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PubMed2000

Competitive binding of bismuth to transferrin and albumin in aqueous solution and in blood plasma.

Several bismuth compounds are currently used as antiulcer drugs, but their mechanism of action is not well established. Proteins are thought to be target sites. In this work we establish that the competitive binding of Bi(3+) to the blood serum proteins albumin and transferrin, as isolated proteins and in blood plasma, can be monitored via observation of (1)H and (13)C NMR resonances of isotopically labeled [epsilon-(13)C]Met transferrin. We show that Met(132) in the I132M recombinant N-lobe transferrin mutant is a sensitive indicator of N-lobe metal binding. Bi(3+) binds to the specific Fe(3+) sites of transferrin and the observed shifts of Met resonances suggest that Bi(3+) induces similar conformational changes in the N-lobe of transferrin in aqueous solution and plasma. Bi(3+) binding to albumin is nonspecific and Cys(34) is not a major binding site, which is surprising because Bi(3+) has a high affinity for thiolate sulfur. This illustrates that the potential target sites for metals (in this case Bi(3+)) in proteins depend not only on their presence but also on their accessibility. Bi(3+) binds to transferrin in preference to albumin both in aqueous solution and in blood plasma.

Albumins↗
PubMed2006

Determination of enantiomeric excess and concentration of unprotected amino acids, amines, amino alcohols, and carboxylic acids by competitive binding assays with a chiral scandium complex.

A practical UV-vis sensing method for enantioselective microanalysis of unprotected amino acids, amines, amino alcohols, and carboxylic acids in aqueous solution has been developed. Both concentration and enantiomeric composition of a wide range of chiral substrates can be determined with high accuracy by two simple competitive binding assays using a scandium complex derived from 1,8-bis(3-(3',5'-dimethylphenyl)-9-acridyl)naphthalene N,N'-dioxide.

Amines↗
PubMed1994

General treatment of competitive binding as applied to the potentiometric ion probe technique: application to the interaction of nonsteroidal anti-inflammatory drugs with bovine serum albumin.

The binding of naproxen, ketoprofen, phenylbutazone, salicylic acid, azapropazone, and indobufen to bovine serum albumin was studied by applying the potentiometric ion probe technique. An ion-selective electrode for the ion probe 1-anilino-8-naphthalene-sulfonate was utilized for the purposes of this study. A modified site-oriented competitive binding model was used for the estimation of the drugs' binding parameters, considering different number of binding sites on the competing binding class(es) for the probe and the drug. Calculations were based exclusively on the concentration data of the free probe. The model's ability for accurate estimations of binding parameters was evaluated by simulation studies. The following values of binding parameters were found at 25 degrees C for the drugs under study; naproxen, n1 = 9.1, k1 = 9.4 x 10(5) M-1; ketoprofen, n1 = 8.8, k1 = 10.8 x 10(5) M-1; phenylbutazone, n1 = 3.2, k1 = 1.4 x 10(5) M-1; salicylic acid, n1 = 2.6, k1 = 1.8 x 10(5) M-1, n2 = 21.5, k2 = 1.0 x 10(4) M-1; azapropazone, n1 = 0.5, k1 = 7.8 x 10(5) M-1, n2 = 26.3, k2 = 1.9 x 10(4) M-1; indobufen, n1 = 5.8, k1 = 5.8 x 10(5) M-1, n2 = 19.9, k2 = 3.8 x 10(5) M-1, where ni the number of binding sites of the i class and ki the corresponding association constant.

Anilino Naphthalenesulfonates↗
PubMed1986

A sensitive routine assay for urinary albumin based on the competitive binding to anti-albumin antibodies in solid phase.

The detection of microalbuminuria has a high prognostic value in diabetic patients with no symptoms of clinical nephropathy. Attention has been focused on the development of a simple, reproducible, specific, and, above all, sensitive method to detect albuminuria in the urine. The method is based on the competitive binding between albumin in the urine to be tested and a fixed amount of radiolabelled albumin to anti-albumin immunoglobulins in solid phase. The immunoglobulin C fraction of a rabbit anti-human albumin antiserum was left to coat highly adsorbent polystyrene microtitre tubes. While the aspecific tube binding was overcome by saturation with a solution of gelatine, increasing dilutions of standard albumin or the diluted urine samples to be tested were left to incubate with an equal volume of radiolabelled albumin at room temperature for 1 hour. The cold/hot albumin mixture was added to anti-albumin immunoglobulin-coated tubes, which had been repeatedly washed, and left to incubate. After washing, radioactivity was assessed. This assay has proved to be fast, simple and highly sensitive since it detects up to 25 ng of albumin per ml and is of value in large-scale screening for microalbuminuria in diabetic patients.

