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PubMed1995

Competitive binding assay of src homology domain 3 interactions between 5-lipoxygenase and growth factor receptor binding protein 2.

c-src homology 3 domains (SH3) modulate the formation of a number of protein complexes that are important in cell signaling and cytoskeletal organization. The SH3 domain is recognized by short conserved proline-rich motifs which adopt left-handed polyproline helices on binding. In order to examine molecular determinants of the proline motif:SH3 interaction, an enzyme-linked immunosorbent assay was developed to observe binding of 5-lipoxygenase to SH3 domains of growth factor receptor binding protein 2 (Grb2). The assay makes use of glutathione S-transferase fusion proteins of Grb2 and fragments of Grb2 immobilized onto wells of standard 96-well microtiter plates. Equilibrium binding is monitored colorimetrically and the measured absorbance is proportional to 5-LO concentration. The interactions is specific for the Grb2 portion of the fusion proteins, and 5-LO binds preferentially to Grb2 fragments containing an SH3 domain. Competitive binding assays with a synthetic peptide which mimicked the proline-rich region of 5-LO yielded results that are consistent with previous estimates. Binding was examined in the presence of a number of peptides containing the consensus sequence -PXXP-, in the presence of enzyme activity mediators and in the presence of plant lipoxygenases that lack the proline-rich binding motif. Results suggest that the specificity of the Grb2:5-LO interaction is high.

Adaptor Proteins, Signal Transducing↗
PubMed1978

Microcalorimetric method to determine competitive binding. Action of a psychotropic drug (dipotassium chlorazepate) on L-tryptophan . human serum albumin complex.

A mathematical treatment and an original microcalorimetric method are developed to verify an eventual competitive binding between any two substances for the same macromolecule. To apply this method, a competitive binding of L-tryptophan and one benzodiazepin (dipotassium chlorazepate) for human serum albumin is perfectly demonstrated. The association constants and the enthalpy variations are equal to 14 000 +/- 2000 M-1 and --6.6 +/- 0.2 kcal/mol for human serum albumin . tryptophan complex and 13 000 +/- 1000 M-1 and --10.0 +/- 0.2 kcal/mol for human serum albumin . chlorazepate complex. In all cases the stoichiometry is equal to one. The binding of tryptophan to human serum albumin is partially stereospecific; the association constant and the enthalpy variation for D-tryptophan complex are equal, respectively, to 1000 +/- 200 M-1 and --2.6 +/- 0.3 kcal/mol.

Anti-Anxiety Agents↗
PubMed1999

Development of a theoretical model for chromatographic-based competitive binding immunoassays with simultaneous injection of sample and label.

This study examined the theory and behavior of an HPLC-based chromatographic competitive binding immunoassay with the simultaneous injection of sample and a labeled analyte analogue. Equations based on nonlinear chromatographic theory were derived to describe the calibration curve for this assay in a system with adsorption-limited kinetics and homogeneous binding sites. These equations related the assay response (B/Bo) to the column's binding capacity, the moles of injected analyte or labeled analogue, and the flow rate/adsorption kinetics of the system. There was good agreement between the predicted theoretical response and experimental data obtained for the binding of human serum albumin (HSA) to an immobilized anti-HSA antibody column. This theory was also successful in describing the changes that occurred in the calibration curve when the flow rate or amount of labeled analogue applied to the column was varied. A comparison was made between the results of this study and previous theoretical work that examined the behavior of a related, sequential injection competitive binding method. On the basis of the results reported in this work, several general guidelines were developed for the design and optimization of simultaneous injection methods for use in such areas as clinical testing, pharmaceutical analysis, and environmental monitoring.

Adsorption↗
PubMed1996

Characterization of competitive binding, fluorescent drug immunoassays based on micellar electrokinetic capillary chromatography.

This paper characterizes competitive binding, electrokinetic capillary-based immunoassays for various drugs in human serum using reagents which were commercialized for fluorescence polarization immunoassays. After incubation of serum with the reactants, a small aliquot of the mixture is applied onto a fused-silica capillary and tracers (fluorescein-labeled drugs) and the antibody-tracer complexes are separated and analyzed by micellar electrokinetic capillary chromatography with on-column laser-induced fluorescence detection. Examples studied include serum assays for theophylline, ethosuximide, paracetamol, salicylate and quinidine. With these assays, concentration-dependent peaks produced by the free tracers or the antibody-tracer complexes serve as the basis for quantitation. The sizes of the peaks produced are shown to be dependent on the applied power and the proportions of the reactants and serum employed. The separation medium permits effective characterization of tracers and antibody selectivities. Based on the high selectivity of the antibodies employed, the feasibility of the simultaneous performance of different immunoassays is demonstrated. For capillaries of 50 microns internal diameter (ID), separations are best performed at electric fields < 500 V/cm, this resulting in electrokinetic analyses within 4 to 10 min (capillaries of 20 to 50 cm effective length).

