Such platforms include the scintillation proximity assay, elisa, mobility shift assay, and protein binding assay. 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 compounds. Abstract scintillation proximity assay spa is a versatile homogeneous technique for radioactive assays which eliminates the need for. Compounds that did not compete in the assay are listed as being below the level of detection. Aleksandra szykowska3, nicola burgessbrown3, masoud vedadi1,2 and peter j. The challenge of selecting protein kinase assays for lead. Scintillation proximity assay for total p53 protein as an. 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. The spa technique now plays a key role in hts, in that it can be used in many assay formats including. Two homogeneous proximity assays for tyrosine kinases, scintillation proximity assay spa and homogeneous timeresolved fluorescence htrf, have been developed and compared. These microspheres or beads are constructed in such a way as to bind specific molecules. Usa82 1985 8673 adjustingthe volumeto 2 mlwith waterandthe phto 5. Thetube wasthen capped, spunin a vortex, shaken on a rotary mixer overnight, and centri fuged.
Review basics of enzymatic assays for hts assay guidance manual. The assay is based on the interfunctional behavior of a known biological material with the material whose presence is quantitatively sought. Scintillation proximity assay for measurement of rna methylation matthew r. Pdf realtime analysis of endosomal lipid transport by. Combine the sensitivity of radioactive assays with the convenience of a homogeneous no wash steps assay using spa technology. Pdf scintillation proximity assays in highthroughput. A study of the molecular mechanism of binding kinetics and long. The assay used either dmapp or geranyl pyrophosphate gpp and 3 hisopentenyl pyrophosphate 3 hipp as substrates, and measured the fppscatalyzed. Paralux count mode for scintillation proximity assays fully utilizes the advantage of twin photomultiplier tubes.
Scintillation proximity assay is a method for performing competitive binding assays such as immunoassays and receptor binding assays with a minimum of sample handling. Upon binding of radioactively labeled ligands here l3harginine or d3hglucose to acceptor proteins immobilized. Pdf configuring radioligand receptor binding assays for. This unit identifies potential uses of the technology for the measurement of receptor. A scintillation proximity assay for histone demethylases. Scintillation proximity assay spa technology to study.
A cellfree scintillation proximity assay for studies on lysosometophagosome targeting vishal trivedi, 1 shao chong zhang,2 walter stockinger, 3 axel nohturfft2 published 26 june 2007 protocol 1molecular oncology research institute,tuftsnew england medical center, boston, ma 02111, usa. Department of pharmacology and toxicology, university of toronto, 1 kings college. Scintillation proximity assay of arginine methylation. Scintillation proximity assay technologies provide a rapid non. When 3hatp is used as the substrate in a the spa assay. Spa is a beadbased assay technique that has been applied to radioimmunoassays, receptorbinding assays and enzyme assays. Cds15 is based on scintillation proximity assay spa technology provided by amersham bioscience inc. Obrien chapter 11 scintillation proximity assay spa receptor binding assays 141 jeffrey r. Purinergic signalling scintillation proximity assay spa as a new approach to determine a ligands kinetic prof ile. The application of spa technology to study protein. 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.
Studying phagocytosis by livecell scintillation proximity. Application of scintillation proximity assay in drug. Scintillation proximity utilizing 11labeled ligands. Scintillation proximity assay spa as a new approach to determine a ligands kinetic profile. Evaluation of synthesis conditions for plastic scintillation foils used to measure alpha and. The ultrasensitive, high dynamic range quench parameter, aqpi, provides superior dpm calculations. Preparation of tritium labeled compounds and their. The supernatant was removed and the antibody coated beads abbeads were then washed twice with 5 ml ofphosphatebuffered saline. Spa beads or plates convert the energy from radioactive decay of. A cellfree scintillation proximity assay for studies on. Scintillation proximity assay on the topcount microplate.
From highthroughput screenings for toxicology to clinical trials. Scintillation proximity assay spa is a radioisotopic technology format used to measure a wide range of biological interactions, including drugtarget binding affinity studies. All ki and sem values reported are averages generated from duplicate or triplicate assays. Scintillation proximity assay spa is a radioisotopic technology format used to measure a wide range of biological interactions, including. When designing an automated spa or leadseeker assay several factors must be taken into consideration, for example. Spa results obtained with the topcount microplate scintillation and luminescence counter are comparable to those obtained with a discrete sample scintillation counter.
