Abstract: Composition and method are provided for the pretreatment of serum prior to a thyroxine assay. Prior to carrying out of the assay, particularly an immunoassay or competitive protein-binding assay, the serum sample is combined with cyclodextrin preferably in combination with an hydroxylated aromatic carboxylic acid in a basic medium, in amounts sufficient to minimize interference with obtaining an accurate and reliable determination of polyiodothyronine in the serum. Normally, after the reagents have been added to the serum, a small incubation period is allowed, after which time the additional reagents may be added for carrying out the assay.

Abstract: Derivatives of anesthetics involving anilides, lidocaine being illustrative, are provided, having an annular amino group. A difunctional linking group is provided, which provides a link to the annular amino group and an antigen, with the resulting conjugate being employed for the preparation of antibodies. The antibodies find particular use in competitive protein binding assays. Conjugates to enzymes are prepared which find particular use in homogeneous enzyme immunoassays.

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: Novel conjugated enzyme compositions are provided for use in homogeneous immunoassays. A wide variety of haptenic compounds, such as drugs of abuse, therapeutic drugs, naturally occurring hormones, and the like, are conjugated to the enzyme lysozyme for use as reagents in homogeneous enzyme immunoassays. From 1 to 5 haptens are conjugated to the enzyme, and the enzyme is substantially inhibited when bound to antibodies bound to the conjugated hapten.

Abstract: Derivatives of dibenz[b,f]azepine drugs are provided for preparation of antigens and antibodies, the antibodies finding use in immunoassays for the dibenz[b,f]azepine drugs. Specifically, N-oxoaliphatic substituted carbamoyl dibenz[b,f]azepine compounds are provided which are conjugated with antigenic materials and injected into animals for production of antibodies specific for the azepine drug.

Abstract: Immunoassays for digoxin and its congeners are provided by pre-treating the serum sample with dilute aqueous alkaline solutions under mild conditions and for short periods of time, preferably in combination with a chelating agent, prior to carrying out the immunoassay. The method finds particular application with radioimmunoassays and homogeneous enzyme immunoassays.

Abstract: Novel compositions are provided for the determination of one or a group of organic materials (hereinafter referred to as "ligands"), where the compositions have a ligand or ligand counterfeit bonded to an enzyme, the conjugate referred to as "enzyme-bound-ligand." Specifically, the ligands are benzdiazocycloheptane drugs, which are conjugated to an enzyme, so that upon binding of a receptor, usually an antibody, the activity of the enzyme changes. Determinations of the benzdiazocycloheptane drugs in a physiological fluid are performed by combining the enzyme conjugate, the physiological fluid and receptor under conditions whereby the amount of receptor bound to the conjugate is related to the amount of the benzdiazocycloheptane drug present in the sample. By metering the enzyme activity of the assay mixture and comparing the result to known standards, the amount of benzdiazocycloheptane drug present in the sample may be determined.

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.

Abstract: Compounds are provided for use in the preparation of reagents which can be used in immunoassays for benzdiazepines, particularly as their urinary metabolites. The compounds are non-oxo-carbonyl derivatives conjugated to the 3-position of the benzdiazepine, which in turn are conjugated to antigenic polypeptides for formation of antibodies for use in immunoassays. The antibodies produced, when employed in immunoassays, are found to be able to detect the ingestion of a wide variety of benzdiazepine drugs as determined by positive results with the urine of the patient.

Abstract: Method and enzyme conjugates are provided for determining physiological amounts of polyiodothyronine in physiological fluid, particularly serum.A homogeneous enzyme immunoassay is provided employing an enzyme-bound-polyiodothyronine having less than about 50 percent of the original enzyme activity, whose activity increases upon binding with a receptor. The enzyme-bound-polyiodothyronine, the unknown suspected to containing a polyiodothyronine, and receptor for the polyiodothyronine are combined in an aqueous medium containing substrate for said enzyme and the enzymatic activity of the solution determined. By comparing the result obtained with results obtained from standards having known amounts of polyiodothyronine, the amount of polyiodothyroninein the unknown can be determined.The enzyme-bound-polyiodothyronine employs an enzyme which is reversibly deactivated by polyiodothyronine, so as to be reactived by receptor binding to the polyiodothyronine.

Abstract: Polyiodothyronine conjugates to enzymes are provided which find use in the determination of polyiodothyronine compounds, particularly thyroxine, normally in physiological fluids, such as serum. The enzymes can provide substantial variation in activity when bound to protein receptors for polyiodothyronine as compared to the free or unbound polyiodothyronine conjugated to the enzyme. Various linking groups are provided for linking the polyiodothyronine to the enzyme.

Abstract: Enzyme conjugates are prepared of cardiac glycosides and aglycones for use in homogeneous enzyme immunoassays. The conjugates retain a substantial degree of the original activity of the enzyme and upon binding of receptor to the steroid moiety, a substantial diminution of enzyme activity is obtained. Various linking groups are employed for linking the steroid portion of the molecule to the enzyme, such as non-oxo carbonyl groups.

Abstract: Novel four-membered ring heterocycles are provided having two nitrogen atoms in the ring bonded to each other and being bonded to exocyclic oxygen atoms. Both reduced and oxidized forms of the diazacyclobutane are provided. Depending on the particular compounds, they find use as triplet quenchers, and as antioxidants in light susceptible systems.

Abstract: Novel compounds are provided which are derivatives of 1,2-diphenyl-3-methyl-4-dimethylamino-2-butanol. The indicated alcohol is employed to prepare the monoester of a dibasic acid which is conjugated with an antigen for the production of antibodies which recognize both propoxyphene and its N-desmethyl metabolite in immunoassays.

Abstract: A sensitive method for determining qualitatively and quantitatively the presence of a wide variety of physiologically active organic compounds (ligand) 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 emisson 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 emisson intensity at some wavelength or band of wavelengths.

Abstract: Immunoassays 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: Novel biological assay method for determining the presence enzyme a specific organic material by employing a modified enayme 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: Compounds are provided for use in assays of organic compounds, where organic compounds of biological interest are determined at extremely low concentrations by combining in a medium, the composition to be determined, hereinafter referred to as ligand, a high molecular weight material of at least 10,000 molecular weight, which has a site spatially characteristic of the ligand, hereinafter referred to as receptor, and an analog of the ligand having a free radical functionality, hereinafter referred to as "ligand analog". The ligand analog and ligand in the medium compete for the receptor site, the amount of ligand analog bound to the receptor, being dependent on the amount of ligand present in the medium. By following the change in electron spin resonance spectrum of the ligand analog and comparing it to the change in spectrum which would be obtained in the absence of any ligand, the amount of ligand can be determined.

Abstract: Compounds are provided for use in assays of organic compounds, where organic compounds of biological interest are determined at extremely low concentrations by combining in a medium, the composition to be determined, hereinafter referred to as ligand, a high molecular weight material of at least 10,000 molecular weight, which has a site spatially characteristic of the ligand, hereinafter referred to as receptor, and an analog of the ligand having a free radical functionality, hereinafter referred to as "ligand analog". The ligand analog and ligand in the medium compete for the receptor site, the amount of ligand analog bound to the receptor, being dependent on the amount of ligand present in the medium. By following the change in electron spin resonance spectrum of the ligand analog and comparing it to the change in spectrum which would be obtained in the absence of any ligand, the amount of ligand can be determined.

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 material (hereinafter referred to as "ligands") and binding an enzyme to the ligand or ligand counterfeit to provide and "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.