Abstract: A method is disclosed for the detection of a His-tagged target biomolecule while said molecule remains in the gel in which it has been separated from other constituents by electrophoresis. The method involves, while the separated His-tagged biomolecule remains in the gel, forming a chelate between the His-tagged molecule, a lanthanide metal ion which exhibits fluorescence, and a sensitizer which enhances the fluorescence of the ion. The gel is then exposed to a u.v. light source to thereby generate a fluorogenic signal indicating the presence of the chelate and, in turn, the His-tagged biomolecule. The fluorogenic signal can then be detected either by direct visualization and/or by other means, such as photographically, e.g., film or charged coupled device (CCD) imaging.

Abstract: A composition for assaying of peroxidase activity:

Abstract: A method is disclosed for preparing a fluorogenic phenolic compound with improved optical qualities for use in formulating a substrate solution for assay of peroxidase or peroxide activity. The method involves forming a solution under anoxic conditions which contains the phenolic compound and an aminopolycarboxylic acid or aminopolyphosphonic acid, or salt thereof, metal chelating agent and, while the solution is maintained under anoxic conditions, recovering the compound from the solution in an optically enhanced condition. A composition of matter is also disclosed which includes the fluorogenic phenolic compound in crystal form and a trace quantity of the metal chelating agent.

Abstract: Chemiluminescent detection of molecules of synthetic or natural origin such as proteins and nucleic acids (DNA and RNA), as well as other biologic molecules, is increasingly replacing radioactive detection as the method of choice where sensitivity is critical. In such assays, luminescence is customarily achieved by the oxidation of a luminol or isoluminol substrate in the presence of an oxidizing agent such as hydrogen peroxide or hydrogen peroxide source, such as perborate, and a peroxidase catalyst such as horseradish peroxidase. To obtain useful levels of luminescence (e.g., detectable levels) by customary techniques, a luminescent enhancer is also present during oxidation. It has been found in the practice of the present invention that azine enhancers have contained an impurity which reduces the properties of the chemiluminescent assay working solutions.

Abstract: The salt of a sulfonated succinic acid is cyclized, then converted to novel sulfonated hydroxamic acids by reaction with hydroxylamine (which is added or formed in situ), and the novel hydroxamic acid is then cyclized to the sulfo-N-hydroxysuccinimide salt. This synthetic procedure is simple, direct, and more rapid than present procedures for synthesis of the succinimide. Novel sulfo-hydroxamic acid intermediates are formed during this procedure.

Abstract: An improved method is disclosed for the lysis of host cells and extraction of proteins of interest therefrom. The method involves, subsequent to expressing the protein in the host cell by a recombinant DNA procedure, using a reagent solution containing an alkylglycoside or an alkylthioglycoside to lyse the cell to release the protein, and to extract the protein of interest from other host cellular components.

Abstract: An improved method is disclosed for assaying peroxidase or peroxide activity utilizing a substrate solution containing a fluorogenic phenolic compound, hydrogen peroxide and a metal chelating compound. The improvement inclosed including within the assay, in an amount sufficient to enhance the effective working range of the assay, a boron acid or salt thereof, or a phosphine-based or hydride-based reducing agent.

Abstract: In accordance with the present invention, there is provided an improved device for the dialysis of small fluid samples, particularly biological fluids. The device of the present invention contains a cylindrical outer sleeve open at both ends. The sidewall portion of the sleeve near the top end contains a channel in and around the inner surface and the sidewall continues to extend upwardly from the channel to the open top end. An inner sleeve is positioned within and in axial alignment with the outer sleeve. The inner sleeve has a cylindrical flange at an open top end that is inserted in the channel of the outer sleeve. The bottom end of the inner sleeve is open and extends below the bottom end of the outer sleeve. A space exists between the sidewalls of the two sleeves near the bottom end of the outer sleeve. A dialysis membrane covers the open bottom end of the inner sleeve.

Abstract: The salt of a sulfonated succinic acid is cyclized, then converted to novel sulfonated hydroxamic acids by reaction with hydroxylamine (which is added or formed in situ), and the novel hydroxamic acid is then cyclized to the sulfo-N-hydroxysuccinimide salt. This synthetic procedure is simple, direct, and more rapid than present procedures for synthesis of the succinimide. Novel sulfo-hydroxamic acid intermediates are formed during this procedure.

Abstract: The salt of a sulfonated succinic acid is cyclized, then converted to novel sulfonated hydroxamic acids by reaction with hydroxylamine (which is added or formed in situ), and the novel hydroxamic acid is then cyclized to the sulfo-N-hydroxysuccinimide salt. This synthetic procedure is simple, direct, and more rapid than present procedures for synthesis of the succinimide. Novel sulfo-hydroxamic acid intermediates are formed during this procedure.

Abstract: A process is described for rapid preparation of pure sulfo-N-hydroxysuccinimides. The process may comprise the steps of:a) esterifying a sulfo-succinic acid to form a diester first product, andb) reacting said diester first product with hydroxylamine to form a sulfo-N-hydroxy succinimide.