Binding, Competitive↗
PubMed1978

Parallel competitive binding and metabolic endpoint assays for hCG based on rabbit granulosa cells.

A competitive binding assay for hCG is described, in which live rabbit granulosa cells adhering to small tubes provide solid-phase binding sites. Parallel assays, in which the synthesis of progestagens and the incorporation of [3H]acetate into lipid serve as endpoints, are also described. The assays are sensitive, reproducible and precise and should be both economical and convenient for studies on the relationship between the molecular structure and the biological activity of hCG and LH.

Acetates↗
PubMed2004

Analysis of competitive binding of ligands to human serum albumin using NMR relaxation measurements.

The competitive binding of two ligands, ibuprofen (IBP) and salicylic acid (SAL), to human serum albumin (HSA) was studied by using nuclear magnetic resonance (NMR) relaxation measurements. When the concentration of one ligand was increased in the solution containing IBP, SAL and HSA, the fractions of free IBP and SAL were increased because of the competitive binding. The 1H relaxation rates (R1) of both ligands were subsequently decreased. If a ligand is in fast exchanging between the free and bound forms, the observed 1H relaxation rate is a weighted average of that for the free ligand and the protein-ligand complex. The concentrations of the free and bound ligands can be quantitatively derived from the relaxation rates. The results presented in this work revealed that IBP and SAL shared certain low-affinity binding sites on the HSA molecule, in addition to the same high-affinity binding site of AIII.

Humans↗
PubMed2005

Microcapsule biosensors using competitive binding resonance energy transfer assays based on apoenzymes.

This paper reports the first demonstration of a fluorescence resonance energy transfer based glucose sensor, wherein a competitive binding (CB) assay is encapsulated into polyelectrolyte microcapsules. The work supports the concept that microcapsules are superior to hydrogel systems or other matrixes for competitive-binding-based system, as they provide free movement of the sensing elements within the capsule interior while constant total sensing assay concentration is maintained. The transduction approach employed in these preliminary experiments is also a novel CB system based on a model apoenzyme, apo-glucose oxidase (AG), which is highly specific to beta-d-glucose, as the model target-binding protein. The glucose sensitivity of the fluorescein isothiocyanate (FITC)-dextran and tetramethylrhodamine isothiocyanate-AG encapsulated in microcapsules showed 5 times greater specificity for beta-D-glucose over other sugars, with sensitivity (change in intensity ratio) in the range of 2-6%/mM. It was observed that the sensitivity and range of the response can be tailored by controlling the assay concentration using different FITC-dextran molecular weight and total capsule concentration. The findings support the concepts of using microcapsules to encapsulate CB assays for reversible and stable sensors and the use of apoenzymes as specific molecular recognition elements in CB assays. Further, characterization results for microcapsule glucose sensors demonstrate their suitability for monitoring physiological glucose levels.

Apoenzymes↗
PubMed2003

Equilibrium competition binding assay: inhibition mechanism from a single dose response.

Inhibition of a receptor by a small-molecule compound in many cases is achieved via a competitive, uncompetitive or non-competitive mechanism. The receptor-inhibitor interaction is often probed through the displacement of a ligand in an equilibrium competition binding experiment. The previous solutions to receptor inhibition mechanisms were borrowed from steady-state enzyme inhibition mechanisms. The inhibition mechanism is determined by a visual inspection or a global fit of ligand dose response curves at a series of inhibitor concentrations. However these solutions only apply to situations when both the ligand and the inhibitor are not significantly depleted by the receptor. In most published equilibrium receptor binding studies, only the relative potency of the inhibitor is calculated. Ranking inhibitors tested under differing experimental conditions is often not possible. In the current paper, we offer exact mathematical solutions to uncompetitive and non-competitive inhibition, and demonstrate that in most cases both the inhibition mechanism and absolute potency of an inhibitor can be simultaneously determined from a single dose response of the inhibitor at a fixed concentration of the ligand. Therefore, an equilibrium competition assay provides a quick and facile method to determine the inhibition mechanism of a large number of inhibitors. The theory herein described is applicable to equilibrium competition binding experiments such as radioligand assays and fluorescence polarization assays.

Animals↗
PubMed1991

Sensitive competitive-binding ELISAs for quantifying free kappa and lambda light chains in cerebrospinal fluid.

