Abstract: 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:G-D-Rwherein G is a glycone, D is a dye indicator moiety, and R is a linking group through which the label residue is covalently bound to a binding component of a conventional binding assay system, such as the ligand, an analog thereof, or a specific binding partner thereof. The monitored characteristic of the label is the release of a detectable product, usually a fluorogen or chromogen, upon enzymatic cleavage of the glycosidic linkage between the glycone and the dye indicator moiety. The assay method may follow a homogeneous or heterogeneous format. The preferred glycone is a .beta.-galactosyl group and the preferred dye indicator moiety is an umbelliferone residue.

Abstract: 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.

Abstract: 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:G--D--Rwherein G is a glycone, D is a dye indicator moiety, and R is a linking group through which the label residue is covalently bound to a binding component of a conventional binding assay system, such as the ligand, an analog thereof, or a specific binding partner thereof. The monitored characteristic of the label is the release of a detectable product, usually a fluorogen or chromogen, upon enzymatic cleavage of the glycosidic linkage between the glycone and the dye indicator moiety. The assay method may follow a homogeneous or heterogeneous format. The preferred glycone is a .beta.-galactosyl group and the preferred dye indicator moiety is an umbelliferone residue.

Abstract: 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.

Abstract: Flavin adenine dinucleotide-labeled conjugates of the formula: ##STR1## wherein Riboflavin--Phos).sub.2 Ribose represents the riboflavin-pyrophosphate-ribose residue in flavin adenine dinucleotide (FAD), --NH)L is a protein or polypeptide (e.g., an immunoglobulin) bound through an amino group thereof, n is an integer from 2 through 10, m is an integer from 1 through 10, and p is on the average from 1 to the number of available amino groups in L. The FAD-labeled conjugates are useful as reagents in specific binding assays (e.g., immunoassays) to determine the conjugated protein or polypeptide, or a specific binding analog or partner thereof, in liquid media.

Abstract: A .beta.-galactosyl-umbelliferone-labeled conjugate of the formula: ##STR1## wherein --NH)L is a protein or polypeptide, such as an immunoglobulin, bound through an amino group thereof, n is an integer from 2 through 10, m is an integer from 1 through 10, and p is on the average from 1 to the number of available amino groups in L. An intermediate in the synthesis of such labeled conjugate is also provided. The labeled conjugates are useful as reagents in specific binding assays (e.g., immunoassays) for determining the conjugated protein or polypeptide, or a specific binding analog or partner thereof, in liquid media.

Abstract: A flavin adenine dinucleotide (FAD) derivative of the formula: ##STR1## wherein n is an integer from 2 through 10, and preferably is 6. The derivatives are intermediates in the preparation of FAD-labeled conjugates useful as reagents in specific binding assays for determining ligands (e.g., antigens and haptens) in liquid media such as serum. Also provided are certain FAD-labeled conjugates, particularly an FAD-theophylline conjugate.

Abstract: 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.

Abstract: 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.

Abstract: .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.

Abstract: Labeled conjugates of the formula: ##STR1## wherein Riboflavin--Phos).sub.2 Ribose represents the riboflavin-pyrophosphate-ribose residue in flavin adenine dinucleotide (FAD), n=2 through 6, and --CO)L is a specifically bindable ligand, or a binding analog thereof, and is preferably an iodothyronine such as thyroxine, bound through an amide bond; and intermediates produced in the synthesis of such FAD-labeled conjugates. The FAD-labeled conjugates are useful as labeled conjugates in specific binding assays for determining the ligand or a specific binding partner thereto in liquid media such as serum.

Abstract: Conjugates of the formula: ##STR1## wherein Riboflavin-(Phos).sub.2 -Ribose represents the riboflavin-pyrophosphate-ribose residue in flavin adenine dinucleotide (FAD), n equals 2 through 6, Y is hydrogen or trifluoroacetyl, and .beta..sup.1 and .beta..sup.2 are, independently, hydrogen or iodine; and intermediates in the synthesis of such FAD conjugates. The FAD-iodothyronine conjugates, particularly where the iodothyronine is thyroxine, are useful as labeled conjugates in specific binding assays for determining the iodothyronine in liquid media such as serum.

Abstract: 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.