A method for rapid, continuous monitoring of solute uptake and binding

Grüner, S.; Kirk, Gregory; Patel, Lehka; Kaback, H. Ronald

doi:10.1021/bi00256a033

Cited by 11 publications

(4 citation statements)

References 8 publications

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“…However, this requires separation of bound from unbound ligands. To circumvent this, the scintillation proximity assay system (SPA) , was developed, where receptors are immobilized on solid microspheres that contain scintillation fluid. This homogeneous assay provides specific information on radiolabeled ligand binding without the need for separation from unbound ligand, as scintillation is only stimulated by radioligand in close proximity to the microspheres. , As a first attempt to apply SPA to MPs, membranes containing acetylcholine receptor were isolated from the electric organ of Torpedo californica and directly added to polyvinyltoluene fluorophore microbeads previously suspended in Triton X-100.…”

Section: Noncovalent Interactions (Adsorption) With the Membranementioning

confidence: 99%

“…However, this requires separation of bound from unbound ligands. To circumvent this, the scintillation proximity assay system (SPA) 28,29 was developed, where receptors are immobilized on solid microspheres that contain scintillation fluid. This homogeneous assay provides specific information on radiolabeled ligand binding without the need for separation from unbound ligand, as scintillation is only stimulated by radioligand in close proximity to the microspheres.…”

Section: Via Native Membrane Hydrophobic Interactions On Beadssmp: Ac...mentioning

confidence: 99%

See 1 more Smart Citation

How to Catch a Membrane Protein in Action: A Review of Functional Membrane Protein Immobilization Strategies and Their Applications

2010

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Section: Noncovalent Interactions (Adsorption) With the Membranementioning

confidence: 99%

Section: Via Native Membrane Hydrophobic Interactions On Beadssmp: Ac...mentioning

confidence: 99%

How to Catch a Membrane Protein in Action: A Review of Functional Membrane Protein Immobilization Strategies and Their Applications

2010

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“…Another application of SPRIA for small tritiated molecules was made by Gruner et al (7). They coated the same fluorophor beads with a layer of gel that was permeable to small tritiated ligands.…”

Section: Assaysmentioning

confidence: 99%

Scintillation proximity radioimmunoassay utilizing 125I-labeled ligands.

Udenfriend¹,

Gerber²,

Brink³

et al. 1985

Proc. Natl. Acad. Sci. U.S.A.

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A unique type of radioimmunoassay is described that does not require centrifugation or separation. Microbeads containing a fluorophor are covalently linked to antibody. When an 12SI-labeled antigen is added it binds to the beads and, by its proximity, the emitted short-range electrons of the 125I excite the fluorophor in the beads. The light emitted can be measured in a standard scintillation counter. Addition of unlabeled antigen from tissue extracts displaces the labeled ligand and diminishes the fluorescent signal. Application of scintillation proximity immunoassay to tissue enkephalins, serum thyroxin, and urinary morphine is described. Applications ofthe principle to study the kinetics of interaction between receptors and ligands are discussed.The term scintillation proximity assay was coined by Hart and Greenwald in 1979 (1) to describe a unique type of RIA. In their procedure they used two different types of polymer beads to which the same antigen (human serum albumin) had been linked covalently. One of the beads had a fluorophor immobilized in it, whereas the second bead was labeled with 3H. Since electrons emitted during 3H decay have a range of only a few micrometers in water, in dilute suspension few of the 3H beads would be close enough to excite the fluorophor beads so that only a low level of fluorescence would be observed. On addition of specific antibody to the suspended beads, agglutination occurs, bringing many of the 3H beads within sufficient proximity of the fluorophor beads, so as to excite them. The emitted fluorescence can be measured in a scintillation counter. The procedure described by Hart and Greenwald (1) MATERIALS AND METHODSThe fluorophor-containing polyvinyltoluene beads (NE-102A), 1-10 pum in diameter, were purchased from Nuclear Enterprises (Edinburgh, Scotland). The methyl groups on the beads were oxidized to carboxyl groups with potassium permanganate by the following procedure. Seven grams of beads was suspended in 70 ml ofa 3% solution ofTriton X-100 (Packard), shaken, and centrifuged. The bead pellet was washed six times by repeatedly suspending in 70 ml of water, shaking, and centrifuging. The washed beads were resuspended in 36 ml of water and stored in the refrigerator overnight to permit settling. The fine particles were removed the next morning by aspirating off the supernatant fluid and the beads were resuspended in water to a final volume of 70 ml.A 7-ml aliquot of the bead suspension (ca. 700 mg) was transferred to a 100-ml round-bottom flask to which 22 ml of water and 22 ml of a freshly prepared solution of 0.1 M KMnO4 were added. After layering the flask with argon, it was placed in a 60°C bath and incubated for 10 hr with stirring. The flask was then removed from the bath and incubated at room temperature for an additional 12 hr. After oxidation, 5 ml of a sodium bisulfite solution (40 g/100 ml) was added with stirring, followed by addition of 30 ml of 1 M HCl. The suspension was then transferred to a centrifuge tube and after centrifugation the sup...

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“…The light emitted by this interaction could be measured with a standard scintillation counter. Subsequently, Gruner and co-workers [5,6] developed a scintillant-impregnated bead that was encapsulated within a permeable gel. This permitted the diffusion of [ 3 H]-tetraphenylphosphonium (TPP) ions through the gel but restricted entry of Escherichia coli membrane vesicles.…”

Section: Introductionmentioning

confidence: 99%

Scintillation proximity assay in lead discovery

Khawaja

Dunlop

Kowal

2008

Expert Opinion on Drug Discovery

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The SPA approach is well suited to the demands of commercial high volume automation and assay miniaturization for target-based high-throughput screening campaigns on synthetic and natural product libraries as well as for benchtop characterization and confirmation studies. In the near future, innovations in the way SPA and fluorescence-based screening strategies are multiplexed will improve our comprehensive understanding of cellular system biology and dramatically advance the lead discovery process for the treatment of complex target-related disorders.

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A method for rapid, continuous monitoring of solute uptake and binding

Cited by 11 publications

References 8 publications

How to Catch a Membrane Protein in Action: A Review of Functional Membrane Protein Immobilization Strategies and Their Applications

How to Catch a Membrane Protein in Action: A Review of Functional Membrane Protein Immobilization Strategies and Their Applications

Scintillation proximity radioimmunoassay utilizing 125I-labeled ligands.

Scintillation proximity assay in lead discovery

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