Abstract: A method for detecting the presence of a high-molecular-weight analyte, which uses a variation on complementation assays, comprising (1) providing an enzyme donor (ED)complex comprising ED coupled to an analyte-specific binding molecule, wherein the ED complex retains measurable complementation activity such that active .beta.-galactosidase is formed in the presence of an enzyme acceptor (EA); (2) contacting the ED complex and EA in the presence of a sample suspected of containing a high-molecular-weight analyte that reacts specifically with the analyte-specific binding molecule; and (3) relating the presence of the analyte in the sample to the formation of active .beta.-galactosidase enzyme.

Abstract: Novel assays employing enzyme fragments which complex to form an active enzyme are provided. The reagents involved are a member of a specific binding pair bound to a solid surface, a first enzyme fragment conjugated to a member of a specific binding pair complementary or cross-reactive in relation to the analyte or antianalyte and a second enzyme fragment which binds to the conjugate to form an active enzyme, where the first enzyme fragment conjugate may be present in up to substantial excess to ensure at least substantially complete binding of all of the analyte. By distributing the conjugate between the solid surface and the medium in relation to the amount of analyte present, the enzyme activity may be determined in relation to the amount of analyte present, without separating the solid surface and the medium.

Abstract: Diagnostic assays are provided comprising complementary enzyme fragments of .beta.-galactosidase, where one of the fragments is bound to a support and the other fragment is conjugated to an immunologically cross-reactive epitope of the analyte or complementary to an analyte receptor. Binding to the receptor allows for discrimination between complexed enzyme fragment conjugate and uncomplexed enzyme fragment conjugate. The assay medium is then allowed to wick onto a support to which the complementary fragment is substantially uniformly bound in the detection region. The support is then developed, where color formation from the substrate in the detection region is used as a measure of the presence of analyte in the sample.

Abstract: A .beta.-galactosidase complementation assay for determining the presence of an analyte which is a member of a specific binding pair (sbp) in a sample is provided, which assay permits visual discrimination between those samples wherein the analyte is present above a predetermined, threshold concentration. The method comprises combining the sample with a hydrophobic enzyme donor- (ED-) analyte conjugate and the complementary member of the sbp to form an sbp complex-containing assay medium. A small volume of the assay medium is spotted onto an enzyme acceptor (EA) affixed to a bibulous, solid support, followed by development with enzyme substrate solution. A substantially larger area is detectable when analyte in the sample is below as compared to above a threshold concentration. The assay is particularly useful in field testing applications such as determining the presence of antibiotics in milk, toxins in water, or drugs in serum or urine. Kits facilitating the method are also provided.

Abstract: A method is provided for combining normally interacting reagents in a liquid single reagent and preventing complex formation using a surfactant and then reversing the inhibition by adding a cyclodextrin. The method finds particular use in diagnostic immunoassays. Reagents facilitating the invention are also provided.

Abstract: A method of stabilizing .beta.-galactosidase peptide fragments against storage degradation for use in a complementation assay which comprises storing the peptide fragment in a storage medium containing an ionic surfactant or a surfactant derived from a sugar residue and adding a cyclodextrin to the storage medium after storing the peptide fragments but before using them in a complementation assay.

Abstract: Serum samples for vitamin B.sub.12 analysis can be pretreated using a combination of peroxy acid and dithiothreitol. The pretreatment makes vitamin B.sub.12 bound to serum binding proteins available for analysis by any of a variety of currently available assay techniques. The pretreatment finds particular use in an assay using solid phase intrinsic factor as a specific binding protein.

Abstract: Novel fluorogenic peroxidase substrates are provided employing novel water soluble activated capped aromatic fluorescent compounds for use as peroxidase substrates. The novel compounds can be used in a wide variety of ways for determination of peroxides, peroxidase, and in assays for analytes employing peroxidase as a label on a member of a specific binding pair.

Abstract: Chloramphenicol derivatives are provided for use in the preparing of antigen conjugates for the production of antibodies specifically for chloramphenicol. Specifically, the aryl amino group is derivatized to introduce a non-oxo-carbonyl group which is used for amide formation with an antigen. The conjugate is then injected into a vertebrate for production of antisera which is isolated in conventional ways and find particular use in competitive protein binding assays.

Abstract: Novel compositions and methods are provided for detecting the presence of a material of interest in a specimen on a solid surface. A composition useful for detecting the presence of a material of interest in a specimen comprises (1) a detector conjugate comprising a fluorescing moiety bonded to a compound capable of specific binding with the material of interest, or a derivative thereof, and (2) a non-detector conjugate comprising a poly(amino acid) and a compound having substantial structural and charge similarity to the fluorescing compound and no observable fluorescence, or low level fluorescence of a different wavelength than that of the fluoresing compound, in the region of fluorescence of the fluorescing compound. In the method of the invention, a specimen, on a solid surface, is combined with the detector conjugate and the non-detector conjugate, and the combination is incubated.

Abstract: Methods are provided for reducing non-specific interference in competitive protein binding assays employing as the labeled reagent a fluorescent conjugate of a hydrophobic ligand conjugated to a fluorescer, which in turn is bound to a water soluble polysaccharide carrier ("fluorescer conjugate reagent"). In order to reduce non-specific interference from physiologic samples, the fluorescent reagent is combined with a lipid substituted neutral support, under conditions which provides two binding fractions: a first weakly binding fraction which is relatively free of non-specific interference in a competitive protein binding assay employing physiological fluids; and a second fraction, which more strongly binds to the lipid substituted support.

Abstract: Chloramphenicol derivatives are provided for use in the preparing of antigen conjugates for the production of antibodies specifically for chloramphenicol. Specifically, the aryl amino group is derivatized to introduce a non-oxocarbonyl group which is used for amide formation with an antigen. The conjugate is then injected into a vertebrate for production of antisera which is isolated in conventional ways and finds particular use in competitive protein binding assays.

Abstract: Novel polyhalothyronines or immunogenic mimetic analogs are provided conjugated to a fluorescer compound with the resulting conjugate being highly fluorescent. Specifically, the compounds have at least one of the following characteristics: bromine(s) at the 3',5'-positions of the thyronine or mimetic analog; or a rigid linking group between the thyronine and the fluorescer, so as to inhibit heavy atom catalyzed decay of fluorescence. The subject conjugates find a wide variety of uses in detecting for thyroid hormones as well as proteins which specifically bind to thyroid hormones.

Abstract: Novel carboxyalkylated disopyramides (NORPACE.RTM.) are provided as precursors for conjugating proteins, either antigenic for the preparation of antibodies or enzymatic for the preparation of enzyme conjugates, which antibodies and enzyme conjugates find use as reagents in immunoassays. The combination of antibodies and enzyme conjugates provide for sensitive, accurate, rapid assays for disopyramide without interference from closely analogous compounds, such as methadone or disopyramides metabolites.