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PubMed1994

Adipose tissue storage of drugs as a function of binding competition. In-vitro studies with distribution dialysis.

Distribution dialysis was used to study binding competition between homogenates of adipose tissue and of lean tissues. The concentration ratios adipose/X (X = blood, muscle, lung, liver) of eight lipophilic drugs were determined in the absence and in the presence of a competing binding system X. With drugs which do not undergo storage in adipose tissue in-vivo, yet have a high volume of distribution, such as imipramine or desipramine, there was strong binding competition, and the balance of distribution was shifted from adipose to lean tissues. In the case of indomethacin with a low volume of distribution this shift was from adipose tissue to blood. With diazepam there was a marked binding competition which was not, however, sufficient to shift the balance of distribution away from adipose tissue. Binding competition was negligible with thiopentone. In contrast, with the equally lipophilic hexethal a moderate binding competition was observed. This is consistent with a decreased adipose tissue storage of the latter barbiturate. It is concluded that binding competition exists not only between blood and tissues but also among individual tissues. It is suggested that occurrence and extent of adipose tissue storage of drugs are determined by binding competition between lean and adipose tissues and, more generally, that distribution of lipophilic drugs is largely a function of binding competition.

Adipose Tissue↗
PubMed1983

Graphical analysis of competitive binding of comparable concentrations of ligand, inhibitor and protein. Ligand binding to serum albumin.

In contrast to analysis for competitive binding in enzyme kinetics, no linear plot for analysing competitive binding of two ligands to a protein, where the concentrations of the three reactants are comparable, seems to exist. In the present communication it is shown that in this situation a linear plot can be obtained by the use of the simple equation VA/VB = KA/KB X [Af]/[Bf], where VA and VB are the average number of moles of ligand A and ligand B bound per mole of protein, respectively; [Af] and [Bf] are the concentrations of free ligand A and free ligand B, respectively; and KA and KB are the corresponding association constants. The plot is commented on both theoretically and experimentally using ligand binding to human serum albumin as an example.

Binding Sites↗
PubMed1986

Human IgG and murine monoclonal antibodies share common idiotopes as determined by competitive binding to polystyrene and nitrocellulose-bound antigens.

Competitive binding to polystyrene-bound antigens showed that human rye Group I (rye I)-specific IgG recognize the same three distinct epitopes of rye I as three monoclonal antibodies directed against the same antigens. Anti-idiotypic antibodies against one of the monoclonal antibodies (290A-167) inhibit completely the reaction between rye I and the relevant monoclonal antibodies but do not affect the reaction between other monoclonal antibodies (348A-6 and 539A-6) and the antigens. Furthermore, rabbit anti-idiotypic antibodies produced against F(ab' )2 of human rye I-specific IgG could inhibit the reaction between two monoclonal antibodies (290A-167 and 539A-6) and the relevant antigens. Those results were obtained by competitive binding to polystyrene or to nitrocellulose-bound antigens. These data indicate a cross-reactivity of idiotypic determinants between human rye I-specific IgG and mouse monoclonal antibodies, which implies structural similarity in the V gene coding for the variable region of the antibody.

Animals↗
PubMed1987

Characterization of drug distribution and binding competition by two-chamber and multi-chamber distribution dialysis.

Binding competition between blood and tissue, a determinant of drug distribution, can be simulated and quantitated in vitro by distribution dialysis. In a study with a standardized two-chamber system, six model drugs, selected according to their ratio of plasma to tissue binding, were allowed to distribute between blood and eight tissue homogenates of rats. The tissue:blood concentration ratios were 1 for antipyrine, less than 1 for salicylic acid and phenylbutazone, slightly greater than 1 for pentobarbital and thiopental, and much greater than 1 for imipramine. Comparable values of tissue:blood ratios were obtained in rats in vivo. A modified dialysis system was developed which allows the simultaneous distribution of a drug between blood and four tissue homogenates. This multi-chamber system was used for homogenates of liver, lung, muscle, and adipose tissue. The drugs tested reached a first apparent distribution equilibrium after 2.5 to 4 h, comparable with the distribution in the two-chamber system, as a result of tissue:blood binding competition. In the following hours there was a redistribution as a result of binding competition among individual tissues, except in the case of pentobarbital. A minor redistribution from muscle to adipose tissue was observed with phenylbutazone, and from muscle to liver with imipramine. There was a considerable redistribution from all tissues, including blood, to adipose tissue with thiopental. In a sequential mode of operation this multi-chamber system was used to simulate the influence of the different perfusion rates of individual tissues by adding the homogenates of muscle and/or adipose tissue several hours after the other tissues.(ABSTRACT TRUNCATED AT 250 WORDS)