Albumins↗
PubMed2003

Competitive binding inhibition enzyme-linked immunosorbent assay that uses the secreted aspartyl proteinase of Candida albicans as an antigenic marker for diagnosis of disseminated candidiasis.

The secreted aspartyl proteinases (Saps) of Candida albicans have been implicated as virulence factors associated with adherence and tissue invasion. The potential use of proteinases as markers of invasive candidiasis led us to develop a competitive binding inhibition enzyme-linked immunosorbent assay (ELISA) to detect Sap in clinical specimens. Daily serum and urine specimens were collected from rabbits that had been immunosuppressed with cyclophosphamide and cortisone acetate and infected intravenously with 10(7) C. albicans blastoconidia. Disseminated infection was confirmed by organ culture and histopathology. Although ELISA inhibition was observed when serum specimens from these rabbits were used, more significant inhibition, which correlated with disease progression, occurred when urine specimens were used. Urine collected as early as 1 day after infection resulted in significant ELISA inhibition (mean inhibition +/- standard error [SE] compared with preinfection control urine, 15.7% +/- 2.7% [P < 0.01]), and inhibition increased on days 2 through 5 (29.4% +/- 4.8% to 44.5% +/- 3.5% [P < 0.001]). Urine specimens from immunosuppressed rabbits infected intravenously with Candida tropicalis, Candida parapsilosis, Candida krusei, Cryptococcus neoformans, Aspergillus fumigatus, or Staphylococcus aureus were negative in the assay despite culture-proven dissemination. Nonimmunosuppressed rabbits receiving oral tetracycline and gentamicin treatment were given 2 x 10(8) C. albicans blastoconidia orally or intraurethrally to establish colonization of the gastrointestinal tract or bladder, respectively, without systemic dissemination; urine specimens from these rabbits also gave negative ELISA results. Dissemination to the kidney and spleen occurred in one rabbit challenged by intragastric inoculation, and urine from this rabbit demonstrated significant inhibition in the ELISA (mean inhibition +/- SE by day 3 after infection, 32.9% +/- 2.7% [P < 0.001]). The overall test sensitivity was 83%, the specificity was 92%, the positive predictive value was 84%, the negative predictive value was 91%, and the efficiency was 89% (166 urine samples from 33 rabbits tested). The specificity, positive predictive value, and efficiency could be increased to 97, 95, and 92%, respectively, if at least two positive test results were required for a true positive designation. The ELISA was sensitive and specific for the detection of Sap in urine specimens from rabbits with disseminated C. albicans infection, discriminated between colonization and invasive disease, reflected disease progression and severity, and has the potential to be a noninvasive means to diagnose disseminated candidiasis.

Animals↗
PubMed1996

[Competitive binding of tetrabutylammonium and cetylpyridinium cations on C18 surfaces].

The adsorption behavior of tetrabutilammonium (TBA) and cetylpyridinium (CP) cations was studied on Hypersil 5-ODS sorbent. After establishing, that CP has about 5 fold stronger affinity to the C18 surface, than the structurally different, symmetric, spheric molecule of TBA, the adsorption from their common solution was also examined. The data of "competitive binding" seem to show, that TBA should have certain selective binding sites on the C18 surface, from which, it can not be displaced by the CP even in the case the latter is present in a 4 fold excess in the mobile phase. The maximal coverage was found 52.9% (CP) and 12% (TBA) i.e. every second of C18 groups is covered by CP cations and only 1/8th of C18 groups may interact with TBA. The accessible, proton releasing silanol content, related to the total C18 content of the column, was found very low.

Adsorption↗
PubMed2004

Cross-flow micellar-enhanced ultrafiltration for removal of nitrate and chromate: competitive binding.

