Abstract: Amitriptyline functionalized compounds are provided for conjugation to antigenic compositions, particularly poly(amino acids), and enzymes. The antigenic conjugates are employed for the production of antibodies, which find particular use in immunoassays for the determination of amitriptyline, while the enzyme conjugate finds use in a homogeneous enzyme immunoassay for the determination of amitriptyline.

Abstract: A competitive protein binding method is provided for the determination of an analyte which is a member of an immunological pair consisting of ligand and receptor for the ligand. A chemiluminescent source is employed comprised of one or more individual members, one chemiluminescent source member being conjugated to one of the members of the immunological pair, so as to provide chemiluminescence adjacent to the site of conjugation. A quencher molecule is conjugated to a member of the immunological pair. When the members of the immunological pair bind, the quencher molecule is brought within quenching distance of the chemiluminescent source so as to inhibit the emission of light by the chemiluminescent source. The amount of analyte present in the assay medium affects the amount of binding between the members of the immunological pair which results in quenching of the chemiluminescence.

Abstract: Methods and compositions are provided for conjugating a wide variety of compounds, particularly polyfunctional compounds, having a mercapto group, either naturally present or synthetically introduced, to a polyamino compound, particularly a polypeptide (including proteins). The method employs a haloalkylcarbonyl compound, which is conjugated to one or more of the amino groups under mild acylating conditions. This is followed by combining the acylated polyamino compound with a mercapto containing compound, whereby the halogen is displaced by the sulfur of the mercapto group to form a stable thioether linkage. The resulting conjugates, depending on the compounds involved, can find uses in immunoassays, as hapten-antigen conjugates for the production of antibodies, and as ligand analog enzyme conjugates for use as reagents in controlling the distribution of substitution of a mercapto compound to a polyamino compound.

Abstract: Methods and reagents are provided for immunoassays employing as reagents a labelled receptor, where the label is capable of providing a detectable signal, and modifying reagents, capable of modifying the signal obtained from the label. When ligand (ligand analog for monoepitopic ligands) is present in the assay medium, a complex is formed which inhibits interaction between the label modifying reagent and the label. By measuring the signal obtained in the presence of a known amount of ligand in the assay medium and comparing that signal with the signal obtained with an unknown sample suspected of containing ligand, one can qualitatively or quantitatively determine the amount of ligand in the unknown. For determinating anti(ligand) the assay is carried out in substantially the same way, except a source of ligand or ligand analog must be provided.

Abstract: Novel biological assay method for determining the presence of a specific organic material by employing a modified enzyme for amplification. By employing receptors specific for one or a group of materials (hereinafter referred to as "ligands") and binding an enzyme to the ligand or ligand counterfeit to provide an "enzyme-bound-ligand", an extremely sensitive method is provided for assaying for ligands. The receptor when bound to the enzyme-bound-ligand substantially inhibits enzymatic activity, providing for different catalytic efficiencies of enzyme-bound-ligand and enzyme-bound-ligand combined with receptor.The receptor, ligand and enzyme-bound-ligand are combined in an arbitrary order and the effect of the presence of ligand on enzymatic activity determined. Various protocols may be used for assaying for enzymatic activity and relating the result to the amount of ligand present.

Abstract: Immunoassays are provided employing antibodies and a fluorescer-quencher (F-Q) chromophoric pair, wherein one or both of the chromophoric pair are bonded to antibodies. Depending on the particular ligand of interest, or whether antibodies are to be assayed, various reagent combinations can be employed, where the amount of quenching is directly related to the amount of ligand or antibody present in the assay medium.In carrying out the assay for ligands, the unknown and antiody specific for the ligand of interest to which is bound one of the F-Q pair, are combined in an aqueous buffered medium.

Abstract: Nitrogen derivatives of benzoyl ecgonine and cocaine are provided, particularly amino, diazonium, and diazo derivatives, the compounds finding use either directly or as intermediates for the preparation of reagents for use in immunoassays. Diazo compounds can be coupled with antigenic materials for the preparation of antibodies to benzoyl ecgonine and/or cocaine. The amino group can be combined with active non-oxo-carbonyl compounds to form reagents which find use in immunoassays.

