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Journal articles on the topic 'Binding assay'

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Published: 4 June 2021

Last updated: 11 February 2022

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Rozwandowicz-Jansen, Anita, Jonne Laurila, Eija Martikkala, et al. "Homogeneous GTP Binding Assay Employing QRET Technology." Journal of Biomolecular Screening 15, no. 3 (2010): 261–67. http://dx.doi.org/10.1177/1087057109358921.

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Functional cell signaling assays have become important tools for measuring ligand-induced receptor activation in cell-based biomolecular screening. Guanosine-5′-triphosphate (GTP) is a generic signaling marker responsible for the first intracellular signaling event of the G-protein-coupled receptors (GPCRs). [35S]GTPγS binding assay is the classical well-established method for measuring agonist-induced G-protein activation requiring a separation of free and bound fractions prior to measurement. Here a novel, separation-free, time-resolved fluorescence GTP binding assay has been developed based on a non–fluorescence resonance energy transfer (FRET) single-label approach and quenching of a nonbound europium-labeled, nonhydrolyzable GTP analog (Eu-GTP). The quenching resonance energy transfer (QRET) method relies on the use of Eu-GTP, providing a time-resolved fluorescent detection as an alternative to the radiolabel [35S]GTPγS assay. Upon activation of recombinant human α2A-adrenoceptors (α2A-AR) expressed in Chinese hamster ovary cells, guanosine-5′-diphosphate is released from the α-subunit of Gi-proteins, enabling the subsequent binding of Eu-GTP. Activation of α2A-AR with 5 different α2-AR agonists was measured quantitatively using the developed QRET GTP assay and compared to [35S]GTPγS and heterogeneous Eu-GTP filtration assays. Equal potencies and efficacy rank orders were observed in all 3 assays but with a lower signal-to-background ratio and increased assay variation in the QRET assay compared to the Eu-GTP filtration and the nonhomogeneous [35S]GTPγS binding assays.

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Kim, Jeong-Ho. "Immobilized DNA-binding assay, an approach for in vitro DNA-binding assay." Analytical Biochemistry 334, no. 2 (2004): 401–2. http://dx.doi.org/10.1016/j.ab.2004.06.045.

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Bylund, D. B., and M. L. Toews. "Radioligand binding methods: practical guide and tips." American Journal of Physiology-Lung Cellular and Molecular Physiology 265, no. 5 (1993): L421—L429. http://dx.doi.org/10.1152/ajplung.1993.265.5.l421.

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Radioligand binding assays are a relatively simple but extremely powerful tool for studying receptors. They allow an analysis of the interactions of hormones, neurotransmitters, growth factors, and related drugs with the receptors, studies of receptor interactions with second messenger systems, and characterization of regulatory changes in receptor number, subcellular distribution, and physiological function. As a result, these assays are widely used (and often misused) by investigators in a variety of disciplines, including pharmacology, physiology, biochemistry, immunology, and cell biology. This article presents a broad overview of the radioligand binding assay technique, primarily for the investigator who has limited experience with this technique. Practical guidelines for setting up a new assay are presented, including the receptor preparation to be used, choice of appropriate radioligand, optimizing assay conditions, and appropriate methods for data analysis. Tips for avoiding some of the common pitfalls in application of these assays are also included. The primary focus is on radioligand binding assays of membrane-bound receptors studied in membrane preparations. However, similar assay techniques can be used to study receptors on intact cells. The unique advantages and disadvantages of these intact cell binding assays are also discussed. In particular, the occurrence of regulatory changes in receptors during the course of intact cell binding assays is considered, with approaches for circumventing these complications and for using intact cell assays to advantage in studying these regulatory changes.

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Zhu, Zhengrong, Sean Kim, Taosheng Chen, et al. "Correlation of High-Throughput Pregnane X Receptor (PXR) Transactivation and Binding Assays." Journal of Biomolecular Screening 9, no. 6 (2004): 533–40. http://dx.doi.org/10.1177/1087057104264902.

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Pregnane X receptor (PXR) transactivation and binding assays have been developed into high-throughput assays, which are robust and reproducible (Z′ > 0.5). For most compounds, there was a good correlation between the results of the transactivation and binding assays. EC50 values of compounds in the transactivation assay correlated reasonably well with their IC50 values in the binding assay. However, there were discrepancies with some compounds showing high binding affinity in the binding assay translated into low transactivation. The most likely cause for these discrepancies was an agonist-dependent relationship between binding affinity and transactivation response. In general, compounds that bound to human PXR and transactivated PXR tended to be large hydrophobic molecules.

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FERRARA, PASCUAL, та CHOH HAO LI. "β-ENDORPHIN: RADIORECEPTOR BINDING ASSAY". International Journal of Peptide and Protein Research 16, № 1 (2009): 66–69. http://dx.doi.org/10.1111/j.1399-3011.1980.tb02936.x.

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Duensing, Thomas D., and Susan R. Watson. "Simple Multiplexed Antibody-Binding Assay." Cold Spring Harbor Protocols 2018, no. 1 (2018): pdb.prot093781. http://dx.doi.org/10.1101/pdb.prot093781.

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Lee, Jennifer Y., Sheri Miraglia, Xiongwei Yan, et al. "Oncology Drug Discovery Applications Using the FMAT™ 8100 HTS System." Journal of Biomolecular Screening 8, no. 1 (2003): 81–88. http://dx.doi.org/10.1177/1087057102239668.

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High-throughput screening (HTS) for potential anticancer agents requires a broad portfolio of assay platforms that may include kinase enzyme assays, protein-protein binding assays, and functional cell-based apoptosis assays. The authors have explored the use of fluorometric microvolume assay technology (the FMAT™ 8100 HTS System) in three distinct homogeneous HTS assays: (1) a Src tyrosine kinase enzyme assay, (2) a Grb2-SH2 protein-peptide interaction assay, and (3) an annexin V binding apoptosis assay. Data obtained from all three assays suggest that the FMAT system should facilitate the implementation of homogeneous assays for a wide variety of molecular targeted and cell-based screens. ( Journal of Biomolecular Screening 2003:81-88)

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Vainshtein, Inna, Scott Silveria, Poonam Kaul, Riaz Rouhani, Richard M. Eglen, and John Wang. "A High-Throughput, Nonisotopic, Competitive Binding Assay for Kinases Using Nonselective Inhibitor Probes (ED-NSIP™)." Journal of Biomolecular Screening 7, no. 6 (2002): 507–14. http://dx.doi.org/10.1177/1087057102238624.

