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T Lövgren

Publications and source records attributed to T Lövgren.

At least 91 records · Page 5Linked to original sources
PubMed1986

Europium-labelled target cells in an assay of natural killer cell activity. I. A novel non-radioactive method based on time-resolved fluorescence.

The use of a new marker for labelling cells used as targets for natural killer cells is described. The human erythroleukaemic cell line K-562 was used as target. The cells were labelled with europium diethylenetriaminopentaacetate (EuDTPA) chelates. The detection of the released marker is based on time-resolved fluorometry. The results obtained show that the method is sensitive, specific and rapid. The high specific activity of the marker and the sensitivity of the detection apparatus result in numeric values (counts per second) which are 10-20 times higher than the values (counts per minute) obtained with 51chromium. In comparison with 51chromium release assay the labelling of target cells is less time consuming, the marker release more rapid and the detection time of released marker only 1 second per tube. The use of this non-radioactive marker is an alternative way of measuring natural killer cell mediated cytolysis of target cells.

Cell Line↗
PubMed1986

Screening for Duchenne muscular dystrophy: an improved screening test for creatine kinase and its application in an infant screening program.

A kinetic bioluminescence assay with optimized reagent conditions has been developed for application as a screening test for increased creatine kinase (CK) activities in dried blood spots. This test is used for the early detection of Duchenne muscular dystrophy (DMD) in a voluntary CK screening program in West Germany. Of the 176,600 boys tested up to December 31, 1984, 48 who were less than 6 months old had certain or probable DMD (frequency 1: 3679). In 1983 and 1984, the rate of false positive results was 0.016% for a cut-off activity 300 U/liter and 0.061% for a cut-off activity 180 U/liter. Long-term counseling is offered to families of newly detected DMD patients in order to facilitate the aims of the screening program, namely, avoidance of secondary cases in affected families, early professional care for the sick child, and the early opportunity to make the appropriate decisions for a life with an handicapped child. Two types of a benign hereditary blood anomaly were also detected by CK screening (CK-BB inside erythrocytes or thrombocytes).

Age Factors↗
PubMed1986

Two-site time-resolved immunofluorometric assay of human insulin.

We describe a two-site "sandwich"-type time-resolved immunofluorometric assay for human insulin, based on use of two monoclonal antibodies with different specificities. The first antibody is immobilized on the surface of microtiter plate strip wells, the other is labeled with Eu3+. Serum samples can be assayed with one incubation step; two incubation steps are required when plasma samples are assayed. After the immunoreactions are complete, the bound fraction of Eu3+-label is quantified by dissociating it in a fluorescence-enhancement solution and measuring its fluorescence with a fluorometer with time-resolution. The sensitivity of the assay is 0.24 micro-int. units/mL. The standard curve is linear from 0.24 to 2400 micro-int. units/mL.

Animals↗
PubMed1986

Solid-phase immunoassay of digoxin by measuring time-resolved fluorescence.

We describe a time-resolved fluoroimmunoassay for the cardiac glycoside digoxin. The assay depends on the competitive distribution of Eu3+-labeled anti-digoxin antibodies between solid-phase-bound digoxin and the digoxin in the sample or standard. After this immunoreaction, the bound fraction of the Eu3+-label is dissociated from the solid phase, converted into a highly fluorescent beta-diketone chelate, and measured in a fluorometer with time-resolution (1230 Arcus, LKB-Wallac). Sample volume is 20 microL, the incubation lasts 30 min, and the concentration range of the assay for digoxin is from 0.25 to 4.0 ng/mL (0.32-5.1 nmol of digoxin per liter). Correlation with an in-house RIA was good (r = 0.97, n = 43). The sensitivity was equivalent to that of most RIAs: 0.2 ng/mL (0.25 nmol/L).

Antibodies↗
PubMed1985

Monitoring the LH surge by ultrarapid and highly sensitive immunofluorometric assay.

A novel time-resolved immunofluorometric method is described for the estimation of human luteinizing hormone (LH) in serum and urine. The method utilizes two monoclonal antibodies: one reacting with the beta-subunit is adsorbed to the wall of a microtiter well, and the other is labeled with a fluorescent europium chelate and reacts with the alpha-subunit. The method is ultrarapid (15-30 min) and highly sensitive (1 IU/L). A large linear measuring range allows measurement of LH levels from 1 to 250 IU/L. For the monitoring of urinary LH, IFMA gives the same information as the Hi-Gonavis assay, but it has the advantages of greater sensitivity and a shorter assay time. For the determination of serum LH levels, an acceptable correlation was observed between radioimmunoassay and IFMA. Furthermore, IFMA has a greater sensitivity, and is more rapid and not dependent on the handling of radioactive materials.

Antibodies, Monoclonal↗
PubMed1984

Competitive solid-phase immunoassay of testosterone using time-resolved fluorescence.

