Miles Laboratories, Inc.

Serim Research Corporation

Miles Inc.

Robert C Boguslaski

Robert J Carrico

James E Christner

John F Burd

A rapid method and easy to use unitized test device is disclosed for determining the presence of Helicobacter pylori in a biological tissue specimen by detecting the presence of urease in the tissue. The system basically utilizes a multilayer test device for separating and optimizing the various reactions involved, i.e. the urease in the specimen with a substrate and the ammonia generated thereby with an indicator element.

Test device and kit for detecting helicobacter pylori

Test for Helicobacter pylori

The reactant advantageously is an enzymatic reactant such as an enzyme substrate or coenzyme. The activity of the conjugated reactant as a constituent of a predetermined reaction is affected by reaction between the specific binding substance in the conjugate and a specific binding counterpart thereto. The presence of a ligand in a liquid medium may be determined using competitive or displacement binding or sequential saturation techniques wherein the specific binding substance in the conjugate is the ligand or a specific binding analog thereof, or using a direct binding technique wherein the specific binding substance is a specific binding partner of the ligand. The effect of the specific binding reaction on the activity of the conjugated reactant is related to the presence or amount of the ligand in the liquid medium tested.

Homogenous specific binding assay reagent system and labeled conjugates

An improved heterogenous specific binding assay method which employs a substance having reactant activity, i.e., a reactant, as a labeling substance in the detection of a ligand in a liquid medium. The method is carried out using reagent means which comprises, as its labeled constituent, a conjugate formed of a specific binding substance coupled to the reactant. The reactant advantageously is an enzymatic reactant such as an enzyme substrate or coenzyme. The activity of the conjugated reactant as a constituent of a predetermined reaction system is utilized as means for monitoring the extent of binding of the labeled constituent in conventional heterogenous specific binding assay schemes. The presence of a ligand in a liquid medium may be determined following conventional competitive binding manipulative techniques. After the necessary separation of the bound- and free-phases resulting in the specific binding reaction system, the extent of binding of the labeled constituent is determined by contacting either phase with the necessary materials to form the predetermined monitoring reaction system in which the labeling substance is active and assessing reactant activity therein.

Heterogenous specific binding assay employing an enzyme substrate as

An improved specific binding assay method and reagent for determining a ligand in a liquid medium employing, as an enzyme-cleavable substrate label, a residue having the formula:

Beta-galactosyl-umbelliferone-labeled hapten conjugates

A homogeneous specific binding assay which employs a substance having reactant activity, i.e., a reactant, in a chemiluminescent reaction as a labeling substance in the detection of a ligand in a liquid medium. The assay employs a conjugate formed of a specific binding substance coupled to the chemiluminescent reactant. The activity of the conjugated reactant as a constituent of the chemiluminescent reaction is affected by reaction between the specific binding substance in the conjugate and a specific binding counterpart thereto. The presence of a ligand in a liquid medium may be determined using competitive or displacement binding or sequential saturation techniques wherein the specific binding substance in the conjugate is the ligand or a specific binding analog thereof, or using a direct binding technique wherein the specific binding substance is a specific binding partner of the ligand. The effect of the specific binding reaction on the chemiluminescent activity of the conjugated reactant is related to the presence or amount of the ligand in the liquid medium tested.

Homogeneous chemiluminescent specific binding assay

A heterogeneous specific binding assay which employs a substance having reactant activity, i.e., a reactant, in a chemiluminescent reaction as a labeling substance in the detection of a ligand in a liquid medium. The method is carried out using reagent means which comprises, as its labeled constituent, a conjugate formed of a specific binding substance coupled to the chemiluminescent reactant. The activity of the conjugated reactant in the chemiluminescent reaction, i.e., the production of light, is utilized as means for monitoring the extent of binding of the labeled constituent in conventional heterogeneous specific binding assay schemes. The presence of a ligand in a liquid medium may be determined following conventional competitive binding manipulative techniques. After the necessary separation of the bound- and free-phases resulting in the specific binding reaction system, the extent of binding of the labeled constituent is determined by contacting either phase with the necessary materials to form the chemiluminescent monitoring reaction and measuring the light produced.

Heterogenous chemiluminescent specific binding assay

Chemiluminescent-labeled conjugates of the formula: ##STR1## wherein one of R.sup.1 and R.sup.2 is hydrogen and the other is -NR.sup.3 R.sup.4 ; R.sup.3 is hydrogen or straight chain alkyl containing 1-4 carbon atoms and R.sup.4 is ##STR2## wherein n=1-3 and L(CO-- is the ligand or analog bound through an amide bond. Intermediates produced in the synthesis of such conjugates are also disclosed.