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A novel competitive binding assay for protein kinase inhibitors has been developed for high-throughput screening (HTS). Unlike functional kinase assays, which are based on detection of substrate phosphorylation by the enzyme, this novel method directly measures the binding potency of compounds to the kinase ATP binding site through competition with a conjugated binding probe. The binding interaction is coupled to a signal amplification system based on complementation of β-galactosidase enzyme fragments, a homogeneous, nonisotopic assay technology platform developed by DiscoveRx Corp. In the present study, staurosporine, a potent, nonselective kinase inhibitor, was chemically conjugated to a small fragment of β-galactosidase (termed ED-SS). This was used as the binding probe to the kinase ATP binding pocket. The binding potencies of several inhibitors with diverse structures were assessed by displacement of ED-SS from the kinase. The assay format was specifically evaluated with GSK3α, an enzyme previously screened in a radio-active kinase assay (i.e., measurement of [33P]-γ-ATP incorporation into the kinase peptide substrate). Under optimized assay conditions, nonconjugated staurosporine inhibited ED-SS binding in a concentration-dependent manner with an apparent potency (IC50) of 11 nM, which was similar to the IC50 value determined in a radioactive assay. Furthermore, 9 kinase inhibitors with diverse structures, previously identified from chemical compound library screening, were screened using the competitive binding assay. The potencies in the binding assay were in very good agreement with those obtained previously in the isotopic functional activity assay. The binding assay was adapted for automated HTS using selected compound libraries in a 384-well microtiter plate format. The HTS assay was observed to be highly robust and reproducible (Z factors > 0.7) with high interassay precision ( R2 > 0.96). Interference of compounds with the β-galactosidase signal readout was negligible. In conclusion, the DiscoveRx competitive kinase binding assay, termed ED-NSIP™, provides a novel method for screening kinase inhibitors. The format is homogeneous, robust, and amenable to automation. Because there is no requirement for substrate-specific antibodies, the assay is particularly applicable to Ser/Thr kinase assay, in which difficulties in identifying a suitable substrate and antibody preclude development of nonisotopic assays. Although the nonselective kinase inhibitor, staurosporine, was used here, chemically conjugating the ED fragment to other small molecule enzyme inhibitors is also feasible, suggesting that the format is generally applicable to other enzyme systems.

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Calvo, David, María Jesús Vázquez, Charlotte Ashby, and Juan Manuel Domínguez. "Kinetic Considerations on the Development of Binding Assays in Single-Addition Mode." Journal of Biomolecular Screening 17, no. 8 (2012): 1041–49. http://dx.doi.org/10.1177/1087057112452318.

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The development of assays in single-addition mode is of great interest for screening purposes given the multiple advantages of minimizing the number of intervention steps. Binding assays seem to be more prone to this attractive format because no functional biological activity is taking place but instead a biophysical process, whose dynamics seem easier to control without introducing significant alterations, is happening. Therefore, single-addition assays based on the displacement of prebound labeled ligands can be conceived, but careful kinetic considerations must still be taken to maximize the sensitivity of the assay and to avoid jeopardizing the identification of compounds with slow-binding kinetics. This article shows the development of a single-addition, displacement-based binding assay intended to identify modulators that act by binding to the gabapentin site of the ion channel regulatory protein α2δ1. After studying the kinetics of gabapentin binding and the influence they might have on the assay sensitivity, the best conditions were identified, and the sensitivity was compared with that of the more classical two-additions competition-based assay. Although the present study focuses on α2δ1 and its interaction with gabapentin, the rationale and the methodology followed are of broad purpose and can be applied to virtually every binding assay.

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Wilkins, T. A., J. E. Midgley, R. A. Stevens, I. Caughey, and N. Barron. "Assay performance and tracer properties for two analog-based assays of free triiodothyronine." Clinical Chemistry 32, no. 3 (1986): 465–69. http://dx.doi.org/10.1093/clinchem/32.3.465.

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Abstract We determined binding characteristics of the triiodothyronine (T3) analog tracer used in the Amerlex and Amerlex-M FT3 radioimmunoassay for the three endogenous binding proteins in serum: thyroxin-binding globulin (TBG), thyroxin binding prealbumin (PA), and albumin. Both T3 and its analog bind to the same sites on TBG and PA. However, the analog has significantly lower association constants (1.0% and 3.8%, respectively, of T3 binding affinity) and it binds to different sites on albumin. Analog binding is characterized by two (weak) specific binding sites [K = 0.46 (SD 0.03) X 10(5) L/mol]; T3 is bound at about 28 very weak, nonspecific sites [K = 0.41 (SD 0.03) X 10(4) L/mol]. Sera from healthy subjects with a wide range of concentrations of binding proteins showed no interference from analog binding in the FT3 assay. In contrast, in vitro studies of albumin binding revealed a weak dependence of both assays on albumin concentration (0.05 pmol of FT3 per gram of albumin per liter), an interference probably unimportant for most laboratory samples. Nonesterified fatty acids (NEFA) and the T3 analog apparently bind to different sites on albumin; thus the Amerlex FT3 assay is insensitive to moderately increased concentrations of NEFA in serum.

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Bee, Weilin Tiger, Wensheng Xie, Maggie Truong, et al. "The Development of a High-Content Screening Binding Assay for the Smoothened Receptor." Journal of Biomolecular Screening 17, no. 7 (2012): 900–911. http://dx.doi.org/10.1177/1087057112447872.