Abstract: The subject invention concerns novel compositions for use in immunoassays, as well as immunoassays employing such novel compositions. The compositions comprise discrete charged colloidal particles comprised of small molecules which particles are capable of retaining their discrete character in an aqueous medium and composed of aggregates of lipophilic and/or amphiphilic organic molecules to which are bound non-covalently a label capable of producing a detectible signal and a ligand or an analog of the ligand capable of competing with a ligand for a ligand receptor. The discrete colloidal particle serves as a hub or nucleus for retaining the ligand or its analog and the label within a limited locus.The compositions are prepared by individually covalently bonding the ligand and the label, when not naturally lipophilic, to a lipophilic (includes amphiphilic) compound, normally a phospholipid.

Abstract: Novel conjugated enzyme compositions are provided for use in homogeneous enzyme immunoassays. A wide variety of compounds, particularly drugs, including drugs of abuse, drugs used in repetitive therapeutic applications, hormones, and the like, are conjugated to malate dehydrogenase. The resulting products have a high turnover rate, so as to provide a high multiplication factor when employed in a homogeneous immunoassay, have long storage lives, and allow for the accurate and sensitive detection of compounds of interest.

Abstract: Novel biological assay method for determining the presence of a specific organic material by employing a modified enzyme for amplification. By employing receptors specific for one or a group of materials (hereinafter referred to as "ligands") and binding an enzyme to the ligand or ligand counterfeit to provide an "enzyme-bound-ligand", an extremely sensitive method is provided for assaying for ligands. The receptor when bound to the enzyme-bound-ligand substantially inhibits enzymatic activity, providing for different catalytic efficiencies of enzyme-bound-ligand and enzyme-bound-ligand combined with receptor.The receptor, ligand and enzyme-bound-ligand are combined in an arbitrary order and the effect of the presence of ligand on enzymatic activity determined. Various protocols may be used for assaying for enzymatic activity and relating the result to the amount of ligand present.

Abstract: Immunoassays are provided employing antibodies and a fluorescer-quencher (F-Q) chromophoric pair, wherein one or both of the chromophoric pair are bonded to antibodies. Depending on the particular ligand of interest, various reagent combinations can be employed, where the amount of quenching is directly related to the amount of ligand present in the assay medium.In carrying out the assay, the unknown and antibody specific for the ligand of interest to which is bound one of the F-Q pair, are combined in an aqueous buffered medium. Depending on the protocol, different assay reagents are employed in the aqueous buffered medium: (1) ligand analog bonded to the other of the F-Q pair; (2) antibodies specific for the ligand to which is bound the other of the F-Q pair or; finally, (3) a combination of a plurality of ligands bonded together through linking groups to a hub molecule, usually a polymer, in combination with antibody bound to the other of the F-Q pair.

Abstract: An enzyme-polyidothyronine conjugate for use in determining polyiodothyronine by immunoassay is prepared containing an enzyme which is reversibly deactivated by polyiodothyronine and is reactivated by an antibody binding to the polyiodothyronine of the conjugate. Preferred enzymes are malate dehydrogenase and triose phosphate isomerase.

Abstract: Method and compositions are provided for determining the thyroxine binding capacity of serum, the value finding use in determinations of free thyroxine index. A competitive assay is performed where antibody for T-3 (anti-triiodothyronine) and serum thyroxine binding sites compete for T-3(triiodothyronine) in the assay medium: the T-3 is present as free T-3 added in a predetermined amount and T-3 conjugated to an enzyme. By combining the materials in a buffered aqueous medium with enzyme substrates, and determining the rate of reaction by the changing concentration of a light absorptive substance over a predetermined period of time, the thyroxine binding capacity of the serum can be determined.

