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Biomedical subjects

G A Rechnitz

Publications and source records attributed to G A Rechnitz.

At least 19 recordsLinked to original sources
PubMed1992

Microtiter plate binding assay for cholinergic compounds utilizing the nicotinic acetylcholine receptor.

A receptor-based binding assay for the determination of cholinergic compounds of the nicotinic acetylcholine receptor has been developed. By conducting the assay in a 96-well microtiter plate, the method is suitable for large-scale screening in drug development. Solid-phase extraction of the enzyme label significantly simplifies the assay protocol compared to earlier methods. The assay is based on immobilization of biotin-BSA on the microtiter which takes up avidin-labeled peroxidase due to avidin-biotin interaction. To perform the assay, a ligand (the analyte) and a biotin alpha-bungarotoxin conjugate (alpha Bgt-biotin) sequentially bind to a vesicle bound nicotinic acetylcholine receptor. This is done either in a test tube, assay I, or in a biotinylated microtiter well, assay II. Avidin-HRP is then added to this mixture; free alpha Bgt-biotin conjugate and immobilized biotin-BSA compete for the avidin sites. After the assay solution has been aspirated off, bound enzyme activity is determined which is directly related to the amount of alpha Bgt-biotin added. Dose-response curves of cholinergic compounds and Scatchard plots were generated to evaluate the apparent binding constants. Kinetic studies were conducted for the purpose of optimization. The final assay can be performed in under 4 h with a minimum of sample handling.

Animals↗
PubMed1992

Biotinylated 1012-S conjugate as a probe ligand for benzodiazepine receptors: characterization of receptor binding sites and receptor assay for benzodiazepine drugs.

A nonisotopic receptor assay using the biotin-1012-S conjugate was developed and the usefulness of this conjugate as a probe ligand for the benzodiazepine receptor was evaluated. The conjugate was incubated in a receptor suspension, and then the concentration of free conjugate in the supernatant was determined nonisotopically with a solid-phase avidin-biotin binding assay. Studies on the ligand saturation with the conjugate demonstrated that the conjugate has very high affinity and specificity for the receptors and the biotin labeling does not decrease the affinity of 1012-S. This assay method was applied to the characterization of binding sites of benzodiazepine receptors in cow brain. Competition interactions between the conjugate and benzodiazepine drugs gave well-defined dose-response curves. These results confirm the possibility that this conjugate could serve as a probe for the study of receptor-ligand interactions and provide the basis of a new nonisotopic receptor assay for benzodiazepine drugs.

Animals↗
PubMed1992

Non-isotopic receptor assay for benzodiazepines using a biotin-labeled ligand and biotin-immobilized microtiter plate.

A non-isotopic receptor assay for benzodiazepine drugs was developed using a biotin-labeled ligand, biotin-1012S. Biotinylated bovine serum albumin (biotin-BSA) was immobilized onto the wall of microtiter plate wells by simple adsorption. Avidin peroxidase conjugate could be extracted from solution owing to its strong interaction with biotin. The amount of avidin peroxidase taken up on the wall was then determined by measuring the enzyme activity. The competition between immobilized biotin on the wall and free biotin for avidin provided the basis for a solid-phase avidin-biotin binding assay. By this binding assay, not only biotin but also biotin-1012S could be measured sensitively. Because 1012S is a ligand with high affinity to benzodiazepine receptors, biotin-1012S could be utilized as a probe ligand for a non-isotopic receptor assay. Based upon the competition between biotin-1012S and various benzodiazepine drugs for the receptor binding sites, a non-isotopic receptor assay was demonstrated.

Animals↗
PubMed1992

Reagentless enzyme electrode for the determination of manganese through biocatalytic enhancement.

An amperometric enzyme electrode is described for the detection and determination of manganese(II). The biosensor is based on the stimulation by manganese of the aerobic oxidation of substrates by horseradish peroxidase. A mediator, 1,2-naphthoquinone, is used as the substrate and is incorporated with the enzyme into a carbon-paste electrode. The resulting electrode acts as an enzyme-based oxygen sensor, which is sensitive to manganese. Electrochemical control of enzyme activity is achieved through substrate promotion of catalysis. Enzyme modulation by manganese can be switched on and off or adjusted through the appropriate selection of the applied potential. Currents are generated due to the bioelectrocatalytic reduction of oxygen in response to the introduction of manganese sulfate. A sustained current is achieved which is dependent on manganese concentration. Concentrations of 0.5 microM manganese or greater can be measured, and the sensor is reversible, as demonstrated by manganese removal. Biological selectivity for manganese provides a sensor which does not respond to other divalent cations tested, with the possible exception of cobalt. Reagentless, continuous sensing is achieved through substrate cycling.