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In this study, the development of an image-based high-content screening (HCS) binding assay for the seven-transmembrane (7TM) receptor Smoothened (Smo) is described. Using BacMam-based gene delivery of Smo, BODIPY-cyclopamine as a fluorescent probe, and a confocal imaging system, a robust 384-well assay that could be used for high-throughput compound profiling activities was developed. The statistically robust HCS binding assay was developed through optimization of multiple parameters, including cell transduction conditions, Smo expression levels, the image analysis algorithm, and staining procedures. Evaluation of structurally diverse compounds, including functional Smo activators, inhibitors, and related analogs, demonstrated good compound potency correlations between high-content imaging binding, membrane fluorescence polarization binding, and gene reporter assays. Statistical analysis of data from a screening test set of compounds at a single 10-µM concentration suggested that the high-content imaging Smo binding assay is amenable for use in hit identification. The 384-well HCS assay was rapidly developed and met statistical assay performance targets, thus demonstrating its utility as a fluorescent whole-cell binding assay suitable for compound screening and profiling.

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Strange, P. G. "Mechanisms underlying agonist efficacy." Biochemical Society Transactions 35, no. 4 (2007): 733–36. http://dx.doi.org/10.1042/bst0350733.

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Agonist efficacy is a measure of how well an agonist can stimulate a response system linked to a receptor. Efficacy can be assessed in functional assays and various parameters (Emax, KA/EC50, Emax·KA/EC50) determined. The Emax·KA/EC50 parameter provides a good estimate of efficacy across the full range of efficacy. A convenient assay for the efficacy of agonists for some receptors is provided by the [35S]GTP[S] (guanosine 5′-[γ-[35S]thio]triphosphate)-binding assay. In this assay, the normal GTP-binding event in GPCR (G-protein-coupled receptor) activation is replaced by the binding of the non-hydrolysable analogue [35S]GTP[S]. This assay may be used to profile ligands for their efficacy, and an example here is the D2 dopamine receptor where an efficacy scale has been set up using this assay. The mechanisms underlying the assay have been probed. The time course of [35S]GTP[S] binding follows a pseudo-first-order reaction with [35S]GTP[S] binding reaching equilibrium after approx. 3 h. The [35S]GTP[S]-binding event is the rate-determining step in the assay. Agonists regulate the maximal level of [35S]GTP[S] bound, rather than the rate constant for binding. The [35S]GTP[S]-binding assay therefore determines agonist efficacy on the basis of the amount of [35S]GTP[S] bound rather than the rate of binding.

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van der Hee, Regine M., Tanja Deurholt, Cindy C. Gerhardt, and Els M. de Groene. "Comparison of 3 AT1 Receptor Binding Assays: Filtration Assay, ScreenReady™ Target, and WGA Flashplate®." Journal of Biomolecular Screening 10, no. 2 (2005): 118–26. http://dx.doi.org/10.1177/1087057104271330.

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In this article, the study of 3 different angiotensin II type 1 (AT1) receptor binding assays in terms of reproducibility, robustness, and feasibility for high-throughput screening (HTS) is described. The following methods were used: a nonhomogeneous filtration assay in a 96-well format using CHO-AT1 cell membranes and 2 homogeneous assays, which include the commercially available ScreenReady™ Target for the AT1 receptor and the wheat germ agglutinin (WGA) Flashplate®, which was coated “in-house” with the CHO-AT1 cell membranes. Receptors were labeled with [125I]-Sar1-Ile8-angiotensin II, and radioligand binding was displaced using the antagonist losartan and the natural agonist angiotensin II. Reproducible Kd, Bmax, and Ki values and good total binding/nonspecific binding (TB/NSB) ratios were obtained with both the ScreenReady™ Targets and the filtration assay, whereas the WGA Flashplates® showed unacceptably high nonspecific binding and high variation when applied as a homogeneous assay. However, when applied as a heterogeneous assay (i.e., when a wash step at the end of the assay is included), the results were significantly better. Interestingly, ligand affinities were consistently lower in Flashplate®-based assays than in the filtration assay. This may be due to the immobilization of the receptors onto the solid surface of the plate, affecting their conformation. In terms of reproducibility, robustness, and feasibility for HTS, the authors conclude that the ScreenReady™ Target plates are most suitable for AT1 receptor binding screening.

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Lawson, Nicola L., Carly I. Dix, Paul W. Scorer, et al. "Mapping the binding sites of antibodies utilized in programmed cell death ligand-1 predictive immunohistochemical assays for use with immuno-oncology therapies." Modern Pathology 33, no. 4 (2019): 518–30. http://dx.doi.org/10.1038/s41379-019-0372-z.

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Abstract Programmed cell death ligand-1 (PD-L1) expression levels in patient tumor samples have proven clinical utility across various cancer types. Several independently developed PD-L1 immunohistochemical (IHC) predictive assays are commercially available. Published studies using the VENTANA PD-L1 (SP263) Assay, VENTANA PD-L1 (SP142) Assay, Dako PD-L1 IHC 22C3 pharmDx assay, Dako PD-L1 IHC 28-8 pharmDx assay, and laboratory-developed tests utilizing the E1L3N antibody (Cell Signaling Technology), have demonstrated differing levels of PD-L1 staining between assays, resulting in conjecture as to whether antibody-binding epitopes could be responsible for discordance between assays. Therefore, to understand the performance of different PD-L1 predictive immunohistochemistry assays, we aimed to distinguish the epitopes within the PD-L1 protein responsible for antibody binding. The sites at which antibody clones SP263, SP142, 22C3, 28-8, and E1L3N bind to recombinant PD-L1 were assessed using several methods, including conformational peptide array, surface plasmon resonance, and/or hydrogen/deuterium exchange mass spectrometry. Putative binding sites were confirmed by site-directed mutagenesis of PD-L1, followed by western blotting and immunohistochemical analysis of cell lines expressing mutant constructs. Our results demonstrate that clones SP263 and SP142 bind to an identical epitope in the cytoplasmic domain at the extreme C-terminus of PD-L1, distinct from 22C3 and 28-8. Using mutated PD-L1 constructs, an additional clone, E1L3N, was also found to bind to the cytoplasmic domain of PD-L1. The E1L3N binding epitope overlaps considerably with the SP263/SP142 binding site but is not identical. Clones 22C3 and 28-8 have binding profiles in the extracellular domain of PD-L1, which differ from one another. Despite identifying epitope binding variance among antibodies, evidence indicates that only the SP142 assay generates significantly discordant immunohistochemical staining, which can be resolved by altering the assay protocol. Therefore, inter-assay discordances are more likely attributable to tumor heterogeneity, assay, or platform variables rather than antibody epitope.