Feasibility of cross-flow micellar-enhanced ultrafiltration was investigated to remove chromate and nitrate from water system using a cationic surfactant, cetylpyridinium chloride (CPC). Removal of chromate with -2 as valence was higher than that of nitrate with -1 as valence in the conditions of co-existence of chromate and nitrate. Removal of nitrate was significantly inhibited by co-existence of chromate because of competitive binding to CPC micelles between chromate and nitrate, while that of chromate was only slightly inhibited by nitrate.

Binding, Competitive↗
PubMed1990

Competitive binding affinity of carcinogenic aromatic amines to the rat hepatic aromatic hydrocarbon (Ah) receptor in vitro and potency to induce monooxygenase activity in vivo.

The aromatic amides 2-acetylaminofluorene (2-AAF), its isomer 4-acetylaminofluorene, 2-acetylaminophenanthrene (AAP), trans-4-acetylaminostilbene (AAS) and the corresponding amines differ in their carcinogenic effects in the rat. Only 2-AAF and 2-aminofluorene (2-AF) are tumor promoters in rat liver. We have determined the competitive binding affinities of these compounds to the rat hepatic cytosolic aromatic hydrocarbon (Ah) receptor, which has been associated with tumor promotion. Binding affinity was determined by displacement of labelled 2,3,7,8-tetrachloro-dibenzo-p-dioxin (TCDD) or 3-methylcholanthrene. The rank order of affinities and the apparent inhibitor constants measured with the sucrose density gradient centrifugation technique were: AAF (2.3 microM) greater than AAP (2.7 microM) greater than 2-AF (7.0 microM) greater than trans-4-aminostilbene (7.7 microM) greater than 2-aminophen-anthrene (10.4 microM). 4-Acetylaminofluorene, 4-aminofluorene and AAS did not displace TCDD from the receptor protein. A number of other fluorene derivatives did not bind, except 2-nitrofluorene (apparent inhibitor constant (1.4 microM). The in vivo biological potency of the aromatic amines was monitored by measuring the induction of ethoxy-resorufin-O-deethylase (EROD) and aromatic hydrocarbon hydroxylase activities. In vitro binding to the Ah receptor correlates roughly with the induction of EROD activity. The effect of 2-AAF was dose dependent, supporting a receptor-mediated mechanism. The correlation between Ah receptor affinity and tumor-promoting properties of the tested aromatic amines is only limited, but the results are not at variance with the concept of a receptor-mediated process in 2-AAF tumor promotion.

2-Acetylaminofluorene↗
PubMed1991

Scintillation proximity assay: competitive binding studies with [125I]endothelin-1 in human placenta and porcine lung.

Scintillation proximity assay (SPA) technology has been used to investigate the competitive binding of [125I]endothelin-1 (ET-1) by ET-1, endothelin-2 (ET-2), endothelin-3 (ET-3), sarafotoxin 6b (SFTX-b), and big endothelin (big ET) to endothelin receptors in human placenta and porcine lung. Specific binding of [125I]ET-1 to high-affinity receptors was detected in membranes coupled to wheat germ agglutinin (WGA)-coated beads impregnated with scintillant (fluomicrospheres). The binding characteristics of ET-1 were similar in both of the tissues studied. In contrast, the receptor-binding properties of ET-2, ET-3, and SFTX-b were different in human placental and porcine lung membranes. This suggests that different endothelin receptor populations exist in these tissues. In addition, the binding characteristics of ET-3 and SFTX-b differed markedly to those of ET-1 and ET-2 in both tissues. This may be due to the four amino acid residue substitution in the N-terminal loop of both ET-3 and SFTX-b. We have also demonstrated that the ET-1 precursor, big ET, competitively inhibits [125I]ET-1 binding in these tissues, but is 100-fold less potent than ET-1.

Animals↗
PubMed2002

High-throughput NMR-based screening with competition binding experiments.

