Novel scintillation proximity assay for measuring membrane. Here the authors compare an automated highthroughput mass spectrometry htms system rapidfire with the scintillation proximity assay spa. Scintillation proximity assay to detect the changes in. This enzyme is considered to be involved in viral replication, and is expected to be one of the target molecules of antihcv drugs. When the radioactive template bound to the polymer, radiation energy was. Scintillation proximity assay technology is covered by us patent number 4568649, european patent number 0154734, and japanese. Scintillation proximity assay for total p53 protein as an.
Hybrid nanoparticle platform for nanoscale scintillation proximity assay colleen m. By coupling a range of specific molecules to spa particles, receptor binding assays, enzyme assays, molecular interaction assays and radioimmunoassays have been established. The technology is homogeneous in nature, as it relies on a mix and measure format and avoids a filtration step to separate bound from unbound radiolabeled ligand as is the case in a traditional receptorbinding assay 3. Ria approach based on the principle of scintillation proximity 14. Previously, we established a highthroughput screening system for hcv helicase inhibitors using the scintillation proximity assay spa system kyono, k.
A high throughput scintillation proximity imaging assay for protein methyltransferases authors. Spa beads or plates convert the energy from radioactive decay of. The scintillation proximity assay spa technology has been widely used to establish high throughput screens hts for a range of targets in the pharmaceutical industry. Scintillation proximity assay spa as a new approach to determine. The scintillation proximity assay spa is a homogeneous, versatile assay technology for the rapid and sensitive analysis of a wide range of biolo gical processes. A method is described to measure the total level of the tumor suppressor p53 using scintillation proximity assay spa beads and radiolabeled streptavidin.
Scintillation counters are widely used in radiation protection, an assay of radioactive materials and physics research because they can be made inexpensively yet with good quantum efficiency, and can measure both the intensity and the energy of incident radiation. File names can combine several identifiers, such as counter. Scintillation proximity assay spa is an assay development and biochemical screening that permits the rapid and sensitive measurement of a broad range of. Scintillation proximity assay spa as a new approach to. Usually scintillation counters are only used to detect radioactive decay directly. Scintillation proximity assay spa is a radioisotopic technology format used to measure a wide range of biological interactions, including drugtarget binding affinity studies. Assays are performed using radioactive labels that emit electrons with only a short range about. Scintillation proximity assay spa is a homogeneous assay technology that has been successfully applied to many drug screening applications. By applying the basic principles of ligandreceptor binding and enzyme kinetics. The assay is homogeneous in nature, as it relies on a mix and measure format. Scintillation proximity assay an overview sciencedirect. Principles and applications of liquid scintillation counting.
Scintillation proximity assay spa is a technology whereby binding reactions can be assayed with out the washing or filtration procedures normally used to separate bound from free fractions. This unit identifies potential uses of the technology for the measurement of receptor. This automated method enables the bioanalyst to more than halve the handson time involved in ria. Two manually loaded models have sample capacities of up to 16 or 32 plates. Pdf scintillation proximity assays in highthroughput. Conventional phosphors emit blue light 350450 nm which is absorbed by yellow or brown assay components. Application of the scintillation proximity assay technique. Mass spectrometry is an emerging format for labelfree highthroughput screening. It has also been validated in the evaluation of proteinpeptide interactions, proteindna.
The tricarb 2100tr liquid scintillation counter is computercontrolled, benchtop liquid scintillation analyzer for detecting small amounts of alpha, beta and gamma radioactivity. Citeseerx document details isaac councill, lee giles, pradeep teregowda. Assays are performed using radioactive labels that emit electrons with only a short range about 10. Scintillation proximity assay is a method for performing competitive binding assays such as immunoassays and receptor binding assays with a minimum of sample handling. This thesis describes the synthesis of scintillanttaggedcompounds for application in. Counting modes include single and dual label cpm, single and dual label dpm, paralux count mode and luminescence counting. Expression and purification of a hepatitis c virus ns34a. This is a pdf file of an unedited manuscript that has. Pdf version of this page 735k pdf version of this title 33m similar articles in pubmed. The technology is homogeneous in nature, as it relies on a mix and measure format and avoids a filtration step to separate bound from unbound radiolabeled ligand as is the case in a traditional receptorbinding assay.
Scintillation proximity assays spas have become a powerful tool for highthroughput screening hts because they can measure the activity and binding of very diverse classes of drug targets. Citeseerx scintillation proximity assay for measurement. In one form of this assay, dilute aqueous suspensions of tritiated latex particles lh and scintillant latex particles l. Use of scintillation proximity assay to measure radioligand binding.