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Mahony, Mary C., Karen Rice, Erwin Goldberg, and Gustavo Doncel. "Baboon spermatozoa–zona pellucida binding assay." Contraception 61, no. 3 (2000): 235–40. http://dx.doi.org/10.1016/s0010-7824(00)00093-7.

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Davis, Diane L., and Steven J. Soldin. "An immunophilin-binding assay for sirolimus." Clinical Therapeutics 22 (January 2000): B62—B70. http://dx.doi.org/10.1016/s0149-2918(00)89023-2.

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Roy, Partha, and Darren Tan Cherng-Wen. "Microfluidic competition assay via equilibrium binding." Sensors and Actuators B: Chemical 139, no. 2 (2009): 682–87. http://dx.doi.org/10.1016/j.snb.2009.03.077.

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Woolley, Joseph L., Jennifer L. Ringstad, and Carl W. Sigel. "Competitive Protein Binding Assay for Piritrexim." Journal of Pharmaceutical Sciences 78, no. 9 (1989): 749–52. http://dx.doi.org/10.1002/jps.2600780910.

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Holland, J. D., P. Singh, J. C. Brennand, and A. J. Garman. "A Nonseparation Microplate Receptor Binding Assay." Analytical Biochemistry 222, no. 2 (1994): 516–18. http://dx.doi.org/10.1006/abio.1994.1530.

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Peter, H., R. J. Laib, H. Ottenw�lder, H. Topp, N. Rupprich, and H. M. Bolt. "DNA-binding assay of methyl chloride." Archives of Toxicology 57, no. 2 (1985): 84–87. http://dx.doi.org/10.1007/bf00343115.

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Geller, Arthur M., and Malak Y. Kotb. "A binding assay for serine hydroxymethyltransferase." Analytical Biochemistry 180, no. 1 (1989): 120–25. http://dx.doi.org/10.1016/0003-2697(89)90098-5.

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Lacy, Brian E., and Charles B. Underhill. "A protein binding assay for hyaluronate." Analytical Biochemistry 158, no. 2 (1986): 436–42. http://dx.doi.org/10.1016/0003-2697(86)90572-5.

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Lee, Eun-Gyung, and Maxine L. Linial. "Yeast Three-Hybrid Screening of Rous Sarcoma Virus Mutants with Randomly Mutagenized Minimal Packaging Signals Reveals Regions Important for Gag Interactions." Journal of Virology 74, no. 19 (2000): 9167–74. http://dx.doi.org/10.1128/jvi.74.19.9167-9174.2000.

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ABSTRACT We previously showed that the yeast three-hybrid system provides a genetic assay of both RNA and protein components for avian retroviral RNA encapsidation. In the current study, we used this assay to precisely define cis-acting determinants involved in avian leukosis sarcoma virus packaging RNA binding to Gag protein. In vivo screening of Rous sarcoma virus mutants was performed with randomly mutated minimal packaging sequences (MΨ) made using PCR amplification after cotransformation with GagΔPR protein into yeast cells. Colonies with low β-galactosidase activity were analyzed to locate mutations in MΨ sequences affecting binding to Gag proteins. This genetic assay delineated secondary structural elements that are important for efficient RNA binding, including a single-stranded small bulge containing the initiation codon for uORF3, as well as adjacent stem structures. This implies a possible tertiary structure favoring the high-affinity binding sites for Gag. In most cases, results from the three-hybrid assay were well correlated with those from the viral RNA packaging assays. The results from random mutagenesis using the rapid three-hybrid binding assay are consistent with those from site-directed mutagenesis using in vivo packaging assays.

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Kwon, Jung-Hwan, Lynn E. Katz, and Howard M. Liljestrand. "Modeling binding equilibrium in a competitive estrogen receptor binding assay." Chemosphere 69, no. 7 (2007): 1025–31. http://dx.doi.org/10.1016/j.chemosphere.2007.04.047.

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Miller, Katherine R., and David P. Cistola. "Titration calorimetry as a binding assay for lipid-binding proteins." Molecular and Cellular Biochemistry 123, no. 1-2 (1993): 29–37. http://dx.doi.org/10.1007/bf01076472.

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Steiner, G., B. Küllinger, G. Mayer, et al. "Determination of cyclosporine by a competitive binding assay with cyclophilin." Clinical Chemistry 37, no. 3 (1991): 403–10. http://dx.doi.org/10.1093/clinchem/37.3.403.

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Abstract A competitive protein-binding assay for cyclosporine based on use of the intracellular cyclosporine-binding protein cyclophilin (CYP) was used to measure cyclosporin A (CsA) and its bioactive metabolites in whole blood. CYP from cytoplasmic extracts or erythrocyte lysates was applied in the binding assay with use of [3H]CsA as tracer and charcoal adsorption for separating bound from free tracer. Binding affinities of various CsA analogs and metabolites correlated well with their reported in vitro immunosuppressive activities. The assay detected as little CsA as 50 micrograms/L (1 g = 0.832 mmol of CsA), analytical recovery was greater than 80%, and CVs were less than 8% for intra-assay and less than 11% for interassay precision in the range of 150-1000 micrograms/L. We used this assay to measure CsA concentrations in blood and compared the results with those measured by HPLC or by CsA-specific (monoclonal) and CsA-nonspecific (polyclonal) radioimmunoassays. Binding assay results were, in nearly all cases, less than those measured by the nonspecific RIA and frequently greater than 20% above the values determined by the CsA-specific assays. Individual patients had pronounced differences in the relative proportions of CsA, CYP-binding (bioactive) metabolites, and cross-reacting CsA metabolites. Because the presence of bioactive metabolites may considerably contribute to the immunosuppressive activity of CsA, we consider the binding assay clinically useful for measuring CsA in biological fluids.