Simple, sensitive, and fully standardized solid-phase enzyme-linked competitive binding immunoassays to quantify free kappa and lambda immunoglobulin light chains are described. The assays were developed to measure the concentration of free light chains in cerebrospinal fluid (CSF), in part because elevated levels of free kappa light chains have utility as a diagnostic marker for multiple sclerosis (MS). Polyclonal rabbit antibodies raised against pooled Bence Jones proteins are bound to solid-phase Staphylococcal protein A and used as the primary antisera in this assay. A pool of Bence Jones proteins isolated from the urine of 10 individuals with multiple myeloma are used as a biotin-labeled ligand and to develop a standard curve. The assays as described are sensitive to the low nanogram range and are specific for free kappa or lambda light chains. The assays were found to have acceptable precision, and results correlated highly with concentrations determined using competitive-binding radioimmunoassays previously described.

Binding, Competitive↗
PubMed1990

Flow cytometric competitive binding assay for determination of actinomycin-D concentrations.

A single step, separation free competitive binding reaction between the fluorescent antibiotic mithramycin and actinomycin-D for common binding sites on DNA coated 10 microns diameter microspheres is described. The fluorescence of the microspheres is measured with a flowcytometer. In the presence of a constant amount of mithramycin, the microsphere fluorescence is inversely proportional to actinomycin-D concentration.

Binding, Competitive↗
PubMedDate not supplied

Competitive binding assay for quantitative determination of GM1 ganglioside in plasma and cerebrospinal fluid.

A competitive binding assay for the quantitative determination of GM1 ganglioside is described. After extraction from biological fluids, GM1 was incubated with a known amount of cholera toxin B-subunit conjugated with horseradish peroxidase, and exposed to GM1 adsorbed onto polystyrene microwells. Since GM1 in solution blocks the binding of toxin B-subunit to GM1 adsorbed onto the solid phase, enzyme activity serves as a reciprocal measure of GM1 concentration in the sample. The assay was used to determine the basal level of GM1 in plasma and cerebrospinal fluid in different populations.

Adult↗
PubMed1987

Ligand dissociation constants from competition binding assays: errors associated with ligand depletion.

The dissociation constant of a ligand at a binding site (e.g., receptor, antibody) is often determined indirectly by competitive displacement of a radioligand. It is well known that such a determination may be seriously in error unless the free concentrations of both the radioligand and the unlabeled ligand can be measured. By means of computer simulations we have explored the conditions under which this error may occur and its magnitude. We offer guidelines for recognizing a probably inaccurate dissociation constant, and we show how, in some cases, a correction can be made. The problem addressed here is not only a theoretical one; it can arise in the ordinary performance of competition binding assays.

Binding, Competitive↗
PubMedDate not supplied

Competition binding studies with biotinylated echovirus 11 in cytofluorimetry analysis.

Competition binding studies between viruses are usually performed with radiolabelled probes. In this report, a cytofluorimetric method using biotinylated echovirus (EV) 11 is described for the study of competition of enteroviruses for a common cell receptor site. An N-hydroxysuccinimide ester biotin spacer arm was used for biotinylation of CsSO4-purified EV 11. Biotinylation did not change the infectivity of the virus (attachment to and replication in susceptible cells). With the exception of EV 22 and EV 23, all the echovirus serotypes and also coxsackievirus A9 (CA 9) were able to inhibit the absorption of biotinylated EV 11 onto cells. The taxonomic implications of these findings are discussed.

Binding, Competitive↗
PubMed1999

Competitive binding of Mg2+, Ca2+, Na+, and K+ ions to DNA in oriented DNA fibers: experimental and Monte Carlo simulation results.

Competitive binding of the most common cations of the cytoplasm (K(+), Na(+), Ca(2+), and Mg(2+)) with DNA was studied by equilibrating oriented DNA fibers with ethanol/water solutions (65 and 52% v/v EtOH) containing different combinations and concentrations of the counterions. The affinity of DNA for the cations decreases in the order Ca > Mg >> Na approximately K. The degree of Ca(2+) and/or Mg(2+) binding to DNA displays maximum changes just at physiological concentrations of salts (60-200 mM) and does not depend significantly on the ethanol concentration or on the kind of univalent cation (Na(+) or K(+)). Ca(2+) is more tightly bound to DNA and is replaced by the monovalent cations to a lesser extent than is Mg(2+). Similarly, Ca(2+) is a better competitor for binding to DNA than Mg(2+): the ion exchange equilibrium constant for a 1:1 mixture of Ca(2+) and Mg(2+) ions, K(c)(Ca)(Mg), changes from K(c)(Ca)(Mg) approximately 2 in 65% EtOH (in 3-30 mM NaCl and/or KCl) to K(c)(Ca)(Mg) approximately 1.2-1.4 in 52% EtOH (in 300 mM NaCl and/or KCl). DNA does not exhibit selectivity for Na(+) or K(+) in ethanol/water solutions either in the absence or in the presence of Ca(2+) and/or Mg(2+). The ion exchange experimental data are compared with results of grand canonical Monte Carlo (GCMC) simulations of systems of parallel and hexagonally ordered, uniformly and discretely charged polyions with the density and spatial distribution of the charged groups modeling B DNA. A quantitative agreement with experimental data on divalent-monovalent competition has been obtained for discretely charged models of the DNA polyion (for the uniformly charged cylinder model, coincidence with experiment is qualitative). The GCMC method gives also a qualitative description of experimental results for DNA binding competitions of counterions of the same charge (Ca(2+) with Mg(2+) or K(+) with Na(+)).

