Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “Binding, Competitive”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 613 records · Page 34Linked to original sources
PubMed1976

Semi-automated competitive protein binding analysis of serum thyroxine on reusable Sephadex columns and its advantages over radioimmunoassay.

Competitive protein-binding analysis of serum thyroxine on small, reusable, Sephadex columns has been further studied and improved. The improved, semi-automated procedure results in reduced working time and costs. It has also been established that triiodothyronine crossreacts only 1/6 to 1/9 as well as thyroxine, and can be ignored because it represents only about 1/80 of the total serum iodothyronine content. The economic and methodological advantages of the improved method over radioammunoassay and other displacement assays are discussed.

Anion Exchange Resins↗
PubMed2006

Competitive displacement binding assay on rat brain sections and using a beta-imager: application to mu-opioid ligands.

A new approach of competitive displacement binding assay using brain sections and a beta-imager is presented to estimate binding parameters such as affinity and selectivity of new compounds or to characterize receptor families or subtypes of receptors in small brain regions. This method includes a preliminary saturation assay intended to define the optimal concentration of displaceable radio-labeled ligand followed by the determination of displacement constants (IC(50) and K(i)) in cerebral regions rich in studied receptor. The technique application was demonstrated in seven rat brain structures, using displacement of the selective tritiated mu-opioid ligand [(3)H]-DAMGO by six opioid ligands: a specific agonist (DAMGO), less specific agonists (morphine, remifentanil), a non-specific antagonist with good affinity for mu receptors (naloxone) and ligands specific of other opioid subtypes (naltrindole, U50.488). Radioactivity counts were collected during 48 h. The assay-validation was performed by measuring intra- and inter-assay variation on determinations and by comparing presently obtained K(i) values with data from recognised methodologies. Both prove the accuracy of the proposed method.

Analgesics, Opioid↗
PubMed1975

[Quality control of testosterone and digoxin radioimmunoassays and cortisol competitive protein binding assay by the reference sample method].

The inclusion of a minimum of 2 reference samples in the radioimmunoassay of testesterone and digoxin, and the competitive protein binding assay of cortisol served as a simple and practical index of overall assay performance. Statistical parameters, the mean plus or minus 2 standard deviation limits as well as the % coefficient of variation were determined at 3 clinically significant values for these samples. While a 50% intercept served mainly to indicate standard curve sensitivity, quality control sera provided a valid assessment of annay precision.

Binding, Competitive↗
PubMed1993

Antagonistic effects of lipopolysaccharide binding protein and bactericidal/permeability-increasing protein on lipopolysaccharide-induced cytokine release by mononuclear phagocytes. Competition for binding to lipopolysaccharide.

Serum proteins play an important role in LPS-induced cell activation. The LPS binding protein (LBP) enhances cellular responses to LPS, whereas the polymorphonuclear leukocyte product bactericidal/permeability-increasing protein (BPI) inhibits LPS-induced cell activation. In this study the influences of LBP and BPI, two proteins with opposite effects, but with considerable sequence homology, on LPS-induced mononuclear phagocytic cell cytokine release was studied. LBP was shown to enhance LPS-induced TNF-alpha, IL-6, and IL-8 release by mononuclear phagocytic cells, whereas BPI inhibited the release of these cytokines. Furthermore, the effects of LBP and BPI on LPS-induced cytokine release by mononuclear phagocytic cells were shown to be counteractive. BPI interfered with the enhancing effect of LBP on the LPS-induced cytokine release. At high LBP to BPI ratios, BPI could no longer inhibit LBP-induced enhancement. In accordance, increasing concentrations of BPI abrogated the LBP effect. Next, it was shown that LBP and BPI compete for binding to LPS by using an assay system that detects binding of free BPI to an anti-BPI mAb. LPS prevented binding of BPI to anti-BPI mAb, whereas preincubation of LPS with LBP prevented the LPS-induced inhibition. Also, it was observed that both BPI and LBP inhibited LPS activity in the chromogenic LAL assay. We conclude from this study that LBP and BPI have counteractive effects on LPS-induced mononuclear phagocytic cell cytokine release by competing for binding to LPS.