Catalysis↗
PubMed1992

Flavonoid-specific staining of Arabidopsis thaliana.

Crop yields may be threatened by increases in UV-B radiation resulting from depletion of the ozone layer. In higher plants, the presence of flavonols provides a protective mechanism, and we report a novel staining procedure for the visualization of such protectants in plant tissue. It is shown that the proposed technique provides sensitive and specific fluorescence of flavonoids in chlorophyll-bleached tissue of Arabidopsis thaliana.

Arabidopsis↗
PubMed1991

Nonisotopic receptor-binding assay for benzodiazepine receptors utilizing a fluorophore labeled ligand.

A nonisotopic receptor-binding assay method provides a new approach for the study of receptor-ligand interactions and a possible receptor assay for benzodiazepine drugs. The proposed method is based upon the use of fluorescence-labeled drugs and a chromatographic system which accepts samples without deproteinization. The effectiveness of the technique is illustrated in a study of benzodiazepine receptor-drug-binding interactions.

Affinity Labels↗
PubMed1990

Enzyme-amplified receptor assay screening test for chlorpromazine, trifluoperazine, and phencyclidine.

A new receptor based assay is described for the determination of classes of drugs which have high affinities for the acetylcholine receptor. The method is based upon the inhibition of the enzyme activity of an enzyme-drug conjugate by the binding to the receptor protein, and competition between free drugs and the enzyme-drug conjugate for a limited number of receptor sites. The activity of the enzyme marker system, glucose-6-phosphate dehydrogenase covalently conjugated to desipramine, is monitored by colorimetric detection of the rate of NADH formation at 340 nM. The procedure proposed is designed to provide a simple drug screen which can be done in a minimally equipped laboratory while achieving the required sensitivity. The technique is illustrated for three acetylcholine channel binding compounds: the hallucinogen phencyclidine (PCP) and the antipsychotic agents chlorpromazine and trifluoperazine. This procedure yields calibration curves with detection limits at nanomolar levels of drug, with binding responses dependent on the amounts of receptor and enzyme-labeled drug used. Aspecific binding responses of unlabeled enzyme to drug or receptor to compounds with low affinity for the receptor are shown to have minimal effect on the assay.

Binding Sites↗
PubMed1979

Antibody-selective membrane electrodes.

Direct antibody-sensing membrane electrodes have been developed by immobilizing ion-carrier immunogen conjugates in a liquid membrane matrix. The resulting potentiometric probes measure specific antibodies with high selectivity over nonspecific antibodies in the physiological pH range. The electrode response is shown to arise from the selective interaction of the antibody with the membrane-bound immunogen.

Antibodies↗
PubMed1978

Glutamine-selective membrane electrode that uses living bacterial cells.

A novel bioselective membrane electrode for L-glutamine has been constructed by coupling living bacteria of the strain Sarcina flava to a potentiometric ammonia gas sensor. Tests in aqueous standards and human serum show that the electrode combines excellent sensitivity and selectivity with rapid response and a useful lifetime of at least 2 weeks.

Ammonia↗
PubMed1977

Determination of total carbon dioxide in serum and plasma using a carbonate ion-selective membrane electrode.

A new manual method for the determination of carbon dioxide content in serum and plasma, based on the potentiometric measurement of carbonate, is proposed and evaluated. The new method offers good precision and accuracy in the physiological concentration range as shown by detailed studies on synthetic samples, reference standards, and actual patient samples. Moreover, the method yields results which correlate very well with analyses carried out using various conventional techniques. It is expected that the proposed method could be used for manual determinations of CO2 content, especially where rapid start-up time, minimal sample treatment, and low cost are of importance.

Carbon Dioxide↗
PubMed1977

Antibody binding measurements with hapten-selective membrane electrodes.

Direct antibody-hapten binding measurements were carried out with the use of a hapten-selective membrane electrode. Potentiometric titrations of rabbit antibody to the hapten trimethylphenylammonium ion demonstrate the speed and convenience of the new method for the determination of binding equilibria. Average intrinsic binding constants obtained agree well with those measured by equilibrium dialysis techniques.

Antigen-Antibody Reactions↗