Abstract: Reagents for use in binding assays, particularly immunoassays, to determine valproic acid in liquid media such as serum. Such reagents include antibody to valproic acid and .beta.-galactosyl-umbelliferone-valproic acid conjugates. Also provided are compounds used to prepare such reagents, including valproic acid immunogen conjugates and intermediates in the synthesis of such immunogen conjugates and .beta.-galactosyl-umbelliferone-labeled conjugates.

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: A process and test kit for the quantitative determination of the individual and total unsaturated Vitamin B-12 binding capacities of the three transcobalamins TC-I, TC-II, and TC-III in serum. The process comprises incubating a serum sample with a solution of .sup.57 Co Vitamin B-12, contacting the resulting mixture with a TC-II adsorbent and with a DEAE-cellulose adsorbent for TC-I and TC-III, measuring the total radioactivity adsorbed to said adsorbents, selectively desorbing TC-III from the DEAE-cellulose adsorbent by washing with a monopotassium phosphate solution of about 0.05 M and at a pH of about 4.6, measuring the individual radioactivity adsorbed to each of said adsorbents, and calculating the radioactivity that had been washed from the DEAE-cellulose adsorbent by said phosphate solution. The resulting four radioactivity values are functions of the total, TC-II, TC-I, and TC-III unsaturated Vitamin B-12 binding capacities, respectively, of the serum tested.

Abstract: Title compounds of the formula: ##STR1## wherein R is hydroxy substituted lower alkyl. The compounds have advantageous water solubility and spectral characteristics for use as instrument calibrators for certain homogeneous fluorescent immunoassays.

Abstract: An apoglucose oxidase preparation having a residual glucose oxidase activity of less than 0.05%, and preferably less than 0.005%. The preparation is obtained by incubation of glucose oxidase in aqueous solution at a pH of less than about 2 and in the presence of about 20 to about 40, preferably about 30, percent by volume glycerol, and separating resulting dissociated prosthetic group, flavin adenine dinucleotide, from apoglucose oxidase by column chromatography, preferably on cross-linked dextran gel. The purified apoglucose oxidase is useful as a reagent in specific binding assays employing flavin adenine dinucleotide as a label.

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: Reagents for use in binding assays, particularly immunoassays, to determine valproic acid in liquid media such as serum. Such reagents include antibody to valproic acid and .beta.-galactosyl-umbelliferone-valproic acid conjugates. Also provided are compounds used to prepare such reagents, including valproic acid immunogen conjugates and intermediates in the synthesis of such immunogen conjugates and .beta.-galactosyl-umbelliferone-labeled conjugates.

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: 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 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 method and test means for determining a specific binding substance, such as an antigen or hapten, in a liquid sample wherein a solid, nonspecific adsorbent is contacted, preferably in sequence, with the sample and a labeled form of a binding partner for the substance to be determined, such as a labeled antibody. The amount of label which becomes associated with the adsorbent by binding of the labeled binding partner to the adsorbed substance to be determined, i.e., in a sandwich fashion, is a function of the amount of the substance to be determined in the liquid sample. Preferably, the adsorbent is an ion exchange material, and the assay steps are carried out under pH conditions between the isoelectric points (pI values) of the substance to be determined and the labeled binding partner. The method and test means are particularly suited for the detection of hepatitis B surface (HB.sub.s) antigen, especially where the label is a radioactive material and a column format is employed.

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: A specific binding assay for determining a ligand in a liquid medium employing an organic prosthetic group residue, such as a residue of flavin adenine dinucleotide, flavin mononucleotide, or heme, as a label component in the labeled conjugate. Preferably, the label component is the prosthetic group residue alone or is a holoenzyme residue comprising such prosthetic group residue combined with an apoenzyme in the form of a holoenzyme complex. In the former case, the label component preferably is monitored in the assay by adding an apoenzyme after the binding reaction has been initiated and measuring the resultant holoenzyme activity. In the latter case, the label component is monitored simply by measuring holoenzyme activity. The assay method may follow conventional homogeneous and heterogeneous schemes. Preferred apoenzymes for use in the assay are apoglucose oxidase and apoperoxidase. The assay offers the advantages of colorimetric read-out and of being readily adaptable to automated techniques.

Abstract: -Dimethyl 7-[.omega.-N-(phthalimido)alkyl]aminonaphthalene -1,2-dicarboxylates of the formula: ##STR1## wherein R is hydrogen or straight chain alkyl containing 1-4 carbon atoms and n=2-6. The compounds are intermediates in the synthesis of chemiluminescent naphthalene-1,2-dicarboxylic acid hydrazide-labeled conjugates which are useful as reagents in specific binding assays for determining ligands or their specific binding partners in liquid media.

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: Amino-functionalized phthalhydrazides 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 isH.sub.2 N--CH.sub.2).sub.nwherein n=2-8. The compounds are intermediates in the synthesis of chemiluminescent phthalhydrazide-labeled conjugates which are useful as reagents in specific binding assays for determining ligands or their specific binding partners in liquid media.

Abstract: Amino-functionalized naphthalene-1,2-dicarboxylic acid hydrazides of the formula: ##STR1## wherein R is hydrogen or straight chain alkyl containing 1-4 carbon atoms and n=2-6. The compounds are intermediates in the synthesis of chemiluminescent naphthalene-1,2-dicarboxylic acid hydrazide-labeled conjugates which are useful as reagents in specific binding assays for determining ligands or their specific binding partners in liquid media.

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: A method for the combined radioimmunoassay for the hormones triiodothyronine (T-3) and thyroxine (T-4) in serum or plasma, wherein the radiolabeled T-3 and T-4 reagents are T-3 and T-4 labeled with the same radioisotope. The hormones are extracted from the serum or plasma sample and separated from their binding proteins by adsorption to a crosslinked dextran gel at a highly alkaline pH. After washing away the dissociated proteins, a predetermined limiting amount of T-3 antibody is incubated with the gel and thereafter radiolabeled T-3 which has become extracted from the gel by antibody binding is washed away. Excess T-3 antibody and a predetermined limiting amount of T-4 antibody are then incubated with the gel and radiolabeled T-3 and T-4 which has become extracted from the gel by antibody binding are thereafter washed away.

Abstract: Chemiluminescent-labeled conjugates of the formula: ##STR1## wherein R is hydrogen or straight chain alkyl containing 1-4 carbon atoms, n=2-6 and L(CO-- is a specifically bindable ligand, such as an antigenic protein or polypeptide, a hapten or an antibody, or a binding analog thereof, bound through an amide bond; and intermediates produced in the synthesis of such conjugates. The labeled conjugates are useful as reagents in specific binding assays for determining ligands or their specific binding partners in liquid media.