Biomedical subjects

M Kwiatkowski

Publications and source records attributed to M Kwiatkowski.

At least 19 recordsLinked to original sources

PubMed1991

Preparation of europium-labelled DNA probes and their properties.

A chemical method for labelling DNA with a europium chelate is presented. First, primary aliphatic amino groups are introduced onto DNA in a transamination reaction. The transamination reaction is altered by adjusting temperature and duration of the reaction. Subsequently, the modified DNA is reacted with an isothiocyanate derivative of a Eu chelate. The optimum amount of Eu chelates on a DNA probe is 4-8% of total nucleotides. There is a decrease of 0.7 degrees C in the melting temperature of DNA for each incorporated Eu chelate on 100 bases. Hybridization efficiency is lowered by the introduction of Eu chelates but this effect can be partly overcome by using high DNA probe concentrations. The detection limit of the Eu-labelled probe is 0.15 attomoles of target DNA in a mixed-phase hybridization assay on microtitration wells. In addition to high sensitivity the Eu-labelled probes offer convenience in use and results which are quantitative and easy to interpret.

Affinity Labels↗

PubMed1991

The use of europium (Eu3+) labelled primers in PCR amplification of specific target DNA.

The polymerase chain reaction (PCR) has many potential applications in the field of DNA probe diagnostics. Here we describe a method that utilizes PCR and time-resolved fluorometry (TRF) for the detection of specific target DNA. First the DNA segment to be detected is amplified according to standard procedures. Then a pair of europium (Eu3+) and biotin-labelled primers nested within the amplified fragment is incorporated in a few additional PCR cycles. Thus amplified DNA fragments are generated that contain an affinity label (biotin) and a detectable label (europium). The doubly-labelled amplified DNA fragments are collected onto streptavidin coated microtitration strips and the bound Eu3+ is measured in a time-resolved fluorometer. We show here the application of this method to the detection of HIV-1 DNA. As few as five copies of HIV-1 DNA could readily be detected using this assay. The method described here is sensitive, rapid and easy to employ. In addition it lends itself to automation.

Bacterial Proteins↗

PubMed1991

Detection of human immunodeficiency virus type 1 by using the polymerase chain reaction and a time-resolved fluorescence-based hybridization assay.

The polymerase chain reaction (PCR) has many potential applications in the field of nucleic acid diagnostics. In particular, it has been successfully applied to the detection of pathogens present in low copy numbers such as the human immunodeficiency virus type 1. Here we describe a time-resolved fluorescence-based hybridization assay which, combined with the PCR, offers an extremely sensitive method for the detection of nucleic acids. In this assay format, the PCR is run by standard procedures and the subsequent hybridization reaction is carried out in solution by using two oligonucleotide probes, one biotinylated and one labeled with europium (Eu3+). The sandwich hybrids are then collected onto a streptavidin-coated microtitration well, and the bound Eu3+ is measured in a time-resolved fluorometer. This assay is rapid, user friendly, and quantitative and lends itself to automation. The application of this assay to the detection of human immunodeficiency virus type 1 is described.

Acquired Immunodeficiency Syndrome↗

PubMed1990

The Ciba Corning ACS:180 benchtop immunoassay analyzer.

We describe a new clinical laboratory instrument, the ACS:180, used to automate heterogeneous immunoassay testing. The ACS:180 automates immunoassays in which paramagnetic particles are the solid phase and changes in chemiluminescence are measured. The system can accommodate both competitive and sandwich-type assay configurations. The microprocessor-based instrument fully automates each step of the assay, including sample and reagent addition, separation and wash of paramagnetic particles, and generation and acquisition of the chemiluminescent signal. The instrument has the flexibility to operate in random-access or batch mode. The time from application of sample to first result is less than 15 min; throughput is as much as 180 tests per hour.

Autoanalysis↗

PubMed1987

Sensitive detection of genes by sandwich hybridization and time-resolved fluorometry.

Europium has been used as a non-radioactive marker in immunoassays as this metal can be detected with high sensitivity by time-resolved fluorometry. In this work streptavidin labeled with europium was used to detect biotinylated probes in a sandwich nucleic-acid hybridization assay with microtitration strips as the solid phase. pBR 322 plasmids were detected with a sensitivity of 4 x 10(5) molecules. As the sample is added in solution in sandwich hybridization, fast and simple sample pre-treatment can be used without encountering background problems. The method was applied to test bacterial samples of uropathogenic Escherichia coli strains for the presence of the beta-lactamase gene.

Animals↗

PubMed1986

Influence of fibrinolysis activation on leukocyte adhesiveness. Preliminary report.

In patients with thrombophlebitis receiving intravenously a nicotinic acid derivative, pentoxifylline and streptokinase the fibrinolytic activity and leukocyte adhesiveness were studied. The euglobulin lysis time was significantly shortened in all cases; at the same time the adhesiveness of leukocytes and the leukocyte number were reduced. The addition of the drugs to blood in vitro resulted in a significant increase in leukocyte adhesiveness; the fibrinolytic activity was enhanced only upon streptokinase. Leukocytes are believed to promote the thrombolytic effect of fibrinolytic agents.

Adult↗

PubMed1985

Chemical synthesis of a pentaribonucleoside tetraphosphate constituting the 3'-acceptor stem sequence of E. coli tRNAIle using 2'-O-(3-methoxy-1,5-dicarbomethoxypentan-3-yl)-ribonucleoside building blocks. Application of a new achiral and acid-labile 2'-hydroxyl protecting group in tRNA synthesis.

A synthesis of a pentaribonucleotide fragment constituting the residues 59-63 of 3'-terminus of E. coli tRNAIle, 5'-ApGpUpCpC-3', has been carried out using a new, easily accessible and achiral 2'-ketal protecting group. The new 2'-ketal group has an additional advantage in that it is easily functionalized to the diamide with aqueous ammonia in the penultimate step of deblocking of fully protected oligoribonucleotides. Such a functionalization of the 2'-ketal group at the penultimate step of deblocking of the fully protected tRNA molecule enhances its relative rate of removal under an acidic condition with a minimum of damage of the target tRNA molecule.

Escherichia coli↗

PubMed1984