Binding, Competitive↗
PubMed1997

Kinetics of competitive binding with application to thrombin complexes.

The kinetics of competitive binding is treated analytically, allowing the rate constants to be determined accurately from simple experiments. The method is especially suited to situations where traditional approximations and numerical integration fail, e.g., when the dissociation constants are small or when the concentration of one receptor cannot be measured accurately. The method is applied to the competitive binding of hirudin to thrombin and anhydrothrombin and found to be accurate to a few parts in ten million. The fitted rate constants show that anhydrothrombin binds hirudin more weakly than thrombin, with a 2.6-fold increase in its dissociation constant. The small relative difference in binding free energy (0.6 kcal/mol indicates that anhydrothrombin is structurally similar to thrombin.

Binding, Competitive↗
PubMed2002

A high-throughput, nonisotopic, competitive binding assay for kinases using nonselective inhibitor probes (ED-NSIP).

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 beta-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 beta-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 GSK3alpha, an enzyme previously screened in a radioactive kinase assay (i.e., measurement of [(33)P]-gamma-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 (IC(50)) of 11 nM, which was similar to the IC(50) 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 (R(2) > 0.96). Interference of compounds with the beta-galactosidase signal readout was negligible. In conclusion, the DiscoveRx competitive kinase binding assay, termed ED-NSIP trade mark, 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.

Adenosine Triphosphate↗
PubMed1984

Quantitative study of aluminum binding to human serum albumin and transferrin by a chelex competitive binding assay.

Binding of aluminum to human serum albumin and transferrin was investigated using a competitive binding assay incorporating a cation exchange resin, chelex. Both albumin and transferrin were found to produce linear Scatchard plots of aluminum binding data over the aluminum and protein concentration ranges found in humans. Binding constants measured for albumin and transferrin were 1.96 and 0.515 microM, respectively.

Aluminum↗
PubMed1999

The iso-competition point for counterion competition binding to DNA: calculated multivalent versus monovalent cation binding equivalence.

In this paper we introduce an important parameter called the iso-competition point (ICP), to characterize the competition binding to DNA in a two-cation-species system. By imposing the condition of charge neutralization fraction equivalence theta1 = ZthetaZ upon the two simultaneous equations in Manning's counterion condensation theory, the ICPs can be calculated. Each ICP, which refers to a particular multivalent concentration where the charge fraction on DNA neutralized from monovalent cations equals that from the multivalent cations, corresponds to a specific ionic strength condition. At fixed ionic strength, the total DNA charge neutralization fractions thetaICP are equal, no matter whether the higher valence cation is divalent, trivalent, or tetravalent. The ionic strength effect on ICP can be expressed by a semiquantitative equation as ICPZa/ICPZb = (Ia/Ib)Z, where Ia, Ib refers to the instance of ionic strengths and Z indicates the valence. The ICP can be used to interpret and characterize the ionic strength, valence, and DNA length effects on the counterion competition binding in a two-species system. Data from our previous investigations involving binding of Mg2+, Ca2+, and Co(NH3)63+ to lambda-DNA-HindIII fragments ranging from 2.0 to 23.1 kbp was used to investigate the applicability of ICP to describe counterion binding. It will be shown that the ICP parameter presents a prospective picture of the counterion competition binding to polyelectrolyte DNA under a specific ion environment condition.

Binding, Competitive↗
PubMed1979

Competitive binding radioassay for 5-fluorodeoxyuridine 5'-monophosphate in tissues.