A competitive solid-phase immunoassay for the determination of testosterone in serum samples using time-resolved fluorescence is described. The solid phase is a testosterone-3-(O-carboxymethyl)-oxime-ovalbumin conjugate coated to polystyrene microtiter strips. Europium-labelled polyclonal and monoclonal antibodies against testosterone-3-(O-carboxymethyl)-oxime-bovine serum albumin were compared. Their behavior was quite similar although the polyclonal antibody was more sensitive, giving a detection limit of 15 fmol testosterone per assay. Correlation with RIA was very good (r = 0.982 and y = -0.150 + 0.969x).

Animals↗
PubMed1984

Europium as a label in time-resolved immunofluorometric assays.

A nonisotopic immunoassay has been developed based on a sensitive detection of europium (III) in water solution using time-resolved fluorometry. The europium label is bound to the antibody with EDTA derivatives, either diazophenyl-EDTA-Eu or isothiocyanatophenyl-EDTA-Eu. After the immunometric assay has been completed the europium is preferably dissociated from the antibody at low pH and measured by time-resolved fluorescence in a micellar solution containing Triton X-100, beta-diketone, and a Lewis base. The detergent solubilizes the chelating compounds in the solution and excludes water from the fluorescent ligand-europium complex. Europium concentrations as low as 5 X 10(-14)M were measured using a 1-s counting time. The sensitivity of the immunoassay of rabbit IgG used as a model system was 25 pg/ml (6 pg/assay).

Animals↗
PubMed1983

The effect of luciferase and NADH:FMN oxidoreductase concentrations on the light kinetics of bacterial bioluminescence.

The effects of NADH:FMN oxidoreductase and luciferase concentrations on the light kinetics of the bacterial bioluminescent reaction were investigated. Light emission with low decay rates was obtained by regulating the conversion of NADH to NAD+ by controlling oxidoreductase activity. Constant light emission can be obtained when the oxidoreductase activity is below 2.5 U/1 in the assay system. The luciferase concentration affects the light intensity but it has no effect on the decay rate of light emission. The substrate decanal and the end-products NAD+ and capric acid had no effect on the light kinetics. The Michaelis constants of bacterial luciferase for FMNH2 and decanal were 3 X 10(-6) M and 8 X 10(-7) M, respectively, and those of oxidoreductase for FMN and NADH were 6.1 X 10(-6) M and 1.6 X 10(-5) M, respectively.

Bacterial Physiological Phenomena↗
PubMed1983

Detection of hepatitis B surface antigen using time-resolved fluoroimmunoassay.

Conventional fluoroimmunoassay (FIA) methods based on various fluorescence principles have not achieved the sensitivity of radioimmunoassay (RIA) mainly because of problems of background fluorescence arising, for example, from the biological specimen. We now describe an immunoassay of hepatitis B surface antigen (HBsAg) based on time-resolved (TR) fluorescence using a lanthanide as label. The assay initiates the development of a new generation of immunoassays. The fluorescence intensity is measured after a selected delay time which almost completely eliminates background fluorescence, which has a fast decay time. The excitation is performed with a flashing light source. The molecules with a long fluorescent lifetime consist of chelates of rare earth metals (Eu, Tb, Sm, Dy). They absorb strongly the excitation radiation and transfer the energy to the chelated central atom which in turn produces an emission spectrum characteristic of the lanthanide used. A long Stokes' shift (greater than 270 nm) helps to reduce the background in the emission region of the chelate and thus optimizes measurement of the relevant fluorescence. The present TR-FIA uses 2-naphthoyltrifluoroacetone as chelating agent because it creates an intense fluorescence with the rare earth metals. Synergistic agents such as trioctylphosphineoxid further enhance the fluorescence of the chelate. Depending on the instrumentation used for measuring time-resolved fluorescence and the conditions used for chelate formation, lanthanides can be detected at 10(-12)-10(-14)M concentrations.

Fluorescent Antibody Technique↗
PubMed1983

Time-resolved fluoroimmunoassay of human choriogonadotropin.

We describe time-resolved fluoroimmunoassay for human choriogonadotropin involving monoclonal antibodies directed against the beta- and alpha-subunits. The latter antibody was labeled with europium, which was measured by counting for 1 s after the immunoreaction was completed. In the solid-phase sandwich assay, both a one-step and two-step procedure were used; the respective measuring ranges were 0.7-135 and 0.7-350 int. units/L, the latter covering a 500-fold dynamic range. The CV within the assay range was between 4 and 8%, depending on the dose. Cross reactivity with lutropin in the one- and two-step procedures was 1.6% and 1.0%, respectively.

Antibodies, Monoclonal↗
PubMed1982

Development of solid-phase immunoassay using chemiluminescent IgG conjugates.

