Miles Laboratories, Inc.

Miles Inc.

James E Christner

Robert J Carrico

Robert C Boguslaski

Robert C Bolguslaski

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

A test composition, device, and method for their use in a homogeneous specific binding assay 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 test composition and device comprise 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 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.

Homogeneous specific binding assay method

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

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

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

A specific binding assay method employing, as a labeling substance, a reversibly binding enzyme modulator for the detection of a ligand in a liquid medium. The method follows conventional specific binding assay techniques of either the homogeneous or heterogeneous type wherein the liquid medium to be assayed is combined with reagent means that includes a labeled conjugate to form a binding reaction system having a bound-species and a free-species of the conjugate. The amount of conjugate resulting in the bound-species or the free-species is a function of the amount of ligand present in the liquid medium assayed. In the present invention, the labeled conjugate comprises a reversibly binding enzyme modulator covalently linked to a binding component of the binding reaction system. The distribution of the conjugate between the bound-species and the free-species is determined by addition of an enzyme whose activity is affected, either decreased or increased, by said modulator and measuring the resulting activity of the enzyme. The enzyme modulator may be a conventional enzyme inhibitor, preferably of the coompetitive type, or an allosteric effector.

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Ann Arbor, MI, United States of America

James E Christner