The Achilles heel of ligand-based NMR screening methods is their failure to detect high-affinity ligands and molecules that bind covalently to the receptor. We have developed a novel approach for performing high-throughput screening with NMR spectroscopy that overcomes this limitation. The method also permits detection of potential high-affinity molecules that are only marginally soluble, thus significantly enlarging the diversity of compounds amenable to NMR screening. The techniques developed utilize transverse and/or selective longitudinal relaxation parameters in combination with competition binding experiments. Mathematical expressions are derived for proper setup of the NMR experiments and for extracting an approximate value of the binding constant for the identified ligand from a single-point measurement. With this approach it is possible to screen thousands of compounds in a short period of time against protein or DNA and RNA fragments. The methodology can also be applied for screening plant and fungi extracts.

Binding, Competitive↗
PubMed2005

Competitive binding assay for glucose based on glycodendrimer-fluorophore conjugates.

A new fluorescent glucose assay has been created using Alexa Fluor 647-labeled concanavalin A (Con A) and a fourth-generation PAMAM Alexa Fluor 594-labeled glycodendrimer. This assay has been shown to have a large response to glucose within the biological range and to be capable of functioning within a polymer hydrogel. In this paper, the glucose response is shown to be a single fluorophore-based quenching reaction. Data showing that the sensor is fully reversible and specific through competitive binding between the dendrimer and glucose with Con A are presented. Overall, the assay is shown to have potential over the traditional dextran-based assay because it has a larger dynamic response to physiological glucose concentrations, incorporates longer wavelength dyes that improve signal penetration through dermal tissue, and provides an internal reference in the form of a nonreactive fluorescent label.

Binding, Competitive↗
PubMed1987

Analysis of competition binding assays: assessment of the range of validity of a commonly invoked assumption.

A common assumption invoked in the analysis of competition binding assays is that the fractional saturation of sites with the unlabeled ligand is given by 1-(the concentration of bound labeled ligand in the presence of unlabeled ligand)/(the concentration of bound labeled ligand in the absence of unlabeled ligand). This assumption is critically evaluated in the context of several binding models: (a) binding of univalent ligands to multiple classes of equivalent and independent sites, with and without nonspecific binding; (b) cooperative binding of univalent ligands; and (c) binding of multivalent ligands to a single class of univalent acceptors. We show that the conventional assumption is only valid when the labeled ligand is mainly in the free form, occupies a small fraction of the total sites and binds univalently to all sites in an equivalent and independent manner, and when the unlabeled ligand forms 1:1 complexes with the acceptor sites. When these conditions are satisfied, the conventional assumption is valid even if the unlabeled ligand binds to nonequivalent sites or exhibits cooperativity. Finally, we apply the theory derived for case (a) above to the binding of fluoresceinated epidermal growth factor to A431 cells and demonstrate that the analysis of data obtained from both conventional and competition assays provides information which is difficult, if not impossible, to obtain from either assay alone.

Binding Sites↗
PubMed2000

Molecular imprinting of biotin derivatives and its application to competitive binding assay using nonisotopic labeled ligands.

Synthetic biotin-binding polymers were prepared by molecular imprinting. Methacrylic acid (MAA) was copolymerized with ethylene glycol dimethacrylate in the presence of biotin methyl ester (B-Me) in chloroform. Hydrogen-bonding-based complexation of B-Me with MAA generates the binding sites complementary to B-Me after extracting B-Me from the resulting copolymers. Data from NMR titration suggest a one-to-one prepolymerization complex formation of B-Me with MAA in chloroform. A possible complex structure was estimated by docking of the most stable conformers by intermolecular Monte Carlo conformational search under the assumption of a one-to-one association. The selectivity of the imprinted polymers was investigated and an imprinted polymer-based competitive binding assay for B-Me was demonstrated using biotin p-nitrophenyl ester as a nonisotopic-labeled ligand.

Acrylates↗
PubMed1992

Monoclonal antibodies against potato virus A--competitive binding tests.

Six mouse monoclonal antibodies (MoAbs) against potato virus A (PVA) were tested. One of them (PVA 534) reacted only with complete virions and was apparently specific for epitopes dependent on quaternary structure. MoAb PVA 328 recognized the virus antigen only after its dissociation into subunits. MoAb PVA 328 must have reacted with a cryptotope of the antigen. MoAb PVA 151 and 290 appeared to be conformation independent and reacted with exposed regions on native virus particles as well as on the surface of dissociated coat protein subunits. Two other MoAbs (PVA 187 and 634) did not recognize subunits or the virions adsorbed directly to the microtiter plate. This seems to be an evidence of metatope existence. The results of competitive binding tests combined with the reaction patterns of individual MoAbs to different potyviruses indicate that the MoAbs are specific for 6 distinct epitopes.