Chemiluminescent-labeled haptens and antigens

Amino-functionalized phthalhydrazide intermediates of the formula: ##STR1## wherein one of R.sup.5 and R.sup.6 is hydrogen and the other is --NR.sup.7 R.sup.8 ; R.sup.7 is hydrogen or straight chain alkyl containing 1-4 carbon atoms and R.sup.8 is ##STR2## wherein n=1-3. The compounds are intermediates in the synthesis of chemiluminescent-labeled conjugates which are useful as reagents in specific binding assays for determining ligands or their specific binding partners in liquid media.

Amino-functionalized phthalhydrazide intermediates

An improved heterogenous specific binding assay method which employs a substance having reactant activity, i.e., a reactant, as a labeling substance in the detection of a ligand in a liquid medium. The method is carried out using reagent means which comprises, as its labeled constituent, a conjugate formed of a specific binding substance coupled to the reactant. The reactant advantageously is an enzymatic reactant such as an enzyme substrate or coenzyme. The activity of the conjugated reactant as a constituent of a predetermined reaction system is utilized as means for monitoring the extent of binding of the labeled constituent in conventional heterogenous specific binding assay schemes. The presence of a ligand in a liquid medium may be determined following conventional competitive binding manipulative techniques. After the necessary separation of the bound-and free-phases resulting in the specific binding reaction system, the extent of binding of the labeled constituent is determined by contacting either phase with the necessary materials to form the predetermined monitoring reaction system in which the labeling substance is active and assessing reactant activity therein.

Heterogenous specific binding assay employing a cycling reactant as label

Specific binding assay employing an enzyme-cleavable substrate as label

Bis-phthalimide intermediates of the formula: ##STR1## wherein one of R.sup.9 and R.sup.10 is hydrogen and the other is --NR.sup.11 R.sup.12 ; R.sup.11 is hydrogen or straight stain alkyl containing 1-4 carbon atoms and R.sup.12 is ##STR2## wherein n=1-3. The compounds are intermediates in the synthesis of chemiluminescent-labeled conjugates which are useful as reagents in specific binding assays for determining ligands or their specific binding partners in liquid media.

Bis-phthalimide intermediates

A specific binding assay method and reagent means for determining a ligand, such as an antigen or antibody, in, or the ligand binding capacity of, a liquid medium which employs a fluorescent substance, i.e., a fluorescer, as a label. The improvement comprises measuring the fluorescer-label by chemically exciting the label and measuring the resulting light emitted thereby. Chemical excitation of the label is preferably accomplished by exposure to a substance, such as a high energy intermediate produced by the reaction between hydrogen peroxide and highly reactive materials such as oxalyl chloride, oxamides and bis-oxalate esters. The assay may follow conventional homogeneous and heterogeneous formats. The improved assay is more convenient than conventional fluorescent binding assays by obviating the need for photogenic excitation of the fluorescent label.

Fluorescer-labeled specific binding assays

An improved heterogeneous specific binding assay method which employs a substance having reactant activity, i.e., a reactant, as a labeling substance in the detection of a ligand in a liquid medium. The method is carried out using reagent means which comprises, as its labeled constituent, a conjugate formed of a specific binding substance coupled to the reactant. The reactant advantageously is an enzymatic reactant such as an enzyme substrate or coenzyme. The activity of the conjugated reactant as a constituent of a predetermined reaction system is utilized as means for monitoring the extent of binding of the labeled constituent in conventional heterogeneous specific binding assay schemes. The presence of a ligand in a liquid medium may be determined following conventional competitive binding manipulative techniques. After the necessary separation of the bound-and free-phases resulting in the specific binding reaction system, the extent of binding of the labeled constituent is determined by contacting either phase with the necessary materials to form the predetermined monitoring reaction system in which the labeling substance is active and assessing reactant activity therein.

Heterogenous specific binding assay employing a coenzyme as label

.beta.-Galactosyl-umbelliferone labeled aminoglycoside antibiotics, e.g., gentamicin, useful in homogeneous and heterogeneous specific binding assays for such antibiotics in liquid media such as serum. Also provided are a .beta.-galactosyl-umbelliferone-carboxylic acid and salts thereof which are intermediates in a method of preparing the labeled antibiotics.

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Elkhart, IN, United States of America

Robert C Boguslaski