed to test the basic performance of the system. The results show that the DDA system can quantify plutonium of as small as 20 mg and it is not affected by intense neutron background up to 1.57 × 107 s-1 and gamma ray of 4.43 × 1010 s-1. The gamma-ray background counting rate at the PGA detector was reduced down to 3.9 × 103 s-1 even with the use of the D-T neutron generator. The test measurements show that the PGA system is capable of detecting 0.783 g of boron and about 86.8 g of gadolinium in 30 min.

A Generic Time-resolved Fluorescence Assay for Serine/threonine Kinase Activity: Application to Cdc7/Dbf4

  • Xu, Kui;Stern, Alvin S.;Levin, Wayne;Chua, Anne;Vassilev, Lyubomir T.
    • BMB Reports
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    • v.36 no.4
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    • pp.421-425
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    • 2003
  • The serine/threonine protein kinase family is a large and diverse group of enzymes that are involved in the regulation of multiple cellular pathways. Elevated kinase activity has been implicated in many diseases and frequently targeted for the development of pharmacological inhibitors. Therefore, non-radioactive antibody-based kinase assays that allow high throughput screening of compound libraries have been developed. However, they require a generation of antibodies against the phosphorylated form of a specific substrate. We report here a time-resolved fluorescence assay platform that utilizes a commercially-available generic anti-phosphothreonine antibody and permits assaying kinases that are able to phosporylate threonin residues on protein substrates. Using this approach, we developed an assay for Cdc7/Dbf4 kinase activity, determined the $K_m$ for ATP, and identified rottlerin as a non-ATP competitive inhibitor of this enzyme.

NON-RADIO ISOTOPIC ENDPOINT FOR LOCAL LYMPH NODE ASSAY USING IMMUNOHISTOCHEMISTRY

  • Lee, Jong-Kwon;Park, Jae-Hyun;Kim, Hyung-Soo;Jang, Eun-Jung;Hwang, In-Chang;Jung, Seung-Tae;Jun H. Eum;Oh, Hye-Young
    • Proceedings of the Korean Society of Toxicology Conference
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    • 2001.10a
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    • pp.193-193
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    • 2001
  • A murine local lymph node assay (LLNA) has been developed as an alternative to guinea pig models for contact sensitization potential. However, a disadvantage of the LLNA is the need for the use of radioactive material. In this study, we aimed to investigate the development of non-radio isotopic endpoint for LLNA using immunohistochemistry.(omitted)

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Study on the Anti-Cancer Effect of Acanthopanax Sessiliflorus (오가피(五加皮)의 항암효과에 관한 연구)

  • Kim, Young-Chul;Woo, Hong-Jung;Lee, Jang-Hoon
    • The Journal of Korean Medicine
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    • v.20 no.3 s.39
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    • pp.54-65
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    • 1999
  • Objectives: Hepatoma is a very serious disease in Korea and vvorldwiclc. Hepatitis B vims (HBV) has proved the most significant cause of hepatoma. We canied out this study to investigate the effect of Acanthopanax sessilifloms on inhibiting cell proliferation and DNA synthesis in HepG2.2.15 cell line and on inhibiting phosphorilation of oncogene (MAP kinase) in NIT/3T3-HBx ceIl. Methods: To investigate the anti-cancer effect of Acanthopanax sessiliflorus, we did the CellTiter 96 Aqueous Non-radioactive Cell Proliferation assay (Promega); MTS/PMS assay, [$^3H$]-thymicline incorporation assay, and we measured the gene expression through westem blotting. Results: Acanthopanax sessiliflorus showed an inhibiting effect on the increase of HepG2.2.15 in the NTS/PMS assay. It also showed an inhibiting effect on DNA synthesis of HepG2.2.15 in the [$^3H$]-thymidine incorporation assay. Acanthopanax sessiliflorus showed an inhibiting effect of phosphorilation of MAP kinase in HBV - X genes. too. Conclusions: The results suggested that this herb had an anti cancer effect. We may discover an effective anti-cancer herb medicine through further studies on this herb medicine.

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Alternative Immunossays

  • Barnard, G.J.R.;Kim, J.B.;Collins, W.P.
    • Korean Journal of Animal Reproduction
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    • v.9 no.2
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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.

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Uranium Isotopic Ratio Analysis of U-Bearing Particulates By SIMS in CIAE

  • Yonggang, Zhao
    • Proceedings of the Korean Radioactive Waste Society Conference
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    • 2004.02a
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    • pp.257-259
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    • 2004
  • In this paper measurement method of uranium isotope ratio of uranium-bearing particles in swipe samples was introduced; Swipe sample screening program was proposed on the basis of studying various destructive assay and non-destructive assays. Scanning electron microscope(SEM) equipped with an energy dispersive X-ray fluorescence(XRF) system was applied to locate the deposited uranium-containing particles on the graphite support, particle's composition and size can be identified. Some isotope ratio results were compared with those of other bulk analytical methods; By measuring the same prepared sample, we got the U-particle isotopic ratio data similar to those from IAEA NWAL, indicating that our operation parameters and experimental conditions are viable and can be used for measurement of U-particle isotopic ratio from swipe samples.

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Flavonoids inhibit the AU-rich element binding of HuC

  • Kwak, Ho-Joong;Jeong, Kyung-Chae;Chae, Min-Ju;Kim, Soo-Youl;Park, Woong-Yang
    • BMB Reports
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    • v.42 no.1
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    • pp.41-46
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    • 2009
  • Post-transcriptional regulation of mRNA stability by Hu proteins is an important mechanism for tumorigenesis. We focused on the molecular interactions between the HuC protein and AU-rich elements (AREs) to find chemical inhibitors of RNA-protein interactions using RNA electrophoretic mobility shift assay with non-radioactive probes. Screening of 52 natural compounds identified 14 candidate compounds that displayed potent inhibitory activity. Six (quercetin, myricetin, (-)-epigallocatechin gallate, ellagic acid, (-)-epicatechin gallate, and rhamnetin) were categorized as phytochemicals, and their $IC_{50}$ values were low ($0.2-1.8\;{\mu}M$).