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PubMed2006

Glucose sensors based on microcapsules containing an orange/red competitive binding resonance energy transfer assay.

Fluorescent sensing systems offer the potential for noninvasive monitoring with implantable devices, but they require carrier technologies that provide suitable immobilization, accessibility, and biocompatibility while maintaining adequate response characteristics. A recent development towards this goal is a highly specific and sensitive competitive binding assay for glucose using apo-glucose oxidase (apo-GOx) as the recognition element and dextran as the competing ligand; this has been demonstrated as a glucose sensor system by encapsulating the competitive binding assay in semipermeable microcapsule carriers. This paper describes the extension of this sensor design to longer wavelengths in an attempt to increase the applicability to in vivo monitoring. The glucose sensitivity of the tetramethylrhodamine isothiocyanate-dextran (TD) and cyanine Cy5-apo-GOx (CAG) complexes showed five to 10 times greater specificity for beta-D-glucose over other sugars. Microcapsules loaded with TD/CAG complexes exhibited a linear, totally reversible response in the range of 0-720 mg/dL, with a sensitivity (percent change in intensity ratio) of 0.06%/(mg/dL). The decrease in sensitivity observed with the use of longer-wavelength dyes is most likely to be compensated with the deeper penetration of light and reduced tissue scattering. These findings imply that the encapsulation of sensing assay elements in microcapsules is a simple and translatable method for the fabrication of stable biosensors, and optimization of resonance energy transfer pairs and assay component preparation will further improve the response to approach clinically relevant performance.

Apoenzymes↗

PubMed1989

Potentiometric homogeneous enzyme-linked competitive binding assays using adenosine deaminase as the label.

Homogeneous enzyme-linked competitive binding assays for biotin are described that are based on the competition between an enzyme-biotin conjugate and free biotin for a fixed number of binding sites of avidin. Unlike conventional homogeneous enzyme immunoassays, in this system the analyte (biotin) is labeled with adenosine deaminase (ADA), an ammonia-producing enzyme. Consequently, potentiometric rather than photometric methods can be used as means of detection. Several ADA-biotin conjugates were prepared and showed as high as 97% inhibition of the enzymatic activity in the presence of avidin. Addition of free biotin reverses this inhibition in an amount proportional to the concentration of analyte. Relatively steep dose-response curves were observed, leading to a precise and accurate assay for biotin. The detection limits of these curves were as low as 1 x 10(-8) M. Varying the concentration of the reagents in the assay allowed the detection limit and working range to be altered to a desired value. The proposed method was applied in the determination of biotin in a horse-feed supplement.

Adenosine Deaminase↗

PubMed1990

A competition binding assay for determination of the inositol (1,4,5)-trisphosphate content of human leucocytes.

We developed a competition binding assay for estimation of the intracellular inositol (1,4,5)-trisphosphate (Ins(1,4,5)P3) and optimalized it for the measurement of the Ins(1,4,5)P3 content of human blood leucocytes. The present method is considerably cheaper and requires five times fewer cells than the commercial Ins(1,4,5)P3 kit. The mean Ins(1,4,5)P3 content of human blood monocytes, granulocytes, and lymphocytes amounted to 3.3 +/- 1.2 microM, 3.1 +/- 1.4 microM, and 4.6 +/- 1.5 microM, respectively. After stimulation with formyl-methionyl-leucyl-phenylalanine (f-MLP) the Ins(1,4,5)P3 content of human granulocytes and monocytes increased 2-3 times within 10 sec and then gradually decreased, returning to basal values at 60 sec. Lymphocytes did not respond to f-MLP with an increase in their Ins(1,4,5)P3 content.

Binding, Competitive↗

PubMed1980

[Contribution of the IGF (insulin-like growth factors or somatomedins) competitive binding assay in the study of growth disorders in children (results for 124 observations) (author's transl)].