Animals↗
PubMedDate not supplied

Chromatographic competitive binding immunoassays: a comparison of the sequential and simultaneous injection methods.

Two approaches for performing competitive binding immunoassays by HPLC and other flow-based systems are the simultaneous and sequential injection methods. Both these techniques make use of a column with a limited amount of antibody, onto which is injected a sample and a fixed amount of a labeled analyte analog. An indirect measure of the unlabeled analyte in the sample is then obtained by looking at the amount of analog in either the nonretained or retained peaks. In the simultaneous injection mode, the sample and labeled analog are applied at the same time to the column, while in the sequential mode the sample is injected first, followed by the analog. This results in a difference in the analytical characteristics of these two approaches. This study used chromatographic theory and previous data obtained for injections of human serum albumin (HSA) onto an anti-HSA antibody column to compare the response, detection limits, range, and sensitivity of these methods. Under equivalent conditions, it was found that the sequential method always provided the best lower limit of detection and sensitivity. However, the simultaneous mode had a broader dynamic range and higher upper limit of detection. From these observations, several guidelines were developed regarding the use and selection of such assays for new applications.

Binding, Competitive↗
PubMed1990

A new competitive binding enzyme-linked immunosorbent assay for glycocalicin in plasma and platelet concentrate supernatants.

A new competitive binding enzyme-linked immunosorbent assay (CB ELISA) for glycocalicin (GC) was developed using GC coated wells and a monoclonal antibody (MAb) to glycoprotein Ib (AN51). The principal stages of the CB ELISA consisted of coating the plate with GC extract overnight, blocking with 3% BSA, incubating the wells with test or standard sample dilution and AN51, and a final incubation with horseradish peroxidase-conjugated goat anti-mouse IgG. Serial dilutions of purified GC, starting in 2% BSA, yielded standard curves which were linear between 10 and 0.4 micrograms/ml. Parallel curves were obtained for platelet concentrate supernatants and for citrated plasma. We used the ELISA to measure GC levels in platelet concentrates during storage. The results indicated that soluble GC increased progressively during storage from 3.3 to 6.7 micrograms/ml, while GC levels in platelet-poor plasma remained at 1.9-2.2 micrograms/ml. These results show that the new CB ELISA is a simple and short assay for the direct measurement of GC in plasma solutions, and may be of use in clinical studies.

Binding, Competitive↗
PubMed1983

The antigenic structure of HLA-A2: an analysis with competitive binding assays and monoclonal antibodies.

Twelve mouse monoclonal antibodies against HLA-A2 were analyzed by competitive binding assays on cells. The A2 molecule was found to have two topologically distinct regions of highly specific alloantigenic determinants. One region encompasses determinants shared with HLA-A28 and the other contains determinants shared with HLA-B17 and a variant, A28*, of HLA-A28. Each of these two alloantigenic regions is more closely associated with a different broadly polymorphic determinant shared by many HLA gene products. The topographic relationship between the two alloantigenic regions, four monomorphic determinants, and the carbohydrate moiety of the HLA heavy chain was also assessed. All the antigenic determinants preferentially associate with one or other of the two alloantigenic regions. An analysis of these results with reference to the amino acid sequences of A2 and A28 suggests a working hypothesis whereby the A2,A28*,B17 region primarily involves residues of the NH2-terminal or first domain, and that the A2,A28 region primarily involves residues of the second domain of the A2 heavy chain.

Amino Acid Sequence↗
PubMed1978

General treatment of competitive binding of small molecules to macromolecules as applied to dynamic dialysis: theoretical analysis.

A mathematical analysis of the dynamic dialysis process is presented, demonstrating how the process can be applied generally to study competitive and noncompetitive binding between small molecules and macromolecules. A law of mass action model for competitive binding with independent sites and classes with equivalent sites (CIE) is considered as a specific case without loss of generality. The escape profiles of two compounds are calculated to illustrate the effect of an increasing degree of binding competition. Noisy data are generated using the CIE model to test the presented method of estimating competitive binding parameters. The parameters estimated by the nonlinear regression technique came close to the true values, considering the degree of noise added to the exact dialysis data. A transformation approach is presented, enabling initial estimates of the binding parameters in the CIE model to be determined by multiple linear regression, thereby eliminating the main problem in the nonlinear estimation. The presented method of analysis is extended to strongly bound compounds, which also bind significantly to the dialysis membrane.

Binding, Competitive↗