Coulter Corporation

Coulter International Corp.

Beckman Coulter, Inc.

Olavi Siiman

Alexander Burshteyn

John A Maples

Ravinder K Gupta

Norma Sue Kenyon

Wade E Bolton

Cynthia G Healy

Egon Matijevic

Melissa J Stiglitz

Stephen F Healy, Jr

Robert H Raynor

Julie Wilkinson

Cecilia Smith

James Keller Whitesell

Ivan Sondi

Patricia Roth

Ravindra Mylvaganam

Carlos M Rodriguez

Kristie M Gordon

Robert J Schmittling

Meryl Forman

Marilyn Cayer

Orlando Concepcion

Stephen L Ledis

A composition containing polydisperse aminodextran polymer molecules is soluble in an aqueous solution at a concentration of 10 mg/ml. The molecules therein have a narrow size distribution characterized by an average molecule mean hydrodynamic diameter of greater than 115 nm, a polydispersity index of between 0.10 and 0.47, an average molecular weight (MW) greater than 3 million daltons, and an amine content of greater than 50 amines per molecule. Similar soluble compositions contain the polymer molecules with an average MW of greater than 7 million daltons. These compositions are useful in forming reagents by conjugation with proteins for labeling cells. Methods of making these compositions and reagents from conventional mixtures of aminodextran polymers involve fractionation on column chromatography.

Aminodextran compositions and conjugates and method of making and using them

A method of analyzing the concentration of soluble analyte in a sample involves performing a competition assay using a predetermined amount of formed bodies to which are attached at least one analyte, varying known concentrations of an unlabeled ligand that binds to analyte, and a known concentration of ligand labeled with a detectable marker. After analysis in a flow cytometer, the test sample and a plurality of control samples generate data on curves formed by plotting signal vs. concentration of labeled ligand in controls (first), and vs. concentration of total labeled and unlabeled ligand in test samples (second). The concentration of unlabeled ligand that bound to soluble analytes in test samples is determined by evaluating the difference between the concentration that corresponds with the intersection of these two curves and the constant labeled ligand concentration in the test samples. The concentration of said soluble analytes in said sample is the product of that difference and the binding stoichiometry of the ligand to soluble analytes.

Method for the analysis of soluble analytes

A semiconductor nanoparticle for use in analysis of biological samples is described. This semiconductor nanoparticle is composed of an aminodextran which is bound to at least one nanoparticle of the formula (X Y) wherein X is selected from the group comprising Cd , Hg , and Zn and combinations thereof, and Y is selected from the group comprising S , Se and Te and combinations thereof; and n&equals;approximately 50 to 1000. Also provided are methods of making these semiconductor nanoparticles and methods of making conjugates composed of these semiconductor nanoparticles linked to ligands. Also described are uses for the conjugates in a variety of biological assays.

Semiconductor nanoparticles for analysis of blood cell populations and methods of making same

Ligand-aminodextran-(phycobiliprotein or tandem dye) conjugates useful for detection of a desired target biological material by providing an enhanced fluorescent signal are described. Also described is a method for a single-measurement quantification of multiple populations of cells based upon the labeling of different pairs of cell populations, each pair containing mutually exclusive cell receptors which are expressed at substantially similar receptor densities with labeled ligands for each receptor. One cell population is labeled with a ligand capable of binding to a first cell surface receptor which ligand is directly conjugated to a fluorescent phycobiliprotein or tandem dye; and a second cell population is labeled with a ligand capable of binding to a second cell surface receptor, which ligand is cross-linked to an aminodextran to a fluorescent phycobiliprotein or tandem dye.

Ligand-aminodextran-marker conjugates and uses thereof

A semiconductor nanoparticle for use in analysis of biological samples is described. This semiconductor nanoparticle is composed of an aminodextran which is bound to at least one nanoparticle of the formula (X Y),wherein X is selected from the group comprising Cd,, Hg,, and Zn,and combinations thereof; and Y is selected from the group comprising S,, Se,and Te,and combinations thereof; and n&equals;approximately 50 to 1000. Also provided are methods of making these semiconductor nanoparticles and methods of making conjugates composed of these semiconductor nanoparticles linked to ligands. Also described are uses for the conjugates in a variety of biological assays.

A stable protein-coated nickel particle useful in biological assays contains a nickel particle having removed from the surface thereof nickel oxide; a linker attached to said nickel particle, the linker having a free amino group; and a protein attached to said linker by covalently bonding to the free amino group. Methods of producing and using these oxide-free nickel-protein conjugates are disclosed.