A competitive binding assay for IGF (insulin-like growth factors) employing a specific carrier protein produced by rat liver in culture has been used in a study of a variety of growth disorders in children. In 34 subjects with a total somatotropic deficiency, serum IGF levels were significantly (p < 0.001) lower than those in normal children for whom the means were 0.57 +/- 0.05 (SEM) U/ml between the ages of 1 and 5, and 1.01 +/- 0.11 U/ml between the ages of 5 and 15. Within this group of patients, there was also a significant difference in IGF levels depending on whether the deficiency was idiopathic (0.16 +/- 0.03 U/ml) or caused by a tumour (0.49 +/- 0.06 U/ml). IGF levels were particularly low in 2 cases of Laron's syndrome (0.08 and 0.03 U/ml) and 6 cases of coeliac disease (0.19-0.27 U/ml). In the later group, a gluten-free diet resulted in a rise in IGF levels. However, the stunted growth observed in 10 cases of Cushing's syndrome was found to be independent of IGF levels which fell within the normal range (1.02 +/- 0.11 U/ml). For 34 children investigated because of idiopathic retarded growth varying from --2 to --4 SD, the mean IGF levels (0.68 +/- 0.04 U/ml) were significantly lower than controls (p < 0.001). By contrast, abnormally high IGF levels were found in children with tall stature (> 3 SD). In 7 children aged between 1 and 5, the mean was 0.94 +/- 0.05 U/ml and in 17 older patients, 1.47 +/- 0.11 U/ml, with some cases reaching levels similar to those of untreated acromegalics. In both age groups the means were significantly higher than control levels (p < 0.001). Oestrogen treatment led to a progressive drop in IGF levels accompanied by slower growth, and normal levels were reached by the time ossification was established.

Adolescent↗

PubMed1993

Mechanisms of distamycin A/DAPI chromosome staining. I. Competition binding effects of nonintercalative DNA groove-binding agents in situ and in vitro.

The molecular mechanism underlying distamycin A-induced differential DAPI fluorescent staining of metaphase chromosomes was studied in Sus scrofa domestica both cytologically, using, besides DAPI, two isomeric derivatives of DAPI (D288.45 and D288.48), and molecularly, by in vitro competitive-binding studies using S. scrofa satellite DNA and synthetic DNA polymers. Significant differences in heterochromatin staining were observed between D288.45 and D288.48. Distinct distamycin A/DAPI bands were obtained with DAPI and D288.45 but not with D288.48. Circular dichroism measurements were performed to characterize the displacement of DAPI from its DNA binding sites by distamycin A and also netropsin. Distamycin A was most effective in displacing DAPI when DAPI was bound to contiguous clusters of AT base pairs and much less effective in displacing DAPI bound to GC or mixed AT/GC base-pair sequences. The results of these competitive-binding studies provide the basis of a molecular explanation of the quenching phenomenon of distamycin A counterstaining on chromosomal DAPI fluorescence.

Animals↗

PubMed1989

Competitive binding of gangliosides and glycophorin to wheat germ agglutinin.

Gangliosides and glycophorin are receptors for wheat germ agglutinin. The competitive binding of these molecules to wheat germ agglutinin is studied by electron spin resonance spectroscopy with spin labels attached to the oligosaccharide chains of gangliosides. Evidence shows that glycophorin is more accessible to wheat germ agglutinin binding than gangliosides. The interactions of gangliosides and glycophorin in liposomes is disrupted on low level binding of WGA.

Animals↗

PubMed1993

Theory of a sequential addition competitive binding immunoassay based on high-performance immunoaffinity chromatography.

This study examined the theory and behavior of a chromatographic competitive binding immunoassay with sequential injection of sample and a labeled analyte analog. Based on nonlinear chromatographic theory, an equation was derived to describe the calibration curve for this assay in a system with adsorption-limited kinetics and homogeneous binding sites. This equation related assay response (B/Bo) to the column's binding capacity, the moles of analyte and labeled analog injected, and the flow rate/adsorption kinetics of the system. There was good agreement between this equation and experimental data for the binding of human serum albumin (HSA) to an immobilized anti-HSA antibody column. It was found that the amount of labeled analog injected, when applied in excess vs binding sites in the column, had little or no effect on the relative response or position of the calibration curve. The position of the curve was determined mainly by the number of binding sites on the column; however, this position could be shifted over several orders of magnitude by varying the flow rate used for analyte injection. By varying both the injection flow rate and labeled analog load, this method could be designed for use as either a screening test or quantitative assay.

