Syntex (u.s.a.) Inc.

Microgenics Corporation

Syva Company

Boehringer Mannheim Gmbh

Discoverx Corporation

Abbott Laboratories Corporation

Chemtrak Corporation

Syra Company

Pyare L Khanna

Edwin F Ullman

Prithipal Singh

Evan S Snyder

Glenda L Choate

Litai Weng

David Calderhead

Michael Joseph Powell

Joseph L Horecka

Scott J Eisenbeis

Rohan Peries

Floyd W Colvin

Warren Colvin

David Lingenfelter

Wayne B Manning

Robert T Dworschack

Roberta D Ernst

Anne J Stone

Ian Gibbons

Jimmy D Allen

Chiu C Chang

Kirk Schulkamp

Shu-Ling Cheng

Peter Fung

Rueyming Loor

David S Kates

Stephen B Friedman

Cynthia D Stiso

Dennis M Bleile

Dean Jenkins

Fred Pearlman

William A Coty

Theresa Medlin

Bill Coty

Imo-Jean C Ford

Patricia A Porreca

Reuyming Loor

Jeff Shindelman

Phillip Ruprecht

Methods and compositions are provided for assaying for enzymes capable of releasing an enzyme donor fragment (ED) conjugated to a nucleic acid strand bonded to a surface. Conveniently, the beads are magnetic allowing segregation of the beads during the determination. Upon addition of enzyme acceptor fragment (EA) and substrate to the assay mixture, the method allows for discrimination between ED free in solution and ED bound to the bead. The complexing of ED and EA provides an active &#x3b2;-galactosidase enzyme. The method permits the assay of any substance involved in a pathway that can result in a reaction releasing the ED.

Assays for nucleic acid modifying enzymes

Methods and reagents are provided for measuring protease activity. The reagent comprises a surface to which is linked an enzyme donor fragment through a protease recognition sequence, where the enzyme donor fragment complexes with an enzyme acceptor fragment to form an active indicator enzyme when the enzyme donor fragment is cleaved from said surface. Conveniently, the surface is a cell membrane surface where the reagent is expressed in the cell. The method comprises bringing together the reagent, the protease, the enzyme acceptor and substrate for the indicator enzyme and measuring the indicator enzyme activity as a measure of the protease activity. The method finds application in screening for compounds modulating the activity of the protease.

Protease EFC cell surface fusion protein assay

A protein reagent is provided for measuring protease enzyme activity in a sample. The protein reagent comprises two inhibition entities joined by a linker comprised of an indicator enzyme donor and an amino acid sequence susceptible to enzymatic cleavage. The protein reagent is substantially inhibited from binding to the cognate enzyme acceptor fragment, while the product of the enzymatic cleavage binds to the cognate enzyme acceptor fragment to form a functional indicator enzyme. The indicator enzyme activity is related to the protease enzyme activity of the sample.

Enzyme activation protease assay

Formation of an intramolecular cross-link in enzyme donor polypeptide fragments of .beta.-galactosidase, thereby forming a cyclic enzyme donor which is hindered from complementation with an enzyme acceptor fragment to form active of .beta.-galactosidase. The cyclic enzyme donor can be linearized by cleaving to restore complementation ability. Assays in which such cyclic enzyme donors are linearized by specific analytes are disclosed, as well as novel homobifunctional bis-maleimido cross-linking agents of the formula ##STR1## wherein R is hydroxy or acetate.

Cross-linked polypeptide fragments of .beta.-galactosidase

Simplified calibration and improved dose-response linearity of the standard curve of competitive assays are obtained by adding a predetermined amount of unlabeled competitive binding compound (usually just the analyte itself) to a first reagent containing anti-analyte antibodies before the reagent is reacted with a sample containing an unknown amount of analyte.

