Boehringer Mannheim Gmbh

Roche Diagnostics Gmbh

Hans-Joachim Guder

Rupert Herrmann

Harvey Buck

Johann Berger

Werner Guthlein

Herbert Von Der Eltz

Manfred Kuhr

Klaus Muhlegger

Christian Klein

Erhard Kopetzki

Lance Scott Kuhn

Peter Sluka

Wolfgang Knoll

Martina Junius-Comer

Dorothee Ambrosius

Fern DeLaCroix

Dietmar Zdunek

Wolfgang Weckerle

Robert J McEnroe

Rebecca W Muddiman

Mary L Ochs

John G Hurrell

Mary E Brown

Ruppert Herrmann

Bernd Schmidt

Petra Ruger

Franz-Josef Schmitt

Martha Liley

Jurgen Spinke

A diagnostic kit, method, and apparatus for electrochemically determining the presence or concentration of an analyte in a sample. A mixture is formed which includes the sample, an enzyme acceptor polypeptide, an enzyme donor polypeptide, and a labeled substrate. The enzyme donor polypeptide is capable of combining with the enzyme acceptor polypeptide to form an active enzyme complex. The formation of such the active enzyme complex is responsive to the presence or concentration of the analyte in the fluid sample. The active enzyme hydrolyzes the labeled substrate, resulting in the generation of an electroactive label, which can then be oxidized at the surface of an electrode. A current resulting from the oxidation of the electroactive compound can be measured and correlated to the concentration of the analyte in the sample.

Electrochemical enzyme assay

An electrochemical sensor is provided containing a supporting material having a surface of noble metal such as gold or palladium to which is bound an enzyme such as glucose oxidase via binding molecules. The binding molecules are in the form of a homogenous monolayer and are bound to the metal surface by anchor groups such as thiol, disulphide or phosphine groups. The enzyme is linked to the binding molecules by an ionic bond, a covalent bond or a metal-chelate bond. The binding molecules may contain a spacer such as an alkylene chain. Coverage of the binding molecules on the metal surface is less than the maximum coverage to optimize enzyme coverage, decrease response time of the sensor and increase signal yield per surface area. The monolayer may contain diluent molecules which are not linked to an enzyme and which are bound to the metal surface via anchor groups. The diluent molecules lower the concentration of binding molecules in the monolayer, increase conductivity of the monolayer and lower unspecific binding of components from a sample solution in contact with the sensor. The binding molecules can be linked by an ionic bond or a metal-chelate bond, respectively, to a charged amino acid residue or a chelate-forming amino acid residue of the N-terminus, C-terminus, or N- and C-terminus of recombinant enzyme protein.

Electrochemical sensor containing an enzyme linked to binding molecules

The invention concerns a binding matrix containing a carrier material and a solid phase reactant which is adsorbed to this via anchor groups that is capable of binding to at least one free reaction partner wherein the solid phase reactant forms a dilute and essentially laterally homogeneous binding layer on the surface of the carrier material. In addition a method for the determination of an analyte in a sample solution is claimed in which a solid phase reactant is used which is a component of a binding matrix according to the present invention. In this process the specific binding reaction is preferably determined by optical reflection techniques.

Universal binding film

A method of detecting hydrolase activity using as substrates the compounds of the formula ##STR1## wherein each of R.sup.1, R.sup.2 and R.sup.3 is C.sub.1 -C.sub.4 alkyl, or phenyl optionally substituted in the meta- or para- position by C.sub.1 -C.sub.6 alkyl, C.sub.1 -C.sub.6 alkoxy or mono- or di-(C.sub.1 -C.sub.6)-alkylamino, or optionally substituted by an O-X group in which X is a glycosyl, phosphate or acyl moiety of a natural substrate of the corresponding glycosidase, phosphatase or esterase.

Agents for the detection of substrates with hydrolase activity

A process for the detection of substances with hydrolase activity in a sample by mixing the sample with a hydrolase substrate and an oxidising agent, and the evaluating the resultant colour intensity, wherein, as hydrolase substrate, there is used at least one compound of the formula ##STR1## in which R.sup.1 is hydrogen or an alkoxy radical,

Agent for the detection of substances with hydrolase activity

The present invention provides compounds of the general formula: ##STR1## wherein R is a trifluoromethyl radical or a cyano group. The present invention also provides processes for the preparation of these compounds, as well as reagents containing them. The present invention is also concerned with the use of these compounds as .beta.-galactosidase substrates, especially in a CEDIA system.

.beta.-galactosidase substrates for cedia

An apparatus for determining an analyte in a sample, as well as a method for carrying this out, are disclosed. The apparatus utilizes a first zone containing a labeled substance, and a second zone which permits separation of a mobile detectable moiety from unreacted reaction component. Detectable moiety is the product of reaction between reaction component and labeled substance.

Apparatus for determining an analyte and method therefor

A process for the detection of substances with hydrolase activity in a sample by mixing the sample with a hydrolase substrate and an oxidizing agent, and the evaluating the resultant color intensity, wherein, as hydrolase substrate, there is used at least one compound of the formula ##STR1## in which R.sup.1 is hydrogen or an alkoxy radical,

Process for the detection of substances with hydrolase activity

In order to increase the enzymatic reactivity of .beta.-galactosidase, an azide, thiocyanate, cyanate and/or thiosulphate is added to the reaction mixture.

Method for increasing the enzymatic reactivity of .beta.-galactosidase

Reagents for the determination of hydrolase comprises compounds of the formula ##STR1## wherein Z is an organic or inorganic acid residue or a sugar residue, A is an organic or inorganic acid residue, and B is an organic acid residue. Each of R.sup.2 and R.sup.5, is hydrogen, halogen or lower alkyl. Each of R.sup.1, R.sup.3, R.sup.4 and R.sup.6, is hydrogen, halogen, cyano, lower alkyl, lower alkoxy, carboxyl, lower alkoxycarbonyl, carboxy lower alkyl or lower alkoxycarbonyl lower alkyl, or carboxamido groups optical substituted once or twice, or a radical of the formula

Hydrolase substrates

Reagents for the determination of hydrolase comprises compounds of the formula ##STR1## wherein Z is an organic or inorganic acid residue or a sugar residue, A is an organic or inorganic acid residue, and B is an organic acid residue. Each of R.sup.2 and R.sup.5, is hydrogen, halogen or lower alkyl. Each of R.sup.1, R.sup.3, R.sup.4 and R.sup.6, is hydrogen, halogen, cyano, lower alkyl, lower alkoxy, carboxyl, lower alkoxycarbonyl, carboxy lower alkyl or lower alkoxycarbonyl lower alkyl, or carboxamido groups optional substituted once or twice, or a radical of the formula

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Weilheim, Germany

Hans-Joachim Guder