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Lee, Paul H., Steven C. Miller, Carlo van Staden, and Evan F. Cromwell. "Development of a Homogeneous High-Throughput Live-Cell G-Protein-Coupled Receptor Binding Assay." Journal of Biomolecular Screening 13, no. 8 (2008): 748–54. http://dx.doi.org/10.1177/1087057108317835.

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The measurement of ligand receptor binding parameters for G-protein-coupled receptors is indispensable in the drug discovery process. Traditional ligand receptor binding assays require scale-up of cells and membrane preparations, which is an expensive and time-consuming process. In this report, the authors describe the development of a homogeneous live-cell binding assay for GPCRs using a fluorophore-labeled nonpeptide ligand. The model assay used Cy3B-labeled telenzepine and Chinese hamster ovary cells expressing M1 muscarinic acetylcholine receptors. This homogeneous live-cell fluorescence binding assay format is superior to the traditional binding methods because it measures binding of a ligand to intact receptors on living cells. The assay requires no washing or separation steps, thereby allowing a real-time kinetic readout for the determination of ligand association and dissociation from the intact receptors. The results also suggest that miniaturization is feasible without compromising the data quality. ( Journal of Biomolecular Screening 2008:748-754)

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Guo, Dong, Erika J. H. van Dorp, Thea Mulder-Krieger, et al. "Dual-Point Competition Association Assay." Journal of Biomolecular Screening 18, no. 3 (2012): 309–20. http://dx.doi.org/10.1177/1087057112464776.

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The concept of ligand-receptor binding kinetics is emerging as an important parameter in the early phase of drug discovery. Since the currently used kinetic assays are laborious and low throughput, we developed a method that enables fast and large format screening. It is a so-called dual-point competition association assay, which measures radioligand binding at two different time points in the absence or presence of unlabeled competitors. Specifically, this assay yields the kinetic rate index (KRI), which is a measure for the binding kinetics of the unlabeled ligands screened. As a prototypical drug target, the adenosine A1 receptor (A1R) was chosen for assay validation and optimization. A screen with 35 high-affinity A1R antagonists yielded seven compounds with a KRI value above 1.0, which indicated a relatively slow dissociation from the target. All other compounds had a KRI value below or equal to 1.0, predicting a relatively fast dissociation rate. Several compounds were selected for follow-up kinetic quantifications in classical kinetic assays and were shown to have kinetic rates that corresponded to their KRI values. The dual-point assay and KRI value may have general applicability at other G-protein-coupled receptors, as well as at drug targets from other protein families.

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Tirri, Marko E., Roope J. Huttunen, Juha Toivonen, Pirkko L. Härkönen, Juhani T. Soini, and Pekka E. Hänninen. "Two-Photon Excitation in Fluorescence Polarization Receptor-Ligand Binding Assay." Journal of Biomolecular Screening 10, no. 4 (2005): 314–19. http://dx.doi.org/10.1177/1087057104273334.

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Fluorescence polarization is one of the most commonly used homogeneous assay principles in drug discovery for screening of potential lead compounds. In this article, the fluorescence polarization technique is combined with 2-photon excitation of fluorescence. Theoretically, the use of 2-photon excitation of fluorescence increases the volumetric sensitivity and polarization contrast of fluorescence polarization assays. The work in this report demonstrates these predictions for an estrogen receptor ligand binding assay.

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DeLeeuw, Lynn W., Robert C. Monsen, Vytautas Petrauskas, et al. "POT1 stability and binding measured by fluorescence thermal shift assays." PLOS ONE 16, no. 3 (2021): e0245675. http://dx.doi.org/10.1371/journal.pone.0245675.

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The protein POT1 (Protection of Telomeres 1) is an integral part of the shelterin complex that protects the ends of human chromosomes from degradation or end fusions. It is the only component of shelterin that binds single-stranded DNA. We describe here the application of two separate fluorescent thermal shift assays (FTSA) that provide quantitative biophysical characterization of POT1 stability and its interactions. The first assay uses Sypro Orange™ and monitors the thermal stability of POT1 and its binding under a variety of conditions. This assay is useful for the quality control of POT1 preparations, for biophysical characterization of its DNA binding and, potentially, as an efficient screening tool for binding of small molecule drug candidates. The second assay uses a FRET-labeled human telomeric G-quadruplex structure that reveals the effects of POT1 binding on thermal stability from the DNA frame of reference. These complementary assays provide efficient biophysical approaches for the quantitative characterization of multiple aspects of POT1 structure and function. The results from these assays provide thermodynamics details of POT1 folding, the sequence selectivity of its DNA binding and the thermodynamic profile for its binding to its preferred DNA binding sequence. Most significantly, results from these assays elucidate two mechanisms for the inhibition of POT1 –DNA interactions. The first is by competitive inhibition at the POT1 DNA binding site. The second is indirect and is by stabilization of G-quadruplex formation within the normal POT1 single-stranded DNA sequence to prevent POT1 binding.

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Hamel, Martine, Martin Henault, Huda Hyjazie, et al. "Discovery of Novel P2Y14 Agonist and Antagonist Using Conventional and Nonconventional Methods." Journal of Biomolecular Screening 16, no. 9 (2011): 1098–105. http://dx.doi.org/10.1177/1087057111415525.

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P2Y14 is a member of the pyrimidinergic GPCR family. UDP-Glc has been previously shown to activate human P2Y14, whereas UDP was unable to activate the receptor. In this study, the authors used conventional and nonconventional methods to further characterize P2Y14 and its ligands. Conventional calcium mobilization and nonconventional cellular impedance functional assays revealed that UMP and UDP selectively activated HEK cells coexpressing P2Y14 and Gαqi5. In the impedance assays, the presence of exogenous Gαqi5 resulted in agonist-induced Gq signaling, whereas in the absence of exogenous Gαqi5, the signal was indicative of Gi. The authors established the first P2Y14 membrane filtration binding assay using a novel optimized expression vector and [3H]UDP as radioligand. UDP-Glc, UMP, and UDP dose dependently inhibited [3H]UDP binding in the binding assay, and saturation analysis revealed that UDP bound P2Y14 with a KD = 10 nM and a Bmax = 110 pmol/mg. The authors screened a phosphonate library and identified compound A, which inhibited UDP-Glc–mediated calcium signaling in the fluorometric imaging plate reader assay (IC50 = 2.3 µM) and competed for [3H]UDP binding in the novel binding assay with a Ki = 1280 nM.