Abstract: A rapid radioactive method of measuring enzymatic activity of a protein kinase is disclosed. The method is an improvement to existing methodology which involves phosphorylating a peptide substrate using .sup.32 P-ATP, adsorbing the phosphorylated peptide to a solid phase, washing the phase to remove non-adsorbed .sup.32 P-ATP, and measuring the radioactivity of the phosphorylated peptide adsorbed to the phase. The disclosed improvement uses a membrane as the solid phase and positions the membrane within a chamber to separate the chamber into a first and second region. Washing is accomplished with centrifugal force; the washed solution being forced through the membrane from the first region into the second region.

Abstract: Trifunctional cross-linking compounds are disclosed. The compounds contain the biotin moiety, the NHS active ester, and a photoactivatable aryl azide. The presence of a disulfide bond in association with either the ester or azide permits the compound to be cleaved.

Abstract: A method of measuring enzymatic activity of a protein kinase is disclosed. The method is an improvement to existing methodology which involves phosphorylating a peptide substrate, adsorbing the phosphorylated peptide to a solid phase, washing the phase to remove non-adsorbed constituents, and measuring the amount of phosphorylated peptide adsorbed to the phase. The disclosed improvement uses a membrane as the solid phase and positions the membrane within a chamber to separate the chamber into a first and a second region. Washing is accomplished with centrifugal force; the washed solution being forced through the membrane from the first region into the second region. Radioactive and non-radioactive assays are disclosed. The latter uses a support containing the Fe.sup.+3 ion chelated to the support through imminodiacetic acid groups. The peptide contains a dye and measurement is spectrophotometric or fluorometric.

Abstract: A device for the dialysis of a sample. The device embodies a hermetically sealed sample chamber formed by a gasket with dialysis membranes affixed to each side in substantially parallel relationship. The gasket is impermeable to the sample being dialyzed, but is penetrable and reusable such that a needle or the like can be inserted through the gasket into the chamber and then withdrawn without sample being permitted to leak. The gasket is of sufficient thickness to accommodate insertion of a needle. In a further embodiment, the device is fitted into a rigid housing containing windows exposing the dialysis membranes and further containing a channel parallel to the dialysis membranes for directing a needle into the gasket in a direction substantially perpendicular to the edge of the gasket so that the needle can access the chamber without contacting the surfaces of the membranes.

Abstract: A process is described for isolating an enzyme-antibody conjugate, wherein the enzyme is horseradish peroxidase or alkaline phosphatase, from an aqueous mixture of said conjugate and unconjugated enzyme. The process involves contacting the mixture with a water insoluble stationary phase having the Ni.sup.+2 ion chelated thereto and binding said conjugate to the stationary phase. The phase containing bound conjugate is then washed to remove unbound enzyme. Thereafter the conjugate is eluted from the stationary phase and recovered in a form substantially free of the unconjugated enzyme.

Abstract: An apparatus for use in a ligand-receptor assay procedure for the quantitative determination of the concentration of a target ligand in a liquid sample. The apparatus contains top and middle plates having holes therethrough. The holes in the two plates are in axial alignment and the holes in the middle plate have sidewalls that extend below the bottom surface of the middle plate. The apparatus contains a bottom chamber having an opening on the upper side thereof facing the bottom surface of said middle plate. The chamber contains at least one port extending through a surface thereof to permit a vacuum to be created within said chamber and means for accepting a microtiter plate containing a plurality of wells. When the microtiter plate is positioned within the chamber, the ends of the holes extending beneath the bottom surface of the middle plate are located within the wells.

Abstract: A conjugate of a protein carrier and an antigen is disclosed. The carrier protein is cationized and the conjugate has enhanced immunogenic properties over those of the antigen alone. Cationization can be accomplished by derivatization of native carboxyl groups on the protein with an aklyl diamine, e.g. ethylene diamine, resulting in the formation of side chain aminoalkylamide groups, e.g. aminoethylamide.

Abstract: Preparation of a composite of mannan binding protein attached to an insoluble, support matrix is accomplished by reacting cyanogen bromide activated beaded agarose with a buffered solution of mannan binding protein isolated from rabbit serum. The composite has utility as an affinity sorbent for IgM when divalent metal ions are incorporated in the binding buffer. The composite does not show cross-reactivity (binding) with immunoglobulins of the G class.

Abstract: Cu.sup.+ produced during the reaction of protein with alkaline Cu.sup.++ can be monitored by measuring the absorbance at 562 nm of the intense purple complex formed with the ion of bicinchoninic acid (BCA). The color produced is stable and increases in a linear fashion over a broad working range of increasing protein concentration. Since BCA is stable, it is incorporated in the reagent formulation at the start of the reaction. Thus, the method offers mechanical simplification over the method described by Lowry et al.