Acute-Phase Proteins↗
PubMed2004

Characterization of the non-competitive antagonist binding site of the NMDA receptor in dark Agouti rats.

The ability of non-competitive NMDA antagonists and other selected compounds to inhibit [3H]MK-801 binding to the NMDA receptor in brain membranes was evaluated in female, dark Agouti rats. In homologous competition binding studies the average apparent affinity (KD) of [3H]MK-801 for its binding site was 5.5 nM and the binding site density (Bmax) was 1.83 pmol/mg protein. Inhibition of [3H]MK-801 binding by non-competitive NMDA antagonists was best described with a one-site competition model and the average Hill coefficients were -1. A series of eight non-competitive NMDA antagonists inhibited [3H]MK-801 binding with the following rank order of affinity (K(i), nM): MK-801 (5.5) > dexoxadrol (21.5) > or = TCP (24.2) > phencyclidine (100.8) > (+)-SKF 10,047 (357.7) > dextrorphan (405.2) > ketamine (922.2) > dextromethorphan (2913). These inhibition binding constants determined in dark Agouti rat brain membranes were significantly correlated (P = 0.0002; r2 = 0.95) with previously reported values determined in Sprague-Dawley rats [Wong et al., 1988, J. Neurochem. 50, 274-281]. Despite significant differences in metabolic capability between these strains, the central nervous system NMDA receptor ion channel shares similar characteristics.

Animals↗
PubMed1997

Competitive electrostatic binding of charged ligands to polyelectrolytes: practical approach using the non-linear Poisson-Boltzmann equation.

We have developed a practical analytical treatment of the non-linear Poisson-Boltzmann (P-B) equation to characterize the strong but non-specific binding of charged ligands to DNA and other highly charged macromolecules. These reactions are notable for their strong salt dependence and anti-cooperativity, features which the theory fully explains. We summarize analytical results for concentration profiles and ion binding in various regimes of surface curvature and ionic strength, and show how counterion size and charge distribution may influence competitive binding. We present several practical applications of the formalism, showing how to estimate the ligand concentration needed to effectively compete with a given buffer salt, and how to calculate the amounts of counterion species bound at various distances from the DNA surface under given bulk solution conditions. We cast our results into the form of a Scatchard binding isotherm, showing how the apparent binding constant K(obs) and S = -dlog K (obs )d log[M (+)] can be predicted from the basic theory. Anti-cooperativity arises naturally without steric repulsion, and binding curves can be fitted with K(obs) and effective charge as the only free parameters. We extend the analytical P-B analysis to an arbitrary number of counterion species, and apply the results to fit and predict three-ion competition data.

Journal Article↗
PubMed1980

Trimethoprim interferes with serum methotrexate assay by the competitive protein binding technique.

Administration of Bactrim (a combination of trimethoprim and sulfamethoxazole) to a patient who also was receiving methotrexate caused a significant increase in apparent plasma methotrexate concentrations as determined by competitive protein binding assay with use of dihydrofolate reductase (EC 1.5.1.3) from Lactobacillus casei as the binding protein. This spurious increase was caused by trimethoprim in the patient's plasma. A plasma trimethoprim concentration of 0.1 mg/L inhibited binding of radiolabeled methotrexate to dihydrofolate reductase by 50%. In contrast, radioimmunoassay for methotrexate was not affected by concomitant administration of trimethoprim. The competitive protein binding assay for methotrexate should not be used in patients being treated with Bactrim or Septra (a similar combination). However, the L. casei competitive protein binding assay technique can be used to assay plasma trimethoprim concentrations with sensitivity to 0.02 mg of trimethoprim per liter.

Adult↗
PubMed1983

Syngeneic humoral immune responses to tumor-associated antigens expressed by K-1735 UV-induced melanoma and its metastases.

