Abstract: An improved immunoassay method, reagent means, test kit, and test device for determining an iodothyronine, e.g., thyroxine (T-4), in a biological fluid, usually serum or plasma, wherein 2-hydroxy-4-methoxybenzophenone-5-sulfonic acid (HMS), or a salt thereof, is employed as a blocking agent for the binding of iodothyronines to thyroxine binding protein (TBP). The present invention is particularly advantageous as applied to homogeneous competitive binding iodothyronine immunoassays employing labels which are participants in enzyme-catalyzed reactions. Such labels include enzyme substrates, coenzymes, enzyme inhibitors, enzyme prosthetic groups, and enzymes.

Abstract: A method for increasing the ability of apoglucose oxidase to combine with flavin adenine dinucleotide (FAD) and derivatives thereof to form active glucose oxidase by interacting apoglucose oxidase with an immunologically derived binding substance, e.g., an antibody or a fragment thereof, having a specific binding affinity for glucose oxidase. The apoglucose oxidase/anti-glucose oxidase immune complex is characterized by enhanced ability to combine with FAD and FAD-derivatives to yield glucose oxidase activity. The activation is particularly significant at temperatures elevated from room temperature, e.g., between 30.degree.-45.degree. C. An improved homogeneous specific binding assay method is provided for determining ligands wherein an FAD label is used and is monitored by its ability to combine with apoglucose oxidase to form active glucose oxidase by including anti-glucose oxidase in the reaction mixture.

Abstract: An improved immunoassay method, reagent means, and test kit for determining an iodothyronine, e.g., thyroxine (T-4), in a biological fluid, usually serum or plasma, wherein fenclofenac and related phenylacetic acids, or salts thereof, are employed as novel blocking agents for the binding of iodothyronines to thyroxine binding protein (TBP). The present invention is particularly advantageous as applied to homogeneous competitive binding iodothyronine immunoassays wherein a spectrophotometric response is generated in the assay reaction mixture at a wavelength greater than about 300 nm, the blocking agents of the present invention having been found to have no substantial absorbance at wavelengths above 300 nm. Such homogeneous immunoassays include those which employ labels such as fluorescers, enzyme substrates, enzyme prosthetic groups, enzymes, and enzyme inhibitors.

Abstract: A method of preparing an enzyme-labeled ligand for use in specific binding assays for determining said ligand or a specific binding partner thereof, by covalently coupling said ligand or a binding analog thereof to an organic prosthetic group and combining the resulting coupled product with an apoenzyme to form a conjugated enzyme complex with said prosthetic group in said coupled product. Preferably, the conjugated enzyme complex in the resulting labeled conjugate exhibits holoenzyme activity which is affected by binding of said labeled conjugate by a specific binding partner of said ligand. Preferred prosthetic groups are flavin mononucleotide, heme, and, especially, flavin adenine dinucleotide, with preferred apoenzymes being apoperoxidase and, especially, apoglucose oxidase.

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: 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: 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: 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: 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.