Abstract: Methods, reagents, and kits are provided that permit flow cytometric determination of the phosphorylation status of retinoblastoma susceptibility gene protein (pRB) in individual cells. Methods are described that permit the hypophosphorylated, active, form of pRB to be measured either as an absolute quantity or as a proportion of total cellular pRB. Further described are methods that permit pRB phosphorylation status to be correlated with cell cycle phase and with protein components of the cell cycle. Screening of chemical compounds for antiproliferative and antineoplastic activity using the flow cytometric assays is demonstrated. Reagent kits that facilitate the subject methods are also provided.

Abstract: Improved methods, reagents, and kits for quantitation of HLA-DR and/or CD11b expression on peripheral blood cells are presented. Inclusion of a lysosomotropic amine, such as chloroquine, during staining stabilizes HLA-DR and CD11b expression. Use of a novel anti-CD14 conjugate, anti-CD14-PerCP/CY5.5, permits the ready discrimination of monocytes. The improved methods, reagents, and kits may be used to assess immune competence, and to direct and monitor immunostimulatory therapies in septic patients exhibiting monocyte deactivation.

Abstract: The present invention provides instruments for analyzing a multiplicity of fluorescent dyes using a multiplicity of amplifying photodetectors, methods for using the instruments, methods for setting the instrument parameters, and methods for resetting the instrument parameters following a changed in photodetector amplification. The present invention is particularly applicable in the field of flow cytometry.

Abstract: A sort block for use with sorting flow analysis system. The sort block includes a fin extending into a sort stream in the sort block. Unsorted particles and aerosol are aspirated into the open top of the fin. When error is detected, collection baskets associated with the fin extend into the sort stream, block passage of particles to the collection containers and aspirate all droplets and aerosol to a waste or retrieval container. This sort block may include error monitors, such as a laser and camera combination, which monitor the flow stream and control the extension of the fin and basket type collector.

Abstract: Methods, reagents, and kits are provided that permit flow cytometric determination of the phosphorylation status of retinoblastoma susceptibility gene protein (pRB) in individual cells. Methods are described that permit the hypophosphorylated, active, form of pRB to be measured either as an absolute quantity or as a proportion of total cellular pRB. Further described are methods that permit pRB phosphorylation status to be correlated with cell cycle phase and with protein components of the cell cycle. Screening of chemical compounds for antiproliferative and antineoplastic activity using the flow cytometric assays is demonstrated. Reagent kits that facilitate the subject methods are also provided.

Abstract: An apparatus and method for examining particles in a flow stream of a flow cytometer, employing incoherent light sources, such as light emitting diodes (LEDs), and detectors. The light emitting diodes operate as the excitation light sources and emit light toward said flow stream, and the detectors detect light, in particular, fluorescent light, emanating from the particles in response to the excitation light striking the particles. A controller controls each of the light emitting diodes to emit their excitation light for a predetermined period during which the excitation light radiates onto particles of interest. The controller evaluates the detected light to ascertain characteristics of the particles, such as particle size, density and granularity. The apparatus and method can further employ one or more coherent and homogenous light emitting devices, such as a laser, as an additional excitation light source.

Abstract: A method and device is disclosed for rapidly identifying a large number of proteins by placing a protein mixture in a sample chamber of an electrophoresis gel, and performing electrophoresis to separate the mixture by molecular weight, in a direction of separation, into a two-dimensional separation pattern in the gel. The separation pattern is transferred to a membrane, such as a sheet of nitrocellulose, and a plate with a set of separate, side-by-side slots is then applied to the membrane. A different antibody mixture is introduced into each of the slots by perfusing each antibody mixture under pressure through the slots. The antibody mixture that is perfused through each slot recognizes several different proteins of sufficiently different molecular weights that different protein bands can be resolved by the antibody mixture in each slot.

Abstract: The expression of various cell adhesion molecules and growth factor receptor was examined on human progenitor cells (i.e., CD34+/CD38+). Certain changes in the expression if one or more of these molecules and receptors correlates with the progression of cell from non-lineage committed to commitment to a specific lineage. Using one or more markers for these molecules and/or receptors in combination with markers for CD34 and CD38 will enable one to identify and isolate each of the different progenitor cell populations.

Abstract: Mutant, chimeric thermostable DNA polymerases are provided, along with purified DNA sequences that encode the enzymes. Also provided are methods for producing and using the enzymes.

Abstract: A process of detecting a target nucleic acid using labeled oligonucleotides uses the 5′ to 3′ nuclease activity of a nucleic acid polymerase to cleave annealed labeled oligonucleotide from hybridized duplexes and release labeled oligonucleotide fragments for detection. This process is easily incorporated into a PCR amplification assay.

Abstract: The present invention is directed to a process for amplifying any target nucleic acid sequence contained in a nucleic acid or mixture thereof using a thermostable enzyme. The process comprises treating separate complementary strands of the nucleic acid with a molar excess of two oligonucleotide primers, extending the primers with a thermostable enzyme to form complementary primer extension products which act as templates for synthesizing the desired nucleic acid sequence, and detecting the sequence so amplified. The steps of the reaction can be repeated as often as desired and involve temperature cycling to effect hybridization, promotion of activity of the enzyme, and denaturation of the hybrids formed.