An enzyme-linked immunoassay (ELISA) was developed to study syngeneic humoral immune response to a primary tumor and its metastases in the K-1735 ultraviolet light (UV)-induced C3H murine melanoma system. Binding of sera from syngeneic animals previously immunized with primary tumor or metastatic tumor tissue (M-3, M-4) to corresponding 3 M KCl extracts of tumor was significantly greater than binding of control C3H mouse serum. Antibody binding was not significantly reduced by competitive binding with syngeneic murine muscle or liver extracts, indicating the presence of tumor antigen(s) not shared by normal murine tissue. Antibodies to the tumor-associated antigens were selectively removed by competitive binding with syngeneic K-1735 tumor extracts but not by the unrelated 102 murine sarcoma from C57BL/6. However, EL-4 extracts (C57BL/6) did inhibit antibody binding to the primary and both metastases. Further competitive binding studies demonstrated the presence of a common antigen(s) present on the primary tumor and both metastases. We conclude that the K-1735 UV-induced melanoma primary tumor and its metastases express serologically detectable shared antigenic determinate.

Animals↗
PubMed1993

Interaction of monoclonal antibodies with growth hormone-binding protein and its complex with growth hormone.

The properties of four independent lines of monoclonal antibodies (MAbs) specific to rat GH-binding protein (GHBP) were examined. Three MAbs, designated GHR-12, GHR-13 and GHR-16, were raised against the entire GHBP molecule. The fourth MAb, designated as GHBP4.3, was raised against the 17 amino acid residues at the C-terminal end of rat GHBP. The interaction of these antibodies with GHBP and their effect on GH binding to GHBP were analysed by conventional competition binding assays and surface plasmon resonance, i.e. with a Biospecific Interaction Analysis (BIAcore) instrument. The binding affinity of these MAbs to GHBP ranged from 29 nmol/l to 30.9 pmol/l. The pair-wise antibody binding to GHBP on BIAcore suggested that GHR-13 and GHR-16 recognized different antigenic determinants while part of the GHR-12 epitope might be shared with the other antibodies. The antibodies inhibited the interaction of GH with GHBP in the competition binding assay. However, in sequential binding on the BIAcore instrument, they were able to bind GHBP after its interaction with GH, indicating that the inhibition observed in the competition binding assay resulted from steric hindrance rather than direct interference with the GH-binding site of GHBP. The present findings, therefore, suggest that these antibodies are useful for investigating GHBP and its interaction with GH.

Animals↗
PubMed1984

Characterization of verapamil binding sites in cardiac membrane vesicles.

Specific, saturable, and reversible binding of verapamil has been demonstrated in crude cardiac sarcolemmal membranes. These receptors possess a Kd of approximately 50 nM for verapamil as determined by either equilibrium binding studies, competition binding analysis, or kinetic analysis of on and off rates and display an average density of 1.25 pmol/mg of protein. Specificity of binding is indicated by several criteria. Competition studies with the verapamil analog D-600 indicate that (-)D-600 is 200-fold more potent than the (+)-isomer in displacing bound verapamil. Likewise, several other aryl alkyl amine Ca2+ entry blockers effectively displace bound ligand. In addition, dihydropyridines and diltiazem promote partial (25-35%) displacement of bound verapamil with Ki values similar to the Kd values for their respective receptors. Characterization of nitrendipine binding in this preparation indicates an average density of 0.3 pmol of receptors/mg of protein suggesting that the verapamil:nitrendipine binding site ratio is approximately 4:1. Binding characteristics of verapamil and nitrendipine receptors in highly purified sarcolemmal vesicles are similar to those in the crude preparation except that the ratio of verapamil:nitrendipine sites approaches 1 and nitrendipine and diltiazem promote almost complete displacement of bound verapamil. Fractionation studies of crude sarcolemmal membranes indicate that excess verapamil receptors, insensitive to the action of dihydropyridines or diltiazem, are located in a high-density, nonmitochondrial, non-sarcolemmal membrane fraction. Thus, verapamil receptors exist in two locations in cardiac tissue but only in the sarcolemmal membrane are these receptors coupled to the dihydropyridine receptor.