Antibodies↗

PubMed1995

A novel application of a competitive binding model in dioxin risk assessment.

The EPA-recommended toxicity equivalence factor (TEF) approach to estimating the lifetime incremental cancer risks for dioxins does not address (a) differences in the severity of toxicity according to the composition of chemical mixture and (b) potentials for modification of tissue-level doses of congeners in mixtures and consequently the cancer risk estimates. Our earlier efforts to model the binding of congeners to the Ah receptor in the low-dose range and to develop quantitative estimates for the formation of fractions of Ah receptor-congener complexes resulted in the definition of a unique parameter, defined as competitive binding ratio (CBR), to adjust tissue-level doses for mixture exposure. We made an effort to incorporate CBR values in the dose-response analysis and risk characterization of congeners in two distinct exposure scenarios. The modified approach to estimating tissue-level doses of congeners in mixtures by the use of a competitive binding model indicated that (a) the Ah receptor affinity is an important criterion in the determination of tissue-level dose of congeners, (b) the TEF doses calculated by using the model algorithms modified the tissue-level doses for congeners in mixture exposures, and (c) the combined lifetime incremental cancer risks for all congeners were generally lower when model algorithms were used in the dose-response analysis. However, the percentage contribution of toxic congeners was significantly higher when model algorithms were used. The percentage contribution of higher congeners with low toxicity was considerably reduced when model algorithms were used.(ABSTRACT TRUNCATED AT 250 WORDS)

Adipose Tissue↗

PubMed1998

Modulation of cytokine-induced HIV gene expression by competitive binding of transcription factors to the coactivator p300.

The host response to viral infection involves the secretion of multiple cytokines which alter immune function and viral replication. These proteins activate several signal transduction pathways in infected cells which must be integrated to regulate cellular and viral gene expression. In this report, we demonstrate that specific transcription factors induced by distinct cytokines regulate HIV transcription by competitive binding to the p300 coactivator. Interferon-alpha (IFN-alpha) was found to inhibit NF-kappaB-dependent HIV gene expression stimulated by tumor necrosis factor-alpha (TNF-alpha). This inhibition was mediated by binding of the IFN-alpha signal transducer and activator of transcription 2, Stat2, to a specific domain of p300 which also binds to the RelA (p65) subunit of NF-kappaB. p300 was found to be limiting with respect to RelA (p65) and Stat2, and this effect was reversed by overexpression of p300. Inhibition by Stat2 was specific for NF-kappaB and was not mediated by Stat1, which is also induced by IFN-alpha. Gene activation induced by the Stat2 transcription domain was also inhibited by expression of RelA. These results demonstrate that HIV transcription can be regulated in the nucleus by competitive binding of specific cytokine-induced transcription factors to a discrete domain of a transcriptional coactivator.

Binding, Competitive↗

PubMed2004

[Microbial receptor competitive binding assay of basic macrolide antibiotics].

OBJECTIVE: To establish a biological method for determining the macrolide content in various matrices. METHODS Human serum, urine and tissue homogenate samples were diluted or extracted with MSU buffer, and the specimens of grain and premixed feed extracted with methanol-H(3)PO(4) buffer, before microbial receptor competitive binding assay was carried out on these various specimens, with the elimination of interference from methanol with the M8 buffer. RESULTS: The method was sensitive, class-specific and precise, and the recommended screening concentrations for specimens of the serum, urine, tissue and grains were 200, 200, 100 and 1 200 ng/g (ng/ml), respectively, with a relative standard deviation less than 8%. CONCLUSION: Microbial receptor competitive binding assay is accurate and rapid for efficient qualitative and quantitative assay of the total macrolide content in various matrices.

Anti-Bacterial Agents↗

PubMed2002

Fluorine-NMR competition binding experiments for high-throughput screening of large compound mixtures.