Abstract: DNA sequences and corresponding amino acid sequences from the HLA class II beta region of the human genome that are associated with insulin-dependent diabetes mellitus (IDDM) and Pemphigus vulgaris (PV) have been identified. Specifically, marker DNA sequences which detect either directly or indirectly the identity of the codon encoding for the amino acid at position 57 of the DQ&bgr; protein sequence are disclosed as well as sequences from the DR&bgr; region. These sequences may be used to generate DNA hybridization probes and antibodies for assays to detect a person's susceptibility to autoimmune diseases, such as IDDM and PV. Such antibodies and peptides encoded by said DNA sequences can be used therapeutically or prophylactically.

Abstract: This invention relates to improved methods for nucleic acid detection using methods such as the polymerase chain reaction (PCR). More specifically, the invention provides methods for simultaneous amplification and detection to enhance the speed and accuracy of prior methods. The methods involve the introduction of detectable DNA binding agents into the amplification reaction, which agents produce a detectable signal that is enhanced upon binding double-stranded DNA. In a preferred embodiment, the binding agent is a fluorescent dye. The methods also provide means for monitoring the increase in product DNA during an amplification reaction.

Abstract: The subject matter of the invention concerns three cosmid vectors which are suitable for fragment cloning of a size between 7 and 36 kb. These vectors consist of an E. coli ColE1 replica, an ampicillin resistance gene, a multiple cloning cassette, cos sites for in vitro packaging with the lambda packaging extracts as well as fragments from the genome of the bacteriophage lambda. The lambda sequences were selected in a way to prevent lytic processes, vector instabilities (deletions) and unwanted recombination events between lambda DNA and the fragment to be cloned. Depending on the length of the lambda fragment inserted in the corresponding vector heterologous fragments of different lengths can be cloned.

Abstract: 2'-Deoxyisoguanosine, isosteric analogues and isoguanosine derivatives of formulae I-V, processes for their production via compounds of the general formulae a or b and reaction with aroyl isocyanates or from compounds of the general formulae VI-IX by photochemical irradiation. A further production process is the conversion of deoxyguanosines or guanosines by means of persilylation, reaction with ammonia and deamination in the 2 position. The compounds are suitable as pharmaceutical agents with antiviral efficacy.

Abstract: A purified thermostable nucleic acid polymerase is obtained that has unique characteristics. Preferably the nucleic acid polymerase is DNA polymerase isolated from a Thermus aquaticus species and has a molecular weight of about 86,000-95,000 daltons. The thermostable nucleic acid polymerase may be native or recombinant and may be used in a temperature-cycling chain reaction wherein at least one nucleic acid sequence is amplified in quantity from an existing sequence with the aid of selected primers and nucleotide triphosphates. The nucleic acid polymerase is preferably stored in a buffer containing non-ionic detergents that lends stability to the nucleic acid polymerase. A preferred buffer contains glycerol, polyoxyethylated sorbitan monolaurate, ethoxylated nonyl phenol and gelatin.

Abstract: The present invention relates to oligonucleotides which hybridize specifically to the cytosine DNA methyltransferase gene of Neisseria gonorrhoeae and distinguish the cytosine DNA methyltransferase gene of N. gonorrhoeae from highly homologous sequences which have been discovered in some strains of other species of the genus Neisseria. The oligonucleotides are useful as primers for the polymerase chain reaction (PCR) amplification of a nucleic acid sequence from the cytosine DNA methyltransferase gene of N. gonorrhoeae and in N. gonorrhoeae amplification/detection assays.

Abstract: The present invention is directed to a process for amplifying any target nucleic acid sequence contained in a nucleic acid or mixture thereof using a thermostable enzyme. The process comprises treating separate complementary strands of the nucleic acid with a molar excess of two oligonucleotide primers, extending the primers with a thermostable enzyme to form complementary primer extension products which act as templates for synthesizing the desired nucleic acid sequence, and detecting the sequence so amplified. The steps of the reaction can be repeated as often as desired and involve temperature cycling to effect hybridization, promotion of activity of the enzyme, and denaturation of the hybrids formed.

Abstract: The present invention provides modified primers for use in the amplification of a nucleic acid sequence. Amplifications carried out using the modified primers result in less non-specific amplification product, in particular, primer dimer, and a concomitant greater yield of the intended amplification product compared to amplifications carried out using unmodified primers.

Abstract: This invention relates to improved methods for nucleic acid detection using methods such as the polymerase chain reaction (PCR). More specifically, the invention provides methods for simultaneous amplification and detection to enhance the speed and accuracy of prior methods. The methods involve the introduction of detectable DNA binding agents into the amplification reaction, which agents produce a detectable signal that is enhanced upon binding double-stranded DNA. In a preferred embodiment, the binding agent is a fluorescent dye. The methods also provide means for monitoring the increase in product DNA during an amplification reaction.