Animals↗
PubMed1993

Identification of binding proteins for nuclear localization signals of the glucocorticoid and thyroid hormone receptors.

Nuclear entry of proteins the size of the glucocorticoid and thyroid hormone receptors appears to be mediated by an interaction of nuclear localization signals (NLSs) within the proteins and specific NLS-binding proteins. NLSs have been identified in the hinge region of both receptors. We have identified the cellular binding proteins of the glucocorticoid receptor NLS and the thyroid hormone receptor NLS after cross-linking of radiolabeled signal peptides to subcellular fractions. Two S49 lymphoma cytosolic polypeptides of 60 and 76 kilodaltons (kDa) were specifically bound to either the glucocorticoid or thyroid hormone receptor NLS. The two binding sites demonstrated saturable binding. A competitive binding assay showed that the binding sites were specific for NLSs and that a mutated NLS was a poor competitor for the binding of labeled glucocorticoid receptor NLS. However, competition studies with peptides unrelated to NLSs, yet resembling NLSs in that they had a net positive charge, revealed that the 60-kDa entity demonstrated greater specificity for binding to NLSs than did the 76-kDa polypeptide. Glucocorticoid receptor NLS and thyroid hormone receptor NLS-binding proteins of 60 and 76 kDa were also identified in nuclear fractions. Although the unoccupied glucocorticoid receptor resides in the cytoplasm, while the unoccupied thyroid hormone receptor is always found in the nucleus, the hinge NLS interactions do not specify these different localizations of the unoccupied receptors. Rather, the data support roles for the hinge NLS in general steps of nuclear import and the 60-kDa cross-linked product as a chaperone of both receptors into the nucleus. Its cellular localization also suggests a role for the 76-kDa cross-linked product as a chaperone, but its relatively less stringent binding specificity may indicate that this polypeptide has a different physiological function.

Adrenalectomy↗
PubMed1987

Comparison of agonist-induced changes in beta- and alpha 1-adrenergic receptors of DDT1 MF-2 cells.

Agonist-induced changes in beta- and alpha 1-adrenergic receptors (BARs and AARs) were compared in the DDT1 MF-2 smooth muscle cell line. During equilibrium competition binding assays with intact cells at 37 degrees, agonists induced conversion of both BARs and AARs from a native form with high affinity for agonists to a form with much lower affinity for agonists. The native high affinity form of both receptors could be detected either in short-time competition binding assays at 37 degrees or in equilibrium competition binding assays on ice. Conversion to the low affinity form was nearly complete for BARs, but only about half of the AARs were converted to the low affinity form. For BARs, the high affinity form of the receptor observed in short-time assays with intact cells was similar to that observed in membrane preparations, whereas for AARs this form exhibited much higher affinity than was seen in membrane assays. None of these changes were observed during competition binding assays with antagonists. Both short-time competition binding assays with hydrophilic competing ligands and sucrose density gradient centrifugation assays were consistent with the occurrence of agonist-induced internalization of BARs. These same assays for AARs were consistent with the presence of some AARs in an intracellular compartment in the native state, but no agonist-induced increases in intracellular AARs were detected. During more prolonged exposure (13 hr) to agonists, about 80% down-regulation of BARs occurred, whereas only about 20% down-regulation of AARs was detected. These results may indicate that internalization and down-regulation are not involved in conversion of these receptors to the low affinity form observed in intact cell binding assays.

Adrenergic alpha-Agonists↗
PubMedDate not supplied

A powerful, yet easy to use, computerized analysis of competitive protein binding and radioimmunoassay data.