A competitive binding radioassay has been developed for 5-fluorodeoxyuridine 5'-monophosphate, based on the tight binding of this potent inhibitor to thymidylate synthetase (EC 2.1.1.45). Unbound ligand may be separated from that bound to enzyme by precipitating the intact inhibitor-enzyme complex with trichloroacetic acid. Scatchard plot analysis using a two-site model for binding yielded apparent dissociation constants of 1.2 x 10(-11) and 1.7 x 10(-10) M from a least-squares computer fit of the data. 5-Fluorodeoxyuridine 5'-monophosphate could be detected in the range of 0.02 to 2.0 pmol with no apparent interference by other substances. Assay of 5-fluorodeoxyuridine 5'-monophosphate levels in L1210 ascites tumor following 5-fluorouracil in vivo revealed peak levels occurring within the first hr with a subsequent disappearance half-life of 3.9 hr. Close agreement was found between the previously described enzyme inhibition assay and the more rapid and sensitive competitive binding method.

Animals↗
PubMed1997

Mathematical theory of competitive binding assays: an exact and practical model.

In the usual formulation of the equations for competitive binding assays, the free concentrations of the various unlabeled ligands are approximated by the known values of their total concentrations, since the free concentrations are not easily determined. Although equations have been derived previously that give the exact solution with the free concentrations of unlabeled ligands treated as variables, these have not been useful in practice. We have devised a mathematical model for the competitive binding that which is both exact and practical for the general case of one labeled ligand, any number of unlabeled ligands, and any number of classes of binding sites. In this model, the total concentrations of unlabeled ligands are the explicit variables, instead of their free concentrations. The free concentrations of unlabeled ligands can be estimated from the model. The model is based on the law of mass action and the dilution principle, as well as a new concept, called the equivalent competitive binding principle.

Binding Sites↗
PubMed2001

Use of a biomimetic peptide in the design of a competitive binding assay for biotin and biotin analogues.

A competitive binding assay for biotin, biocytin, and desthiobiotin utilizing a genetically engineered enzyme-ligand conjugate is described herein. This assay is unique in that the enzyme-ligand conjugate consists of the streptavidin binding peptide Strep-tag II, which mimics the binding of biotin to streptavidin, rather than biotin itself. This allows for the construction of a well-defined, oligosubstituted enzyme-ligand conjugate for which the site of attachment of the ligand on the enzyme is known precisely. The assay has detection limits of 5 x 10(-8) M for biotin, 1 x 10(-7) M for biocytin, and 2 x 10(-6) M for desthiobiotin, and it serves as a model system in that it demonstrates the feasibility of using enzyme-ligand conjugates in which a peptide mimic of the analyte ligand is genetically fused to the enzyme. This avoids the problems associated with covalent attachment of the ligand to the enzyme, such as multiple substitution of the ligand and variability of the site of attachment. To our knowledge, this is the first example of using an enzyme-peptide mimic conjugate to detect a nonpeptide analyte.

Amino Acid Sequence↗
PubMed1977

An automated competitive binding procedure for measuring thyroxine in serum.

A competitive binding assay for serum thyroxine has been automated, with the use of small, reusable Sephadex columns to separate thyroxine from endogenous thyroid-binding globulin and later to separate the bound and free thyroxine. Sixty samples an hour are run through columns, which are held in an aluminum turntable rotated by a fraction collector motor. Reagents and samples are fed to the columns by a proportioning pump. Waste eluates are collected and drained to the sink by a Teflon tray positioned between the columns and counting tubes, also held by the turntable. A cut out area in the tray allows one to collect the bound fraction, which can then be counted in a scintillation counter. Values are calculated and printed by a desk calculator interfaced with the scintillation counter. The "day-to-day" CV for this method is 5%. The accuracy is satisfactory when tested by comparison with other methods, by recoveries, and by linearity of dilutions.

Autoanalysis↗
PubMed1988

Unusual cross-reactions among monoclonal antibodies to bacterial antigens: idiotypic and competitive binding analysis.