Abstract: A novel sensitive method for determining qualitatively and quantitatively the presence of a wide variety of physiologically active organic compounds (ligand) and their receptors is provided. The method employs a reagent which involves bonding a compound having structural similarity to the compound to be determined (ligand analog) to a fluorescing compound. The unknown compound is referred to as a ligand, the conjugate of the structurally similar compound and fluorescer is referred to as ligand analog-fluorescer, and compounds which recognize a specific structure and bind to such structure are referred to as receptors and are normally antibodies.The fluorescer which is chosen will have either a change in quantum yield or a change in its emission and/or absorption spectra or all of them, when bound to antibody, as compared to being unbound. For the purposes of the assay, all that is required is that there be a change in the emission intensity at some wavelength or band of wavelengths.

Abstract: Method and compositions are provided for competitive protein binding assays wherein a non-enzymic catalytically mediated reaction is sterically inhibited by receptors binding to a ligand to which the reaction intermediary is bound. The method employs first and second reactants, which only react with each other either very slowly or not at all. The reaction intermediary or non-enzymic catalyst provides for a two step reaction having as its final result the transformation of the first and second reactants. By conjugating the reaction intermediary to a ligand, whereby the presence of receptor bound to the ligand inhibits the approach of one or both of the reactants, ligand and antiligand can be determined. Alternatively, anticatalyst can be employed which is inhibited from binding to the catalyst when antiligand is bound to ligand. The binding of anticatalyst to catalyst inhibits the catalytically mediated reaction.

Abstract: A novel sensitive method for determining qualitatively and quantitatively the presence of a wide variety of physiologically active organic compounds (ligand) and their receptors is provided. The method employs a reagent whch involves bonding a compound having structural similarity to the compound to be determined (ligand analog) to a fluorescing compound. The unknown compound is referred to as a ligand, the conjugate of the structurally similar compound and fluorescer is referred to as ligand analog-fluorescer, and compounds which recognize a specific structure and bind to such structure are referred to as receptors and are normally antibodies.The fluorescer which is chosen will have either a change in quantum yield or a change in its emission and/or absorption spectra or all of them, when the ligand analog-fluorescer is bound to receptor, as compared to being unbound.

Abstract: Theophylline derivatives are provided for preparing reagents for use in competitive protein binding assays and for use as reagents in competitive protein binding assays. Theophylline is substituted at the 3 position and conjugated to antigens for production of antibodies which specifically recognize theophylline as distinct from structurally similar analogs such as caffeine. Enzyme conjugates are provided which find use for measuring the amount of theophylline in a sample suspected of containing theophylline. A method is provided employing the reagents for the determination of theophylline.

Abstract: A method and apparatus for measuring a predetermined relatively small volume of liquid from a supply liquid and subsequently dispensing that volume of liquid. A long hollow tube having one end sealed is connected at the other end to an open-ended probe. The long tube is curved into at least one C-shaped segment and is generally rigid along its entire curved length. With the probe tip inserted into the supply liquid, the curved tube is flexed about an axis normal to the plane in which it lies, thereby decreasing the cross-sectional area of the tube. Since the tube is generally rigid along its entire curved length, this flexure of the tube increases the internal volume of the tube thereby reducing the pressure within the tube and withdrawing liquid from the supply liquid into the probe. The amount of liquid withdrawn into the probe is determined by the amount of flexure of the tube.

Abstract: A method is provided for the determination of receptors, particularly antibodies, where a polyepitopic reagent is employed, at least one of the epitopes being specific for the receptor of interest. The epitopic sites are situated on the reagent, so that the binding of the receptor of interest to the reagent will sterically hinder binding of receptors other than the receptor of interest.In carrying out the assay, the sample suspected of containing the receptor of interest, the reagent, and a predetermined amount of receptors for the other epitopes are combined. Because of the steric inhibition to the simultaneous binding of the two different receptors, the amount of receptor of interest bound to the reagent will affect the amount of the other receptor which becomes bound to the reagent.

Abstract: Non-oxo-carbonyl substituted derivatives of cocaine are provided for conjugation with other compounds to provide reagents for use in a diversity of immunoassay techniques. Specifically, carboxy or imidoyl derivatives are provided which may be conjugated with antigens for use in the preparation of antibodies to cocaine or conjugated with detector molecules for use in the qualitative or quantitative determination of the presence of cocaine.