A computerized method has been developed and tested for the automatic computation of data obtained from competitive radio-binding assays, that is easily used by computer and non-computer oriented individuals. The program requires a series of only eleven parameter lines coded from the assay protocol, followed by the data exactly as it is produced from a standard sample counter. From the set of parameters the program is able to find standard curves at scattered locations through an assay, check their "log dose-logit response" least squares linear regression equations statistically for homogeneity of slopes and elevations, pool the standard curves and check the pooled least squares linear regression equation statistically for linearity and non-linearity. The results of the pooled standard curve is presented graphically and in tabular form. Using the linear equation for the pooled standard curves, the concentration for each unknown sample is predicted with its corresponding 95% confidence interval and presented in a table of unknowns. Also provided in the table of unknowns is a mean and standard error of the mean for all biological replicates, including footnote flags to warn the user when an unknown concentration: (i) is outside the 0-100% bound range; (ii) is estimated from a range outside that covered by the standards; or (iii) was estimated from the unusable tail regions of the standard curve.

Binding, Competitive↗
PubMed1985

Amobarbital interactions with 25-hydroxycholecalciferol: effects on the extraction, quantification, and competitive protein binding in vitro.

Amobarbital has been found to coelute with 25-hydroxycholecalciferol (25OHD3) on a normal phase high performance liquid chromatographic system and cause subsequent interference in the UV detection and plasma transport competitive protein binding assay for this vitamin D metabolite. Concentrations of 25OHD3 were overestimated by 95% in the presence of 0.4 mg amobarbital in the competitive protein binding assay; as little as 0.1 mg amobarbital caused a 22% overestimation in the concentration of 25OHD3 in the assay. Separation of 25OHD3 from amobarbital on a reverse phase high performance liquid chromatographic system allowed for proper quantification without interference. Because of the similarity of chemical structures, other barbital-based compounds may cause similar interactions with 25OHD3 or other vitamin D metabolites as well.

Amobarbital↗
PubMedDate not supplied

Discrepant cortisol values: comparison of radioimmunoassay and competitive protein binding technics.

Radioimmunoassays are expected to yield values representing the antigen, i.e., insulin, growth, cortisol, etc. employed in producing the antibody. However, in a recent study of a synthetic ACTH which involved the measurement of cortisol levels (1), we found that above 20gamma%, the radioimmunoassay (RIA) yields cortisol values distinctly above those obtained with the competitive protein binding (CPB) technic. We now suggest a possible explanation for this discrepancy and have calculated a correction factor.

Binding, Competitive↗
PubMed2002

Demonstration of competition between endogenous dopamine and [11C]raclopride binding in in vitro brain slices using a dynamic autoradiography technique.

To elucidate the mechanism of in vivo binding competition between radioligand and endogenously released transmitter, we examined the influence of depolarization-induced dopamine (DA) release on [11C]raclopride-specific binding to D2 receptors in slices of living brain tissues using dynamic positron autoradiography. Rat brain slices were incubated in a chamber with [11C]raclopride in oxygenated medium at 34 degrees C for 150 min. Two-dimensional images of radioactivity in the slices were recorded on a storage phosphor screen and dynamic changes were measured. When the brain slices were exposed to the depolarization agents (25 mM K+, 50 mM K+, and 20 microM veratridine), the percentage inhibition of striatal [11C]raclopride-specific binding was 22 +/- 4%, 44 +/- 8% and 54 +/- 7% of the control, respectively. The percentage inhibition of [11C]raclopride-specific binding during each depolarization treatment agreed proportionally with the amount of DA released into the medium. However, preexposure of brain slices to the same depolarization treatment (50 mM K+) did not affect the [11C]raclopride-specific binding, suggesting that the reduction in receptor density and/or affinity was not involved in the decrease of [11C]raclopride-specific binding. [11C]Raclopride-specific binding decreased dose-dependently in the presence of exogenously added DA (range 0.005-3mM). The synaptic DA concentration during each depolarization treatment estimated using three different methods. These results suggest that the decrease of [11C]raclopride-specific binding to striatal slices following evoked DA release is due to competition between endogenous DA and raclopride. It is unlikely that changes in D(2) receptor density or in affinity of the receptors for raclopride are involved. These results provide supportive evidence for in vivo binding competition between radioligand and endogenous neurotransmitter.

Animals↗