Competitive binding assays having improved linearity

Assays using cross-linked polypeptide fragments of .beta.-galactosidase

A method and device for determining the presence of an analyte in a sample suspected of containing the analyte is disclosed. The method involves contacting a test solution containing the sample and a first member of a specific binding pair with an end portion of a strip of bibulous material capable of being traversed by the test solution through capillary action. The first member of a specific binding pair is capable of binding the analyte. The strip contains a second member of a specific binding pair integral therewith for concentrating and non-diffusively binding the first sbp member at a small situs on the strip separated from the end portion of the strip. The detectible signal is produced in relation to the presence of the analyte in the test solution. The test solution passes through the situs as the test solution traverses the bibulous material. After the test solution has been allowed to traverse at least a portion of the strip, the strip is contacted with a developer solution containing members of a signal producing system in a manner that provides contact of the developer solution with the small situs following its contact with the test solution. The strip is then contacted with any remaining members of the signal producing system. The detectible signal produced at the situs is then compared with the signal detectible at a portion of the strip other than the situs to determine the analyte in the sample. In one embodiment of the invention the signal produced at the small situs has a sharp-edged distinctive pattern that provides a sharp contrast to the signal produced at adjacent sites on the strip when analyte is present in the test solution.

Concentrating immunochemical test device and method

A method for reducing or eliminating tyramine interference from amphetamine and methamphetamine immunoassays, comprising treating the sample with aqueous tyramine oxidase for a time and at a temperature and pH sufficient to deaminate any tyramine present in the sample, is provided.

Reducing tyramine interference in immunoassays for amphetamine and

Galactopyranoside derivative compounds for use as substrates for hydrolysis by enzymes with .beta.-galactosidase activity are provided. The concentration of the reaction products can be measured by spectroscopy. The compounds find particular use in diagnostic assays for detection of analyte.

Substrates for .beta.galactosidase

Substrates for .beta.-galactosidase

Compounds are evaluated for their binding to naturally occurring receptors, by employing the natural ligand conjugated to an enzyme donor fragment of .beta.-galactosidase for competing with the sample compound for the natural acceptor binding site or in the absence of competition where the sample compound binds to an allosteric site. By adding the enzyme acceptor fragment of the .beta.-galactosidase and substrate, the binding affinity of the sample compound may be evaluated as a measure of agonist or antagonist capability.

Complementation assay for drug screening

A method for detecting the presence of a high-molecular-weight analyte, which uses a variation on complementation assays, comprising (1) providing an enzyme donor (ED)complex comprising ED coupled to an analyte-specific binding molecule, wherein the ED complex retains measurable complementation activity such that active .beta.-galactosidase is formed in the presence of an enzyme acceptor (EA); (2) contacting the ED complex and EA in the presence of a sample suspected of containing a high-molecular-weight analyte that reacts specifically with the analyte-specific binding molecule; and (3) relating the presence of the analyte in the sample to the formation of active .beta.-galactosidase enzyme.

Determination of high molecular weight analytes using a

The present invention provides reagents for a detectable label for use in specific binding assays. Specifically, modified .beta.-galactosidase enzyme donors (ED) and acceptors (EA) are utilized. Both ED and EA are modified to form separate ED and EA complexes by coupling each with a linking element and a binding moiety which is specific to a binding site in the analyte. The ED and EA complexes are incapable of forming active enzyme in the absence of the analyte. However, when analyte is present, ED and EA complexes which bind to a common analyte in a sample, active .beta.-galactosidase is formed.

Detection of analytes having binding sites for at least two binding

Novel assays employing enzyme fragments which complex to form an active enzyme are provided. The reagents involved are a member of a specific binding pair bound to a solid surface, a first enzyme fragment conjugated to a member of a specific binding pair complementary or cross-reactive in relation to the analyte or antianalyte and a second enzyme fragment which binds to the conjugate to form an active enzyme, where the first enzyme fragment conjugate may be present in up to substantial excess to ensure at least substantially complete binding of all of the analyte. By distributing the conjugate between the solid surface and the medium in relation to the amount of analyte present, the enzyme activity may be determined in relation to the amount of analyte present, without separating the solid surface and the medium.

Solid phase non-separation enzyme complementation assay

Enzyme-donor fragments and other unstructured peptides are stabilized against proteolytic degradation by including a mixture of soluble, random-sequence peptides in the assay medium or in the medium in which the enzyme-donor fragment or other peptide is stored.

Stabilization of polypeptide fragments against proteolytic degradation

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Fremont, CA, United States of America

Pyare L Khanna