Tnks1 enzyme scintillation proximity assay spa presented in tables 15 inhibitory potency ic 50, concentration of a compound inhibiting 50% of tankyrase1 enzymatic activity of compounds on tankyrase1 enzymatic activity was evaluated using a scintillation proximity assay spa. A scintillation proximity assay spa has been used successfully to detect and quantify, in realtime, the kinetic progress of hydrolysis of. Methylation of rna by methyltransferases is a phylogenetically ubiquitous posttranscriptional modification that occurs most extensively in transfer rna trna and ribosomal rna rrna. Spa is a beadbased assay technique that has been applied to radioimmunoassays, receptorbinding assays and enzyme assays. The type of beads that are involved in the spa are microscopic in size and within the beads itself, there is a scintillant which emits light. Arrangement of total spa to measure the amount of p53 protein. Scintillation proximity assays for the realtime detection and quantification of the progress of reactions upon solid supports. Thelightemitted canbemeasuredin astandardscintillation counter. Compared to all other methods, counting efficiency is increased by up to 500%. Miniturization of scintillation proximity assays to 384 well.
Compounds with radioactive labels are imaged in the presence of beads infused with scintillators compounds that glow when exposed to ionizing radiation that are coated with targets intended to mimic a protein or biological compound of interest. Application of scintillation proximity assay in drug. Investigation of their potencies requires not only cellfree but also cellbased assays in which intracellular accumulationdepletion of s1p could be monitored. Scintillation proximity assay spa as a new approach to determine a ligands kinetic profile. Since the scintillation proximity assay produces mostly visible light, it can be detected strictly speaking with photomultipliers in spectrophotometers rather than with photomultipliers in scintillation counters. Review basics of enzymatic assays for hts assay guidance manual. Scintillation proximity assay spa is a homogeneous assay technology which is beadbased and removes the need for a filtration step to separate bound from free ligand in a receptorbinding assay.
Glorymar ibanez, david shum, gil blum, bhavneet bhinder, constantin radu, christophe antczak, minkui luo, hts core facility, memorial sloankettering cancer center, new york, ny 10065, usa. By applying the basic principles of ligandreceptor binding and enzyme kinetics, it is possible to build a large variety of miniaturized, highthroughput assays and screen millions of. This problem is addressed by the use of phosphors that emit radiation of longer wavelength 480900 nm. Aug 15, 2012 scintillation proximity assay spa, characterized by its speed, sensitivity, reliability, and the fact that no separation step is required, has become an important technique in highthroughput screening hts for new drugs, and for investigating their biological interactions. The main limitation of mass spectrometry is throughput, due to the requirement to purify samples prior to ionization. Compounds that modulate the activity of sphingosine 1phosphate s1pmetabolizing enzymes are expected to be potential therapeutic agents for various diseases. Measurement of radioligand binding by scintillation. The success of a particular assay format may be limited by the need to introduce molecular labels and by label interference with the primary reaction. The established ranitidine radioimmunoassay technique ria has been adapted to a scintillation proximity assay spa format by use of microtitre plates.
Addition ofunlabeled antigen fromtissueextracts displaces thelabeled ligand and diminishes the fluorescent signal. Lumb mass spectrometry is an emerging format for labelfree highthroughput screening. The assay gives fewer false positives, is widely recognized as the gold standard for radiometric highthroughput screening. Three different cell extracts were used, with one used to develop the standard curve for the amount of p53. Liquid scintillation counting a primer for orientation national diagnostics laboratory staff principles 1. Microbeta2 and microbeta2 lumijet microplate counters. Scintillation proximity assays spa are used for drug discovery. Scintillation proximity assay spa is a technology whereby binding reactions can be assayed without the washing or filtration procedures normally used to separate bound from free fractions. Scintillation counter principle instrumentationtools.
For example, scintillation proximity assay spa technology requires a capture label such as biotin or a histag and a. Scintillation proximity assay spa technology to study. Scintillation proximity assay on the topcount microplate. Scintillation proximity assay as a highthroughput method to identify slowly dissociating nonpeptide ligand binding to the gnrh receptor christopher e. The invention concerns scintillation proximity assays performed in multiwell plates where a charge coupled device is used to image the wells. Comparison with the scintillation proximity assay with a focused file screen of akt1pkb. Scintillation proximity assay as a highthroughput method. Proprietary timeresolved liquid scintillation counting.