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Sevenler, Derin, Jacob Trueb, and M. Selim Ünlü. "Beating the reaction limits of biosensor sensitivity with dynamic tracking of single binding events." Proceedings of the National Academy of Sciences 116, no. 10 (2019): 4129–34. http://dx.doi.org/10.1073/pnas.1815329116.

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The clinical need for ultrasensitive molecular analysis has motivated the development of several endpoint-assay technologies capable of single-molecule readout. These endpoint assays are now primarily limited by the affinity and specificity of the molecular-recognition agents for the analyte of interest. In contrast, a kinetic assay with single-molecule readout could distinguish between low-abundance, high-affinity (specific analyte) and high-abundance, low-affinity (nonspecific background) binding by measuring the duration of individual binding events at equilibrium. Here, we describe such a kinetic assay, in which individual binding events are detected and monitored during sample incubation. This method uses plasmonic gold nanorods and interferometric reflectance imaging to detect thousands of individual binding events across a multiplex solid-phase sensor with a large area approaching that of leading bead-based endpoint-assay technologies. A dynamic tracking procedure is used to measure the duration of each event. From this, the total rates of binding and debinding as well as the distribution of binding-event durations are determined. We observe a limit of detection of 19 fM for a proof-of-concept synthetic DNA analyte in a 12-plex assay format.

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Lebakken, Connie S., Hee Chol Kang, and Kurt W. Vogel. "A Fluorescence Lifetime–Based Binding Assay to Characterize Kinase Inhibitors." Journal of Biomolecular Screening 12, no. 6 (2007): 828–41. http://dx.doi.org/10.1177/1087057107304480.

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The authors present a fluorescence lifetime—based kinase binding assay that identifies and characterizes compounds that bind to the adenosine triphosphate (ATP)—binding pocket of a range of tyrosine and serine/threonine kinases. The assay is based on displacement of an Alexa Fluor® 647 conjugate of staurosporine from the ATP-binding site of a kinase, which is detected by a change in the fluorescence lifetime of the probe between the free (displaced) and kinase-bound states. The authors screened 257 kinases for specific binding and displacement of the Alexa Fluor® 647-staurosporine probe and found that approximately half of the kinases tested could potentially be assayed with this method. They present inhibitor binding data against 4 selected serine/threonine kinases and 4 selected tyrosine kinases, using 6 commonly used kinase inhibitors. Two of these kinases were chosen for further studies, in which inhibitor binding data were compared to inhibition of kinase activity using 2 separate activity assay formats. Rank-order potencies of compounds were similar, but not identical, between the binding and activity assays. It was postulated that these differences could be caused by the fact that the assays are measuring distinct phenomena, namely, activity versus binding, and in a purified recombinant kinase preparation, there can exist a mixture of active and nonactivated kinases. To explore this possibility, the authors compared binding affinity for the probe using 2 kinases in their respective nonactivated and activated (phosphorylated) forms and found a kinase-dependent difference between the 2 forms. This assay format therefore represents a simple method for the identification and characterization of small-molecule kinase inhibitors that may be useful in screening a wide range of kinases and may be useful in identifying small molecules that bind to kinases in their active or nonactivated states. ( Journal of Biomolecular Screening 2007:828-841)

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Bosse, Roger, Russell Garlick, Beverly Brown, and Luc Menard. "Development of Nonseparation Binding and Functional Assays for G Protein-Coupled Receptors for High Throughput Screening: Pharmacological Characterization of the Immobilized CCR5 Receptor on FlashPlate(r)." Journal of Biomolecular Screening 3, no. 4 (1998): 285–92. http://dx.doi.org/10.1177/108705719800300407.

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G protein-coupled receptors (GPCRs) represent a very important class of drug targets. The development of microformatted nonseparation assays constitute a key step in the process of assay development for high throughput drug screening (HTS). We have developed a microformatted nonseparation assay for membrane preparations containing the CCR5 GPCR using FlashPlate® microplates (Packard Instrument Company, Meriden, CT). The pharmacodynamic (radioligand-binding) and functional (agonist-stimulated [35S]GTPγS binding) properties of this receptor observed in FlashPlate-based assays were compared with standard filtration assays. Saturation binding experiments performed using either assay platform revealed identical Kd for [125I]-MIP-1 β (0.7 nM). Comparable signal-to-noise ratios (SNR), similar affinities (Ki), and identical order of potency (RANTES ≅ MIP-1β > MIP-1α) were observed following competition binding assays in both platforms. In functional assays, the order of potency for different agonists were similar in both platforms with RANTES ≅ MIP-1β ≥ MIP-1α, which correspond to the relative affinities determined for the three ligands in competition binding experiments. Because similar pharmacologic properties were obtained in both FlashPlate microplates and standard filtration platforms, we conclude that FlashPlate microplates could provide a valuable nonseparation platform for primary and secondary HTS for this and possibly other GPCRs.

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Tian, Y. Edward, Lan-Hsin Wu, William T. Mueller, and Fu-Zon Chung. "A Screening Strategy Based on Differential Binding of Ligand to Receptor and to Binding Proteins: Screening for Compounds Interacting with Corticotrophin-Releasing Factor-Binding Protein." Journal of Biomolecular Screening 4, no. 6 (1999): 319–26. http://dx.doi.org/10.1177/108705719900400607.