In both formats, the kinase assay was performed using biotinylated peptide substrate, atp 33 patp in the case of spa, and tyrosine kinase in a 96well assay format. Studying phagocytosis by livecell scintillation proximity assay. Colchicine site competitive assay kit cytodynamix screen 15. Hughes chapter 12 radioligand binding filtration assay. L of assay buffer 20 mm hepes, 10 mm mgcl 2, 100 mm nacl and 1 mm edta, containing 0. Scintillation proximity assay spa is a versatile homogeneous technique for radioactive assays which eliminates the need for separation steps.
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 when it is stimulated. Compared to all other methods, counting efficiency is increased by up to 500%. Development of a scintillation proximity assay spa based. Ijzerman 1 division of medicinal chemistry, leiden academic centre for drug research, leiden university, p. Assays are performed using radioactive labels that emit electrons with only a short range about. The use of spa to detect and quantitate the interaction of the transcription factor, nfkb p50 and its dna consensus sequence has been investigated. Scintillation proximity assays in highthroughput screening.
Tritium is ideally suited for scintillation proximity assay, filtration lsc, and microplate because its. Samuel mang, hannes bucher and peter nickolaus affiliation. The principle of receptorbinding scintillation proximity assays involves. Pdf protein methyltransferases pmts orchestrate epigenetic modifications through posttranslational methylation of various protein. Rife 1, 2, 1 department of medicinal chemistry and 2 department of biochemistry, institute for structural biology and drug discovery, virginia commonwealth university, richmond, va 2329803, usa. Scintillation proximity assays spa are a beadbased assay technology for radioligand binding studies in drug research 1, 2.
The scintillation proximity assay spa is a rapid radioligand binding assay. 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. 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. It does not involve a filtration step to separate bound from free ligand as is the case in a traditional receptorbinding assay. Tag lite, ligand binding assay, htrf, opioid, chemokine and cholecystokinin. Structural requirements of ligands for the oxysterol liver.
Crowe1 many nonpeptide antagonists of the gonadotropinreleasing hormone gnrh receptor, as well as other drug targets, possess. The challenge of selecting protein kinase assays for lead discovery. Development of a scintillation proximity assay spa based, high throughput screening feasible method for the identification of pde12 activity modulators volume. The tox21 and toxcast collaboration is combining technology. The speed and efficiency of the rf can allow for experiments that would otherwise be deemed untenable under normal circumstances.
Realtime analysis of endosomal lipid transport by live cell scintillation proximity assay. 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 as a highthroughput method to identify slowly dissociating nonpeptide ligand binding to the gnrh receptor christopher e. 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. Baker 1 department of medicinal chemistry and 2 department of biochemistry, institute for structural biology and drug discovery, virginia commonwealth university, richmond, va 2329803, usa. Cds15 assay is based on a publication by tahir et al. In spa, scintillant is incorporated into small fluomicrospheres. They combine either constructively or destructively leading to the rapid and. For example, we recently designed a singlestep fluorescent assay for sensitive measurement of prmt1mediated methylation.
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. Assay concepts a number of concepts can be envisaged to study proteindna interactions by spa. Scintillation proximity assay spa is a homogeneous assay technology 1 which is beadbased and removes the need for a filtration step to separate bound from free ligand in a receptorbinding assay. The rf system has been validated as suitable for many drug discoveries,1521, and adme absorption. Taglite, ligand binding assay, htrf, opioid, chemokine and cholecystokinin. Wong chapter nuclear receptors as drug targets 173 patricia d.
Scintillation proximity assay for measurement of rna. The assay is homogeneous in nature, as it relies on a mix and measure format. Scintillation proximity assay spa technology and leadseeker homogeneous imaging system are used extensively in high throughput screening. It is economical when compared to the standard microtubule polymerization assay but it is limited by being restricting to the location of colchicines site. Scintillation proximity assay as a highthroughput method. 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 basic components and operating principle of a scintillation detector are. Scintillation proximity assay spa as a new approach to. An indirect, nondestructive, quantitative assay for the presence of antigens or antibodies in a biological fluid. A simplified scintillation proximity assay for fatty acid.
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