Stable protein-nickel particles and methods of production and use thereof

A method for a single-measurement quantification of multiple populations of white blood cells (WBC) is based upon the labeling of different pairs of cell populations, each pair containing mutually exclusive cell receptors which are expressed at substantially similar receptor densities with labeled ligands for each receptor. One cell population is labeled with a ligand capable of binding to a first cell surface receptor which ligand is directly conjugated to a fluorescent phycobiliprotein or tandem dye; and a second cell population is labeled with a ligand capable of binding to a second cell surface receptor, which ligand is cross-linked by an aminodextran to a fluorescent phycobiliprotein or tandem dye. The phycobiliproteins upon laser excitation produce a different detectable fluorescence intensity for each cell population. Use of such pairs of conjugates enable two populations of cells with similar receptor densities to be distinguished with the use of a single color marker. Further use of additional ligands to other cell surface receptors and additional phycobiliprotein or tandem dye, or other markers in the same manner, enables the simultaneous quantification of up to 45 different cell populations with one laser line and four fluorescent colors.

Simultaneous analyses of white blood cell subsets using multi-color,

A stable colloidal particle comprises a colloidal-sized core substrate having amine-reactive functional groups thereon with an aminodextran coating over its peripheral surface and a layer of colloidal-sized metallic solid overlaying the aminodextran coating. A linker comprising aminotrithiolate attached to the metallic solid has a free amino group to which a protein is attached by covalent bonding to the free amino group. Such novel particles are useful in flow cytometry, particularly useful in the simultaneous analyses of subpopulations of leukocytes. Methods of preparation of such particles, as well as methods of use are provided. These conjugates enable the simultaneous analyses of at least two different mutually exclusive subsets of white blood cells, based on the binding affinity of the conjugated protein.

Protein-colloidal metal-aminodextran coated particle and methods of

The present application discloses dextran crosslinked, antibody-phycobiliprotein conjugates containing up to twenty phycobiliprotein per dextran molecule which conjugates are able to achieve amplification of fluorescence intensity over direct antibody-phycobiliprotein labeled cells of two fold or greater.

Antibody-aminodextran-phycobiliprotein conjugates

This invention relates generally to using fluorescent markers (labelled antibodies) and flow cytometry to enumerate the average number of receptors (antigen) on formed bodies (cells) in whole blood, and to evaluate the specific binding constant of the marker for the particular receptors. Mean channel fluorescence intensities of equilibrated marker-cell suspension mixtures, total concentrations of marker, and targeted cell counts obtained by established procedures are used to complete the analyses. The invention further describes a competitive binding assay between marker and unlabelled antibody for receptors shed from the surface of formed bodies into solution and receptors on the formed bodies to analyze for receptors in solution. Moreover, competitive binding assays between marker and unlabelled antibody for receptors on formed bodies in whole blood are described for determination of relative, specific, and average binding constants of unlabelled/labelled antibody for targeted receptors.

Methods of enumerating receptor molecules for specific binding partners

The invention provides a method for enriching a sample in tumor cells and determining the DNA ploidy and proliferative index of the tumor cells present in the sample. The method uses a pan-leukocyte monoclonal antibody conjugated to a separable substrate such as magnetic microspheres to conjugate and remove leukocytes from the sample. A fluorescently labelled monoclonal antibody specific to a tumor associated antigen or an antigen arising from the presence of the tumor and a DNA staining reagent are added to the leukocyte depleted sample. The sample is then analyzed by dual color flow cytometry to determine cells having an abnormal DNA content by gating on fluorescently labelled cells.

Method of analyzing tumor cell DNA content through tumor cell enrichment

Gelatin and aminodextran coated polymer core particles useful in immunoassays and methods of making the same are disclosed. The preparation of aminodextrans having varying amounts of amine groups is also described, as is a method of crosslinking gelatin and aminodextran without the use of a stabilizer.

Polymeric particles having a biodegradable gelatin or aminodextran

Preparation of non-infectious control cells from normal, non-infectious, non-disease altered blood by depletion or augmentation of one or more cell types found in normal blood to reflect a specific disease state. The control cells so produced are preserved and thereafter reconstituted for use in immunological assays.

Preparation of preserved, non-infectious control cell for use in the

The invention relates generally to colloidal particle having a core material and a gelatin/aminodextran coating with pendent functional groups attached thereto. Biological substances or molecules, especially monoclonal antibodies, may be attached to said particles. The monoclonal antibody containing particles are useful in a variety of positive and negative biological assays.

Biodegradable gelatin-aminodextran particle coatings of and processes

The invention describes the use of novel aminodextran compounds containing about 5-20% by weight amine groups to bind a plurality of monoclonal antibodies. The resulting antibody-aminodextran compounds may be used to induce the activation and proliferation of selected mammalian cells. Specific examples are given using an anti-CD3 monoclonal antibody conjugated two aminodextrans containing about 5% and 16%, respectively, by weight amine groups as an agent for inducing the activation and proliferation of T cells.

Growing community of inventors

Davie, FL, United States of America

Olavi Siiman