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The ligand-receptor interaction has been commonly used in development of high throughput screening assays for new drugs. In some cases, an endogenous ligand interacts not only with membrane receptors but also with soluble binding proteins. Corticotrophin-releasing factor (CRF) is an important stress neurotransmitter/hormone involved in both the central and peripheral nervous systems. CRF exerts its function by interacting with CRFR1 and CRFR2 receptors. In addition, CRF-binding protein (CRF-BP) binds CRF with high affinity. Accordingly, CRF-BP has been suggested to play an important role in modulating CRF function. Based on the potential involvement of CRF-BP in many neurological disorders, it is desirable to develop a screening assay to look for drugs that either mimic or interfere with CRF binding to CRF-BP. An assay was developed to monitor the interactions of radiolabeled CRF with human/rat CRF-BP and the mouse CRFR1 (mCRFR1) receptor. By carefully examining the binding characteristics of radiolabeled CRF to mCRFR1, the assay was able to identify compounds that bind to CRF-BP with high affinity and have little or no affinity for mCRFR1 receptors. Based on a mathematical model, we have verified the screening system with several well-characterized CRF ligands that all have different affinities for CRF receptors and CRF-BP.

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Murray, A., J. F. R. Robertson, and M. R. Price. "Analysis of the Temporal Compressibility of Breast Tumour Marker Assays: Development of a “Near Patient” Assay." International Journal of Biological Markers 10, no. 4 (1995): 200–205. http://dx.doi.org/10.1177/172460089501000402.

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The aim of this study was to investigate whether immunoassays for circulating MUC1 antigen in breast cancer could be compressed in time so that serum level results would be made available during the time of the patient's visit to clinic. Two assays were used: - The EMCA (Euro DPC) is a liquid phase immunoassay and the ELSA CA15-3 (CIS) is a double determinant solid phase immunoradiometric assay. The effects of shortened incubation times were investigated by assaying standards and unknown samples and comparing the results with those using the standard kit protocols. The binding kinetics of the monoclonal antibodies employed in the assays were analysed separately. We conclude that the EMCA assay can be shortened to 35 min and we have attributed this to the fast binding kinetics inherent in a liquid phase assay. This shortened assay may produce the basis for a useful “near patient” assay. By comparison, the solid phase ELSA CA15-3 assay cannot be compressed without loss in assay performance.

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Young, David H., Fernando M. Rubio, and Paul O. Danis. "A Radioligand Binding Assay for Antitubulin Activity in Tumor Cells." Journal of Biomolecular Screening 11, no. 1 (2005): 82–89. http://dx.doi.org/10.1177/1087057105282300.

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The benzamide RH-5854 is shown to be highly potent toward tumor cells and to arrest nuclear division by a highly specific covalent binding to the β-subunit of tubulin in the colchicine binding region. Binding of 3H-RH-5854 to β-tubulin in HCT-116 colon cancer cells is saturable and has been exploited in the development of a cell-based competitive binding assay, which allows antitubulin effects to be detected inwhole cells. 3H-RH-5854 binding is strongly inhibited by preincubating the cells with compounds that bind to the colchicine site andwith paclitaxel. Binding of 3H-RH-5854 is enhanced by preincubating the cells with vinblastine but not by other agents that bind at or near the vinblastine site (ansamitocin P-3 and phomopsin A). Various cytotoxic agents that do not act on tubulin do not affect binding of 3H-RH-5854 in HCT-116 cells, demonstrating specificity of the assay for detection of antitubulin activity. As an alternative to traditional assays that employ isolated brain tubulin, the 3HRH-5854 binding assay enables screening for antitubulin effects directly in tumor cells, providing an assay that accounts for cell-specific criteria that influence sensitivity such as different tubulin isotypes, tubulin mutations, drug metabolism, and efflux mechanisms.

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Grosvenor, Anne L., Agatha Feltus, Richard C. Conover, Sylvia Daunert, and Kimberly W. Anderson. "Development of Binding Assays in Microfabricated Picoliter Vials: An Assay for Biotin." Analytical Chemistry 72, no. 11 (2000): 2590–94. http://dx.doi.org/10.1021/ac991289+.

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Halper, Jaroslava, Douglas S. Colvard, Bernd W. Scheithauer, et al. "Estrogen and Progesterone Receptors in Meningiomas: Comparison of Nuclear Binding, Dextran-Coated Charcoal, and Immunoperoxidase Staining Assays." Neurosurgery 25, no. 4 (1989): 546–53. http://dx.doi.org/10.1227/00006123-198910000-00007.

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Abstract We studied the status of estrogen (ER) and progesterone (PR) receptors in meningiomas removed from 52 patients, comparing dextran-coated charcoal (DCC), nuclear binding (NB), and immunoperoxidase (IP) assays. Each of the assays was performed independently by investigators well-experienced with these assays. The NB assay is a new assay that measures functional steroid receptors—that is, the activation of the receptor and its binding to the nucleus. The assay is very sensitive and requires a relatively small amount of tissue as compared with the DCC assay. In agreement with data from other studies, PR were detected in most meningiomas by all 3 methods: in 69% of the cases by NB, in 76% by DCC, and in 89% by IP. ER were detected in only a few cases: in 33% by NB, in 2% by DCC, and in none by the IP assay. The agreement for PR sites was 62% for all 3 assays, it was 66% between the NB and DCC assays, 67% between the NB and IP assays, and 86% between the DCC and IP assays. Of 26 cases that were positive by the DCC assay, 6 (23%) were negative by NB. The overall agreement for all three ER assays was 65%. The data suggest that the majority of meningiomas contain high-affinity receptors for progesterone, that estrogen receptors are present in only a few meningiomas, and that some of these estrogen and progesterone receptors appear to be functional.

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England, Elizabeth, Philip Newton, Frances Neal, Lisa Kitching, Caroline Colley, and Christine J. Rossant. "Application of the Mirrorball High-Sensitivity Cytometer to Multiplexed Assays for Antibody Drug Discovery." Journal of Biomolecular Screening 20, no. 4 (2014): 536–44. http://dx.doi.org/10.1177/1087057114557776.

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Highly sensitive, high-throughput assay technologies are required for the identification of antibody therapeutics. Multiplexed assay systems are particularly advantageous because they allow evaluation of several parameters within 1 well, increasing throughput and reducing hands-on laboratory time. The mirrorball (TTP Labtech), using high-throughput fluorometric microvolume assay technology, offers simultaneous scanning with up to 3 lasers as well as laser scatter detection. This makes the mirrorball especially suitable for the development of highly sensitive and multiplexed assays. We have developed bead- and cell-based binding assays that demonstrate how the multilaser capability of the mirrorball can be exploited to enhance assay sensitivity. In addition, using the multilaser simultaneous scanning capability, we have established multiplexed cytokine quantitation assays and antibody–cell binding assays. Our results demonstrate the potential utility of this technology to improve the sensitivity and efficiency of biologics screening, resulting in streamlining of the lead antibody selection process.