Scintillation proximity assay spa was used to measure concentrations of human albumin ha and rabbit antihuman albumin raha. Scintillation proximity assay is a technique which enables us with a rapid and automatic throughput of experiments dealing with proteindrug or proteinprotein interactions and several others. Scintillation proximity assay using molecularly imprinted. The proximity scintillation assay described below is particularly useful for biochemical studies on the tethering and docking steps of lysosometophagosome targeting and the regulation of this process by igg. Liquid scintillation counting of samples in solvent resistant picoplatestm or white polystyrene microplates using. Spa results obtained with the topcount microplate scintillation and luminescence counter are comparable to those obtained with a discrete sample scintillation counter. Supplementary appendix this appendix has been provided by the authors to give readers additional information about their work. A high throughput scintillation proximity imaging assay for. Scintillation proximity assays spa are a beadbased assay technology for radioligand binding studies in drug research 1, 2. Development and application of a scintillation proximity. A cellfree scintillation proximity assay for studies on. By using small polymer beads, we were able to place an organic scintillator and an antenna component in close proximity to the imprinted binding sites. For example, an insurmountable effect of slowly dissociating ligands in a functional ip1 assay in spa format on the neurokinin1 nk1 receptor.
Scintillation proximity utilizing 11labeled ligands. Scintillation proximity assay spa is a homogeneous and versatile technology for the rapid and sensitive assay of a wide range of biological processes, including applications using enzyme and receptor targets, radioimmunoassays, and molecular interactions. Scintillation proximity assays for the realtime detection. Scintillation proximity assay spa is a beadbased homogeneous assay technology that. Paralux count mode for scintillation proximity assays fully utilizes the advantage of twin photomultiplier tubes. Scintillation proximity assay how is scintillation. A scintillation proximity assay for histone demethylases.
The scintillation proximity assay spa is a method that is frequently used to detect and quantify the strength of intermolecular interactions between a biological receptor and ligand molecule in aqueous media. Pdf scintillation proximity assays in highthroughput screening. Hsd enzymes has been implicated in the etiology of the metabolic syndrome. Scintillation proximity assay spa is an assay development and biochemical screening that permits the rapid and sensitive measurement of a broad range of biological processes in a homogeneous system. Microbeta is suitable for counting scintillation proximity assays with. This thesis describes the synthesis of scintillanttaggedcompounds for application in a novel cellbased spa. Scintillation medicine scintillation radar scintillation radar scintillation radar scintillation camera. Pyrimidinone nicotinamide mimetics as selective tankyrase. In spa, scintillant is incorporated into small fluomicrospheres. The spa technique now plays a key role in hts, in that it can be used in many assay formats including. Scintillation proximity assay spa beads having immobilized antimouse antibodies bind a mouse monoclonal antibody to p53 that can then bind the p53 protein in an extract. Scintillation proximity assays may be counted in optiplates tm, with up to 80 l total sample volume in the 384well plates, 350 l in the 96well plates, and 1. Topics include the development of nuclear receptor assays using alphascreen and lance technology, the preparation of cells for highcontent screening, scintillation proximity assay of inositol phosphates, aurora assays, and homogeneous multiwell assays for measuring cell viability and apoptosis. Solidphase combinatorial chemistry and scintillation.
Scintillation proximity assay sample types for luminescence. Development of a scintillation proximity assay spa based. Standard instrument features patented trlsc timeresolved liquid scintillation countingusing unique afterpulse rejection technology is featured for high. Pyrimidinone nicotinamide mimetics as selective tankyrase and. Scintillation proximity assay an overview sciencedirect topics. Dec 09, 2015 scintillation proximity assay spa is a radioisotopic technology format used to measure a wide range of biological interactions, including drugtarget binding affinity studies. Scintillation proximity assay technologies provide a rapid non.
In this unit, the application of spa technology to measuring protein. Scintillation proximity assay spa as previously described 24. Scintillation proximity assay spa, highthroughput screening, adenylyladenosine, phosphodiesterase 12 pde12. A high throughput scintillation proximity imaging assay. Each well of the microplate is coated with a thin layer. Biochemical characterization of rna methyltransferase enzymes and their methylated product rna or rnaprotein complexes is. It has also been validated in the evaluation of proteinpeptide interactions, proteindna interactions, and cellular adhesion molecule binding. This chapter describes the development of spa radioligand binding assays detailing the choice of. Application of scintillation proximity immunoassay to tissue.
Prereceptor metabolism of glucocorticoids by the 11. Scintillation proximity assay spa is a versatile homogeneous technique for radioactive assays which eliminates the need for separation steps. Aug 31, 2010 read scintillation proximity assay for total p53 protein as an alternative to elisa, journal of immunological methods on deepdyve, the largest online rental service for scholarly research with thousands of academic publications available at your fingertips. Spa is particularly attractive because it is often used as a high volume screening procedure, typically in drug discovery, and high throughput and ease of. Crowe1 many nonpeptide antagonists of the gonadotropinreleasing hormone gnrh receptor, as well as other drug targets, possess. Microbeta trilux is a multidetector instrument designed for liquid scintillation or luminescence detection of samples in microplates, tubes or on filters.
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