High-throughput ligand-based NMR screening with competition binding experiments is extended to (19)F detection. Fluorine is a favorable nucleus for these experiments because of the significant contribution of the Chemical Shift Anisotropy (CSA) to the (19)F transverse relaxation of the ligand signal when bound to a macromolecular target. A low to moderate affinity ligand containing a fluorine atom is used as a reference molecule for the detection and characterization of new ligands. Titration NMR experiments with the selected reference compound are performed for finding the optimal set-up conditions for HTS and for deriving the binding constants of the identified NMR hits. Rapid HTS of large chemical mixtures and plant or fungi extracts against the receptor of interest is possible due to the high sensitivity of the (19)F nucleus and the absence of overlap with the signals of the mixtures to be screened. Finally, a novel approach for HTS using a reference molecule in combination with a control molecule is presented.

Anisotropy↗

PubMed1986

Absorption of heparin, LMW heparin and SP54 after subcutaneous injection, assessed by competitive binding assay.

Unfractionated heparin, pentosan polysulphate (SP54) and the low molecular weight heparins CY216 and CY222 were injected subcutaneously at a minimum of weekly intervals into 5 healthy volunteers. The dose was 75 mg in all cases. Concentrations of administered glycosaminoglycan in serial plasma samples and voidings of urine were measured using a competitive binding assay, and biological activity was assessed in plasma using APTT and anti-Xa clotting assays. There was wide individual variation in the absorption of unfractionated heparin as indicated both by the maximal plasma concentrations reached 2-3 h after injection and by the area under the concentration vs. time curve. The efficiency of absorption increased and the individual variation decreased with decreasing molecular weight of the administered glycosaminoglycan. Urinary excretion correlated with plasma concentration, and recovery in the urine also increased with decreasing molecular weight. Similar patterns of uptake and clearance were indicated by the APTT and competitive binding assays, but anti-Xa clotting activity could be detected in the plasma after clearance of the administered glycosaminoglycan.

Absorption↗

PubMed2005

Phosphatidylinositol 3,4,5-trisphosphate and Ca2+/calmodulin competitively bind to the regulators of G-protein-signalling (RGS) domain of RGS4 and reciprocally regulate its action.

RGS (regulators of G-protein signalling) are a diverse group of proteins, which accelerate intrinsic GTP hydrolysis on heterotrimeric G-protein a subunits. They are involved in the control of a physiological behaviour known as 'relaxation' of G-protein-gated K+ channels in cardiac myocytes. The GTPase-accelerating activity of cardiac RGS proteins, such as RGS4, is inhibited by PtdIns(3,4,5)P3 (phosphatidylinositol 3,4,5-trisphosphate) and this inhibition is cancelled by Ca2+/calmodulin (CaM) formed during membrane depolarization. G-protein-gated K+ channel activity decreases on depolarization owing to the facilitation of GTPase-activating protein activity by RGS proteins and vice versa on hyperpolarization. The molecular mechanism responsible for this reciprocal control of RGS action by PtdIns(3,4,5)P3 and Ca2+/CaM, however, has not been fully elucidated. Using lipid-protein co-sedimentation assay and surface plasmon resonance measurements, we show in the present study that the control of the GTPase-accelerating activity of the RGS4 protein is achieved through the competitive binding of PtdIns(3,4,5)P3 and Ca2+/CaM within its RGS domain. Competitive binding occurs exclusively within the RGS domain and involves a cluster of positively charged residues located on the surface opposite to the Ga interaction site. In the RGS proteins conserving these residues, the reciprocal regulation by PtdIns(3,4,5)P3 and Ca2+/CaM may be important for their physiological regulation of G-protein signalling.

Acetylcholine↗

PubMed1990

Competitive binding enzyme immunoassay for zonisamide, a new antiepileptic drug, with selected paired-enzyme labeled antigen and antibody.

We assessed the competitive binding between zonisamide (ZNS) in serum samples and beta-galactosidase-labeled ZNS derivatives, using competing antibodies to ZNS derivatives, and selected the best enzyme-labeled antigen and antibody for accurate enzyme immunoassay (EIA) of ZNS in serum without interference from its metabolites or from other antiepileptic drugs. This EIA, based on use of antibody linked to bacterial cell walls, has advantages over HPLC in simplicity, speed (50 samples per hour), and lack of requirement for special equipment. The concentrations of ZNS in serum as measured by the EIA correlated well with those by HPLC (n = 33, r = 0.977).