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Carter, Clare M. Scaramellini, Juliet R. Leighton-Davies, and Steven J. Charlton. "Miniaturized Receptor Binding Assays: Complications Arising from Ligand Depletion." Journal of Biomolecular Screening 12, no. 2 (2007): 255–66. http://dx.doi.org/10.1177/1087057106297788.

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The advent of miniaturized assay formats has made possible the screening of large numbers of compounds against a single target, known as high-throughput screening. Despite this clear advantage, assay miniaturization also increases the risk of ligand depletion, where the actual concentration of free ligand is significantly lower than that added. This, in turn, complicates the interpretation of data from such assays, potentially introducing significant error if not recognized. In this study, the effects of reducing assay volume on radioligand Kd and competitor Ki values have been investigated, using the muscarinic M3 receptor as a model system. It was found that assay miniaturization caused dramatic effects, with up to a 30-fold underestimation of ligand affinity. A theoretical model was developed and shown to accurately predict both the degree of ligand depletion in any given assay volume and the effect of this depletion on affinity estimates for competing ligands. Importantly, it was found that in most cases, errors introduced by ligand depletion could be largely corrected for by the use of appropriate analysis methods. In addition to those previously described by others, the authors propose a simple method capable of correcting errors in competition binding experiments performed in conditions of ligand depletion.

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Nakanishi, Koji, Xuefei Huang, Hong Jiang, et al. "Structure-binding relation of philanthotoxins from nicotinic acetylcholine receptor binding assay." Bioorganic & Medicinal Chemistry 5, no. 10 (1997): 1969–88. http://dx.doi.org/10.1016/s0968-0896(97)00137-5.

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Favaloro, Emmanuel J. "Sulfatide-Binding Assay for von Willebrand Factor." Thrombosis Research 98, no. 2 (2000): 213–19. http://dx.doi.org/10.1016/s0049-3848(99)00232-7.

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Quintino, Luís, Aurélie Baudet, Jonas Larsson, and Cecilia Lundberg. "FACS binding assay for analysing GDNF interactions." Journal of Neuroscience Methods 218, no. 1 (2013): 25–28. http://dx.doi.org/10.1016/j.jneumeth.2013.04.006.

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Lefkovits, H. "Hapten Binding Assay Using Guinea Pig Macrophages." Pathobiology 53, no. 4 (1985): 189–98. http://dx.doi.org/10.1159/000163312.

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Haughton, M. A., and R. S. Mason. "Immunonephelometric Assay of Vitamin D-Binding Protein." Clinical Chemistry 38, no. 9 (1992): 1796–801. http://dx.doi.org/10.1093/clinchem/38.9.1796.

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Abstract An automated immunonephelometric assay was developed to measure vitamin D-binding protein (DBP) in serum. The assay is described for the Behring Nephelometer System, which uses rabbit anti-DBP antiserum and purified human DBP. The detection limit is 0.05 g/L (0.86 mumol/L), and the working range is less than or equal to 1.60 g/L (27.59 mumol/L). Intra- and interassay CVs of 2.0% and 2.8-3.8% compare favorably with alternative methods. When results were compared with those from a immunoradiometric assay, the correlation coefficient was 0.976 (P less than 0.001), and the regression equation [y = 0.866 +/- 0.085x + 0.05 (Syx = 0.042, n = 42)] identified a negative bias. Analysis indicated that both methods appeared to contribute equally to the bias. Although the assay was relatively free from analytical interference, falsely increased values were noted in severely lipemic specimens and in frozen specimens. Interference may be minimized by inclusion of Supplementary Precipitation reagent in a modified assay protocol. The range of concentrations expected in clinical samples was established from normal subjects [0.32-0.46 g/L (5.52-7.93 mumol/L), n = 28], pregnant subjects [0.51-0.70 g/L (8.79-12.07 mumol/L), n = 13], and subjects with liver diseases [0.12-0.33 g/L (2.07-5.69 mumol/L), n = 18].

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Schwenk, Jochen M., Oliver Poetz, Robert Zeillinger, and Thomas O. Joos. "A Miniaturized Ligand Binding Assay for EGFR." International Journal of Proteomics 2012 (April 8, 2012): 1–5. http://dx.doi.org/10.1155/2012/247059.

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In order to study receptor abundance and its function in solutions or in homogenates from clinical specimen, methods such as sandwich or radioimmunoassays are most commonly employed. For the determination of epidermal growth factor receptor (EGFR), we describe the development of a miniaturized bead-based ligand binding assay using its ligand EGF as immobilized capture reagent. This assay was used to analyze lysates from cell lines, and the ligand-bound EGFR was detected using an EGFR-specific antibody combined with a fluorescence-based reporter system. In a proof-of concept study with lysates from breast biopsies, the assay allowed to classify breast cancer samples in accordance to clinically the relevant EGFR cut-off level. The study suggests that such a ligand binding receptor assay could become an integral part of protein profiling procedures to provide additional information about receptor functionality in addition to its abundance.

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KAMINOI, KIMIO. "Assay of protein binding bioactive drug metabolites." Rinsho yakuri/Japanese Journal of Clinical Pharmacology and Therapeutics 19, no. 1 (1988): 353–55. http://dx.doi.org/10.3999/jscpt.19.353.

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ANDERSON, SHONA M. L., and ROBERT J. ELLIOTT. "A dye-binding assay for soluble elastin." Biochemical Society Transactions 19, no. 4 (1991): 388S. http://dx.doi.org/10.1042/bst019388s.

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Nickels, Philipp C., Hans C. Høiberg, Stephanie S. Simmel, Phil Holzmeister, Philip Tinnefeld, and Tim Liedl. "DNA Origami Seesaws as Comparative Binding Assay." ChemBioChem 17, no. 12 (2016): 1093–96. http://dx.doi.org/10.1002/cbic.201600059.

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