Abstract: Method and compositions are provided for performing homogeneous immunoassays. The method involves having a signal producing system, which provides a detectable signal, which system includes a macromolecular member. The determination of the analyte, which is a member of a specific binding pair consisting of a ligand and its homologous receptor, is performed by creating an extensive matrix in the assay medium by having in the assay medium in addition to the analyte, ligand labeled with one of the members of the signal producing system, antiligand either present as the analyte or added, a polyvalent receptor for antiligand, the macromolecular member of the signal producing system, and any additional members of the signal producing system. The labeled ligand, antiligand, and polyvalent receptor for the antiligand create a matrix which modulates, e.g. inhibits, the approach of the macromolecular member of the signal producing system to the labeled ligand.

Abstract: A flow cell for optical analysis of a fluid sample has a cell chamber whose volume is not limited by considerations of fluid residue carryover. A piston movable within the viewing region and having a conduit through its face is used to collapse the volume of the viewing region to substantially zero while maintaining a flow path between inlet and outlet conduits so that residue can be removed from the flow cell and its conduits with a minimum of cleaning fluid.

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: Imipramine functionalized compounds are provided for conjugation to antigenic compounds, 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 imipramine, while the enzyme conjugate finds use in a homogeneous enzyme assay for the determination of imipramine.

Abstract: Method and compositions are provided for performing protein binding assays involving a homologous pair consisting of ligand and receptor for the ligand. The method employs a label conjugated to a member of said homologous pair and a uniformly dispersed discontinuous phase of discrete particles in a continuous aqueous phase, where the discrete particles create microenvironments which allow for discrimination between the label associated with the particle--in a discontinuous phase--and the label in the continuous phase.Various conjugates and particles are provided which find use in the subject method.

Abstract: A method is provided for preparing immunoassay reagents involving labeled members of specific binding pairs substantially enriched relative to contaminating labeled materials. The method involves conjugating a member of a specific binding pair to a support by a covalent bond which is cleavable under mild conditions to provide a binding pair member-support conjugate. Combining the binding pair member-support conjugate with a labeled composition containing the reciprocal member of the binding pair, so that the labeled reciprocal member becomes bound to the support through the binding of the specific binding pair. Separating the support to which is bound the labeled member from the remaining labeled material and then cleaving the bond joining the labeled specific binding pair to the support to provide labeled reagent for immunoassays.

Abstract: Macromolecular compositions are provided which find use as glycosidase substrates, particularly where steric modulation of enzyme activity is involved. The compositions have a macromolecular hub to which is linked by means of a spacer arm a chromophore, which in turn is bonded to a sugar by an enzymatically labile glycosidyl linkage. The chromophore is chosen so as to undergo a substantial change in its chromophoric properties upon cleavage of the glycosidyl linkage. The enzyme activity can be determined spectrophotometrically due to the change in the absorptive or fluorescent properties of the chromophore upon cleavage of the glycosidyl linkage. Varying turnover rates for the enzyme are observed depending upon the nature of the macromolecular hub, the spacer arm, and the chromophore.

Abstract: Immunoassays are provided employing antibodies and 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: 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: Novel immunoassays are provided employing discrete particulate reagents for determining an analyte which is a member of a specific binding pair-ligand and homologous receptor. The assay employs as a first reagent, a member of said pair bound to an insoluble particle (particle conjugate); as a second reagent, a label which is part of a signal producing system, bound to a member of said pair (signal label conjugate); and as a third reagent, a signal repressor comprising an insoluble particle, where the signal repressor is obstructed from interacting with said label of said signal label conjugate, when said signal label conjugate is bound to said particle conjugate.In performing the assay, the analyte, the reagents, and any ancillary materials are combined in an aqueous assay medium and the signal determined as compared to an assay medium having a known amount of analyte.

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 as referred to as receptors and are normally antibodies.The fluorescer which is chosen will have either a change is 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: Background fluorescence which interferes with the measurement of an analyte in fluorescent immunoassays in serum samples is substantially diminished by treating the serum sample with a sufficient amount of a peroxidic compound, either organic or inorganic, for a time sufficient to reduce the background fluorescence contributed by serum components. The peroxidic compound is readily quenched, without adversely affecting the assay composition.

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: Compounds are provided which can be used in the preparation of reagents useful for quantitatively determining the presence of .beta.-adrenergic blocking agents. These compounds contain a derivative of the blocking agent which is linked to an antigen or enzyme, the antigen conjugates being useful for preparing antibodies, and the enzyme conjugates being useful as detectors in an immunoassay as well. Additionally carboxylic acids, esters and aldehydes which are derivatives of the blocking agents are provided which are useful in preparing the above conjugates.

Abstract: .alpha.-substituted derivatives of valproic acid 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 valproate, while the enzyme conjugate finds use in a homogeneous enzyme immunoassay for the determination of valproate. The compounds are synthesized by alkylating valproate at the tertiary carbon atom by an aliphatic chain with a terminal double bond which is cleaved to provide an acid or aldehyde group.

Abstract: Methods and compositions are provided for improved protein binding assays by preparing compositions having indirectly labeled ligands substantially free of label conjugated to materials other than the indirectly labeled ligand. The method for preparing the compositions involves employing a monovalent receptor to which the label is conjugated and combining the monovalent receptor labeled conjugate to the ligand, either in pure or impure form. The mixture is then segregated according to molecular weight and the ligand conjugated to the labeled receptor isolated. This conjugate may then be directly used as a reagent in a protein binding assay, where the assay mixture is substantially free of label other than labeled receptor bound to ligand.The labels will be for the most part of relatively low molecular weight, while the receptor is preferably a Fab fragment.

Abstract: Compounds are provided for use in the preparation of reagents which can be used in immunoassays for the determination of benzamides of N,N-dialkylethyleneamines. A linking group is provided, at a particular site of the drug, which provides a link between the above compounds 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: Method and compositions are provided for chemical analysis of an analyte which is a member of a specific binding pair of organic substances consisting of ligand and ligand receptor (antiligand). The method involves bringing together the following reagents with the analyte in an aqueous assay medium under mild conditions.The first reagent is a conjugate of a member of the specific binding pair with a chemical entity which provides a means for chemically changing the concentration of a compound which acts as a signal mediator. The second reagent is the signal mediator precursor. The third reagent is a conjugate of a member of the specific binding pair with a component of a signal producing system of which system the signal mediator is a member.The amount of signal which can be detected is affected by the local concentration of the signal mediator.

Abstract: Methods and reagent combinations are provided for competitive protein binding assays for determining a member of an immunological pair (ligand and receptor) whereby an enzyme-ligand conjugate is employed in combination with an enzyme inhibitor, conveniently an antibody to said enzyme. When ligand is the analyte, receptor for ligand is also included in the assay medium, while supplemental amounts of receptor need not be added when receptor is the analyte. The assay is carried out in an aqueous buffered medium, normally at constant temperature, by combining in the assay medium the unknown sample suspected of containing the analyte, enzyme-bound-ligand, ligand receptor (antiligand), enzyme inhibitor (antienzyme), and enzyme substrates, and the enzymatic activity in the assay medium determined. By comparing the observed enzymatic activity with an unknown to the enzymatic activity observed in an assay medium with a known amount of analyte, the amount of analyte can be quantitatively determined.

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.