Solid-phase luminescent immunoassay (LIA) was studied using mainly aminobutyl-ethylisoluminol-IgG conjugates. Different solid-phase supports such as immunobeads, polystyrene balls and tubes gave comparable results although tubes were preferred in most of the assays because of the better linearity and reproducibility obtained and their ease of handling. The properties of the conjugates were tested using both direct and sandwich LIA. Direct LIAs performed using sheep-anti-rabbit IgG coated balls provided information on the antigenicity of the conjugates while the sandwich LIA was the actual working system. A sensitivity of 1 ng was obtained in this system. The stability, easy use, safe handling and low cost of the conjugates coupled with a short assay time make this luminescent system a potential alternative to RIA.

Animals↗
PubMed1982

Increase in the estrogen binding capacity of breast cancer cytosols following limited proteolysis with trypsin.

When small amounts of trypsin were added to prelabelled estrogen receptors in 24 human breast cancer cytosols there was a substantial increase in the binding capacity [79 +/- 11 (SE)%]. At the same time the affinity of the hormone receptor interaction was maintained at a very high level or even increased. This finding is discussed in relation to previous results where a diisopropylfluorophosphate (DFP) inhibitable protease activity was shown to cause a similar augmentation of estrogen binding sites in human myometrial cytosols. Addition of sodiummolybdate at or immediately after homogenization led to a similar increase in estrogen binding sites. Because these two effects were not additive we propose that the limited trypsin treatment reactivates the binding sites previously inactivated through a mechanism which can be inhibited by sodiummolybdate.

Adult↗
PubMed1982

The measurement of serum cortisol by a solid-phase chemiluminescence immunoassay.

A solid-phase chemiluminescent immunoassay (LIA) for the determination of cortisol in serum specimens has been developed. The separation of the cortisol onto the solid-phase distinguishes this particular assay from other chemiluminescence immunoassays. The method can be used without prior extraction. Cortisol molecules were made chemiluminescent by conjugation to aminobutylethyl-isoluminol. The detection limit of the label in the chemiluminescent reaction was 0.1 pmol. The LIA was compared with a RIA for cortisol in 36 serum specimens and the correlation between the assays was good (r = 0.97). The time course of the chemiluminescent reaction is fast and a 20's integration time gave reproducible results. The peak light intensities were not reliable.

Humans↗
PubMed1982

Bioluminescent assay of total and brain-specific creatine kinase activity in cerebrospinal fluid.

A bioluminescent assay based on the firefly luciferase reaction has been used for determination of creatine kinase activity in CSF. Activities as low as 0.1 U/L can be measured. The coefficient of variation at an activity level of 0.3-0.4 U/L was between 5 and 6%. The assay conditions optimized for serum specimens can be used for CSF. The adenylate kinase activity is almost completely inhibited, which simplifies the procedure. The creatine kinase/(CK) isoenzyme distribution was obtained using the bioluminescent assay in combination with immunoinhibition or ion exchange chromatography. All specimens contained both MM and BB activity, but no MB was found. The study indicates that the bioluminescent assay is useful in the determination of CK isoenzymes in CSF. The clinical importance of the observed CK levels will be reported in a separate communication.

Adenosine Diphosphate↗
PubMed1982

Optimized bioluminescence assay of creatine kinase and creatine kinase B-subunit activity.

Spectrophotometry of CK-B subunit activity based on immunoinhibition is rapid and convenient. However, the low sensitivity limits clinical applications to serum samples with CK-B activities considerably above normal. A sensitive, but not optimized, bioluminescent assay of CK-B, also based on immunoinhibition, has been described. We improved this method by the use of an ADP concentration saturating the CK reaction and by the combined use of AMP and diadenosine pentaphosphate for inhibition of adenylate kinase. The optimized bioluminescent method is similar to the corresponding spectrophotometric method (reaction conditions, analytical procedure, numerical results, etc.) and results by the two methods correlate well. However, the measuring time is shorter and, more important, the sensitivity of the bioluminescent method allows accurate determinations of CK-B, even in serum from healthy individuals (CV approximately 3%). Kits and instrumentation (manual and automatic) for determination of CK and CK-B are now commercially available.

Adenylyl Cyclases↗
PubMed1979

Effect of substrate properties on the activity of lysosomal cholesteryl ester hydrolase.

The effects of the substrate properties on the catalytic activity of lysosomal cholesteryl ester hydrolase from rat liver have been examined with three standard substrate types: vesicle, micelle and emulsion. The pH optimum of the enzyme coincided to 4.5--5.0 with the substrate types employed. The apparent Km values were 15.3, 14.3 and 7.3 microM for vesicle, micelle and emulsion substrates, respectively. In the systems used in this study reaction products, cholesterol and oleic acid, and the nonionic surfactant Tween 80 and Triton X-100 Had an inhibitory effect. The emulsifier phosphatidylcholine and the charged phospholipid phosphatidic acid stimulated the activity. The mixed micelle of sodium taurocholate and phosphatidylcholine was the most potent substrate vehicle. With dipalmitoyl phosphatidylcholine vesicles the enzyme showed maximal activity at the gel-liquid-crystalline transition temperature of the phospholipid. The possible physiological significance of the lysosomal cholesteryl ester hydrolase is discussed with special reference to the form of the substrate.

Animals↗