In a previous study, we have described unusual cross-reactions among monoclonal antibodies (Mabs) to bacteria and in particular to the Inaba and Ogawa serotypes of Vibrio cholerae. In this study, the extent to which the binding sites of both antibodies and antigens overlap has been investigated by competitive binding and idiotypic analysis. The competitive binding data indicate that the cross-reactive binding of the Inaba Mabs to the Ogawa vibrios can be abolished by incubation with higher affinity Ogawa Mabs. However, rabbit antiserum raised against the Inaba series does not react with the Ogawa series, indicating that anti-Inaba Mabs do not share idiotypic determinants with anti-Ogawa Mabs. The results therefore suggest that the two sets of antibodies recognise different determinants which are closely related in spatial terms, and which consequently do not permit simultaneous binding of the two types of monoclonal antibody.

Animals↗
PubMed1974

Interaction of gelatin with stereospecific binding proteins and its enhancement of competitive binding assays.

The effect of gelatin (0.5 g/l) on binding curves at high dilution of three classes of stereo-specific binding proteins was studied. These included two antibodies (to oestradiol and aldosterone), six transins (horse and dog transcortins, human thyroxine-binding globulin, human sex steroid-binding globulin, and guinea pig transprogestin), and one receptor (bovine adrenal protein kinase). Gelatin increased the apparent binding of all these proteins, particularly at the highest dilutions and sometimes in a striking manner. While much of this action can be attributed to its decreasing the adhesion of the dilute binding protein to glass, gelatin also increased the apparent uptake of some tracers by certain adsorbents. Similar findings were obtained using human gamma globulin (2 g/l). These effects resulted in increased sensitivity and improved reproducibility in the assays employing them.

Aldosterone↗
PubMed1990

Competitive binding of the troponin T-specific pool of caldesmon antibodies and tropomyosin to skeletal troponin T and smooth muscle caldesmon.

The fraction of polyclonal caldesmon antibodies cross-reacting with rabbit skeletal troponin T are shown to compete with smooth muscle tropomyosin for caldesmon and troponin T, as revealed by ELISA method. The epitope recognized by these antibodies was also found in Mr 77 kDa non-muscle caldesmon. These results provide functional confirmation for the suggestion that the regions of amino acid sequence homology in caldesmon isoforms and troponin T belong to the tropomyosin binding sites.

Animals↗
PubMed1986

Determination of an antibody-antigen binding constant by enzyme immunoassay and a theory for analysis of competitive binding of two ligands to heterogeneous receptor.

A method for determining antigen-antibody binding constants by using enzyme-labeled antigens has been developed. In the measurement, enzyme-labeled and unlabeled antigens (Ag* and Ag) were allowed to compete in binding to the antibody (Ab) under conditions where Ag* much less than Ab much less than Ag. The data were analyzed according to a new theory developed for the analysis of competitive binding of two ligands to a heterogeneous receptor. The theory indicates that the binding degree of a labeled ligand measured at various concentrations of the receptor can be used to prepare a standard curve relating the binding degree of the labeled ligand and the average of the concentrations of the free receptor components which are in binding equilibrium with another unlabeled ligand. For homogeneous receptors, the method gives usual binding constants for the unlabeled ligand, but for heterogeneous receptors, it gives a new type of average binding constant for the unlabeled ligand in which the contribution of each receptor component is amplified in proportion to its affinity against the labeled ligand. This average binding constant was named the "affinity-average binding constant." A rabbit anti-blasticidin S (BLS) antiserum analyzed by the present method using beta-galactosidase-labeled BLS as the labeled ligand was found to be fairly homogeneous with respect to the affinity and to have a binding constant of 1.48 +/- 0.24 (S.D.) X 10(8) M-1 for unlabeled BLS.

Animals↗
PubMed1987

Theoretical characterization of ion channel blockade. Competitive binding to periodically accessible receptors.

Competitive ligand binding to periodically activated or accessible receptors is influenced by the interaction between ligand binding kinetics and the interval of time the binding site is accessible. This interaction produces a paradoxical reduction in bound receptors under certain conditions. A mathematical description of multi-ligand binding to a single binding site is presented for both the continuously and transiently accessible cases. The theoretical results predict paradoxical "agonism" and are consistent with the results of studies of lidocaine and bupivacaine binding to cardiac sodium channels.

Binding, Competitive↗