Antibodies, Monoclonal↗
PubMed2001

Competitive binding of triplex-forming oligonucleotides in the two alternate promoters of the PMP22 gene.

Overexpression of the 22-kDa peripheral myelin protein (PMP22) causes the inherited peripheral neuropathy, Charcot-Marie-Tooth disease type 1A (CMT1A). In an attempt to alter PMP22 gene expression as a possible therapeutic strategy for CMT1A, antiparallel triplex-forming oligonucleotides (TFO) were designed to bind to purine-rich target sequences in the two PMP22 gene promoters, P1 and P2. Target region I in P1 and region V in P2 were also shown to specifically bind proteins in mammalian nuclear extracts. Competition for binding of these targets by TFO vs. protein(s) was compared by exposing proteins to their target sequences after triplex formation (passive competition) or by allowing TFO and proteins to simultaneously compete for the same targets (active competition). In both formats, TFO were shown to competitively interfere with the binding of protein to region I. Oligonucleotides directed to region V competed for protein binding by a nontriplex-mediated mechanism, most likely via the formation of higher-order, manganese-destabilizable structures. Given that the activity of the P1 promoter is closely linked to peripheral nerve myelination, TFO identified here could serve as useful reagents in the investigation of promoter function, the role of PMP22 in myelination, and possibly as rationally designed drugs for the therapy of CMT1A. The nontriplex-mediated action of TFO directed at the P2 promoter may have wider implications for the use of such oligonucleotides in vivo.

Animals↗
PubMed1992

Determination of radioligand specific activity using competition binding assays.

Radioligand binding assays are routinely utilized in laboratories throughout the world to study receptors and their related binding sites, carrier proteins, and enzymes. To accurately estimate equilibrium binding parameters, such as the equilibrium dissociation constant (Kd) and maximal number of binding sites (Bmax), the investigator must know the correct value of the specific activity of the radioligand. If the specific activity is overestimated the Kd and Bmax values will be underestimated, while underestimation of the specific activity results in an overestimation of the Kd and Bmax. The present communication describes a simple and rapid method for determining the specific activity of a radioligand using homologous competition binding assays. Performing the competition assays at two or more different concentrations of the radioligand allows the specific activity to be determined from the IC50 values without the need of analytical methods to quantify minute amounts of the radioligand. In addition to providing the specific activity, use of this method estimates the Kd for the radioligand. This method was utilized to determine the specific activity and Kd for two blockers of the dopamine uptake carrier, [3H]GBR-12935 and [3H]-CFT, which share a common binding site in the striatum.

Animals↗
PubMed1997

Competitive binding of viral E2 protein and mammalian core-binding factor to transcriptional control sequences of human papillomavirus type 8 and bovine papillomavirus type 1.

The promoter P7535 of human papillomavirus type 8 and the promoter P7185 of bovine papillomavirus type 1 are negatively regulated by viral E2 proteins via the promoter proximal binding sites P2 and BS1, respectively. Mutations of these E2 binding sites can reduce basal promoter activity. This suggests binding of a transcription-stimulating factor and may indicate that repression by E2 is due to competitive binding of viral and cellular proteins. A computer search revealed putative binding sites for core-binding factor (CBF; also referred to as PEA2, PEBP2, or AML), overlapping with P2 and BS1. Binding of recombinant CBF proteins to these sites was confirmed by band shift analysis. Competition of CBF and E2 protein for DNA binding was shown for both human papillomavirus type 8 and bovine papillomavirus type 1. The importance of CBF-E2 competition in E2-mediated repression could be demonstrated by comparing the E2 effect on P7185 activity in two cell lines containing different amounts of endogenous CBF. In cells with large amounts of CBF, E2 repressed P7185 wild-type constructs to the basal promoter activity of a mutant (50%) that could not bind this protein any more. In contrast, in a cell line containing small amounts of CBF, the promoter activities of constructs with wild-type and mutated CBF binding sites hardly differed and specific repression by E2 was not detectable.

Animals↗