Abstract: 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.

Abstract: 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.

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: 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.

Abstract: N-Aminoalkyl derivatives of iodothyronines, e.g., thyroxine and its lower alkyl esters, immunogen conjugates comprising the derivatives coupled to an immunogenic carrier material, and antibodies raised against such immunogen conjugates.

Abstract: 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.

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: 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 isL(CO--HN--CH.sub.2).sub.nwherein n=2-8 and L(CO)-- is a hapten bound through an amide bond. The labeled conjugates are useful as reagents in specific binding assays for determining haptens or their specific binding partners in liquid media.

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: 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.

Abstract: A homogeneous specific binding assay method and reagent means for determining a ligand, such as a hapten, antigen or antibody, in, or the ligand binding capacity of, a liquid medium employing a labeled conjugate which upon enzymatic cleavage produces a detectable indicator product. The improvement comprises employing as the label component in the labeled conjugate, a residue having the formula:P--X--M--Rwherein P is a photophore, e.g., a fluorescer, which emits light upon exposure to excitation means, X is an enzymatically cleavable linkage, M is a modulator, e.g., a quencher, for said light emission of the photophore, and R is a linking group to the binding component, e.g., the ligand or analog thereof, in the labeled conjugate. The labeled conjugate and the cleaved indicator product emit substantially different amounts of light upon excitation due to modulation of the emission of photophore P by the proximity of modulator M in the labeled conjugate.

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 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: A heterogeneous specific binding assay method and test means for the simultaneous determination of each of a plurality of different ligands (e.g., antibodies, antigens or haptens) in a single liquid test sample. The assay may follow any conventional technique of a heterogeneous type, i.e., which includes a separation of a bound-species from a free-species form of the labeled reagent. The combined assay is accomplished by using solid-phase binding agents for the respective ligands to be determined which are differentially separable from each other, thereby permitting the use of a single label rather than a different label for each ligand to be determined. The assay is particularly suited to the simultaneous determination of different antibodies, particularly antibodies to different viral antigens, in a single serum sample.

Abstract: An immunoassay method for the detection of an antibody (Ab.sub.1) to a viral antigen (Ag) wherein a whole human blood sample diluted with an isotonic aqueous solution is incubated with a solid-phase form of Ag whereby any Ab.sub.1 present in the sample becomes bound to solid-phase Ag, the resulting solid-phase Ag-Ab.sub.1 complexes are separated from the sample, a label-incorporated form of an antibody to Ab.sub.1 (Ab.sub.2 *) is contacted with the separated, solid-phase Ag-Ab.sub.1 complexes, the resulting solid-phase Ag-Ab.sub.1 -Ab.sub.2 * complexes are separated from excess Ab.sub.2 *, and the amount of the label in the separated, solid-phase Ag-Ab.sub.1 Ab.sub.2 * complexes is measured as a function of the presence of Ab.sub.1 in the sample. Preferably the whole blood sample is diluted 1:20 by volume with an isotonic aqueous buffer solution. The method is particularly useful for the detection of cytomegalovirus antibody or Rubella antibody.

Abstract: An improved immunoassay method for determining thyroid stimulating hormone (TSH) in a liquid sample wherein the bound- and free-species of the labelled, preferably radio-labelled, TSH are separated by contacting the liquid reaction mixture with Florisil adsorbent which selectively adsorbs free TSH and physically separating the adsorbent from the liquid mixture. The Florisil adsorbent is preferably contained in a column form. A test kit for performing the improved method is also provided. The assay is more simple and less time consuming than the prior art techniques.

Abstract: A dry radioimmunoassay test composition comprising a mixture of a radiolabelled form of a ligand, i.e., a hapten or an antigen, an antibody for the ligand, and a heavy metal cation, e.g., cupric ion, to inhibit the complex-forming reaction between the radiolabelled ligand and the antibody. A method for preparing the dry test composition is also provided. In use in a radioimmunoassay, the dry test composition is reconstituted with an aqueous liquid and combined with a test sample and a chelating agent to bind the heavy metal cation, permitting reaction among any ligand present in the sample, the radiolabelled ligand, and the antibody. A test kit for performing such radioimmunoassay is also provided.

Abstract: A specific binding assay method, and a test device and test kit for use therein, for determining a liqand, such as an antigen or antibody, in, or the ligand binding capacity of, a liquid medium, particularly a body fluid such as serum, wherein the unknown ligand competes with a labeled component, such as a radiolabeled form of the ligand or of a binding analog of the ligand, for binding with a binding partner, and wherein separation of the resulting bound-species and free-species of the labeled component is accomplished by allowing the liquid reaction mixture to be drawn by capillary action into a column comprising a bed of an adsorbent material selective for one of the two species. The improvement comprises using a column containing at least one additional bed of capillarily absorbent material disposed above said adsorbent bed and which is substantially nonadsorbent relative to the one of said two species which said adsorbent bed selectively binds.

Abstract: 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 isL(CO)--HN--(CH.sub.2).sub.nwherein n=2-8 and L(CO)-- is an antigenic protein or polypeptide bound through an amide bond. The labeled conjugates are useful as reagents in specific binding assays for determining antigenic proteins or polypeptides, or their binding partners, in liquid media.