Antibodies↗

PubMed1986

A competitive binding assay for fructose 2,6-bisphosphate.

A new direct assay method for fructose 2,6-bisphosphate has been developed based on competitive binding of labeled and unlabeled fructose 2,6-P2 to phosphofructokinase. Phosphofructokinase (0.5-1.3 pmol protomer) is incubated with saturating concentrations (5.0-5.5 pmol) of fructose 2,6-[2-32P]P2 and samples containing varying concentrations of fructose 2,6-P2. The resulting stable binary complex is retained on nitrocellulose filters with a binding efficiency of up to 70%. Standard curves obtained with this assay show strict linearity with varying fructose 2,6-P2 in the range of 0.5 to 45 pmol, which exceeds the sensitivity of most of the previously described assay methods. Fructose 2,6-P2, ATP, and high concentrations of phosphate interfere with this assay. However, the extent of this inhibition is negligible since their tissue contents are one-half to one-tenth that examined. This new assay is simple, direct, rapid, and does not require pretreatment of tissue extracts.

Animals↗

PubMed1977

Comparison of toadfish-serum competitive binding and microbiologic assays of vitamin B12.

Toadfish serum (TFS) offers several advantages over other proteins as the binder in a competitive-binding assay for vitamin B12. It is unaffected by pH changes in the range 5.6-9.4 or by the addition of human serum albumin. Prolonged incubation with charcoal does not disrupt the TFS-cyanocobalamin bond, and the addition of albumin as a protein source in the standard tubes was proven unnecessary. The binding capacity of TFS does not increase significantly with increasing concentrations of cyanocobalamin as does the binding capacity of intrinsic factor, normal serum, or transcobalamin I. A single extract was prepared from each of 44 sera and measured for vitamin B12 content simultaneously by the TFS assay and the conventional microbiologic method using Lactobacillus leichmannii. The values obtained with TFS were in each instance higher than those obtained by the microbiologic assay (p less than 0.001).

Animals↗

PubMed1976

[Folic acid determination in the serum and erythrocytes by means of a competitive binding method with 125 iodine labeling].

Folic acid concentration in serum and erythrocytes was measured by radio assay according to the principle of competitive binding to proteins with 125I as marker. The method yielded a linear regression with readily reproducible results. The mean value in controls was 7.05 -+/ 3.4 ng/ml in serum and 386.8 -+/ 215.1 ng/ml in erythrocytes. In chronic alcoholics the respective values were significantly lower: 4.79 -+/ 2.02 ng/ml for serum and 344.4 -+/ 198.9 ng/ml for erythrocytes. The clinical importance of folic acid estimations arises from the general frequency of folic aicd deficits. Latent folic acid deficiency is associated with lowered folic acid concentrations in serum and normal values in red cells. In manifest folic acid deficiency both concentrations are decreased.

Alcoholism↗

PubMed1980

[Competitive binding of histones to DNA and the problem of chromatin self-assembly].

The competitive binding of histones to DNA in a medium of physiological ionic strength (0.15 M NaCl) and in water was studied by an electrophoretic analysis of histones bound to DNA and retaining their free state in the mixtures of DNA with total histone. In the medium of physiological ionic strength the binding sequences of more and less competitive histone fraction molecules to DNA can be represented as follows: H3+H4, H2a+ +H2b and H3, H4, H2a, H2b and H1, which is indicative of the absence of histone complexes in water and their presence in 0.15 M NaCl, in addition to individual molecules. In water the binding sequences of the histone, molecules to DNA were identical, i. E. H3+H4, H2a, H2b+H1. It was concluded that the non-ionic interaction between the histones bound to DNA determines the discrepancy between the values of histone net charges and their relative affinities for DNA by increasing the correlation between the positively charged amino acid residues of the histones. The relations between the histone competition for DNA and the chromatin self-assembly as well as the reproduction of the specific pattern of gene expression in the cell generation are discussed.

Animals↗