Biomedical subjects

K Blomberg

Publications and source records attributed to K Blomberg.

18 recordsLinked to original sources

PubMed2000

Time-resolved fluorometric assay for leukocyte adhesion using a fluorescence enhancing ligand.

A new 96-well microtiter plate, time-resolved fluorometric assay was developed to measure leukocyte adhesion in vitro. The assay is based on loading leukocytes with a fluorescence enhancing ligand 2,2':6', 2"-terpyridine-6,6"-dicarboxylic acid (TDA), which in its acetoxymethyl ester form readily diffuses through the cell membrane. After hydrolysis by nonspecific intracellular esterases, the impermeable TDA accumulates inside the cells. When the TDA-labeled adherent leukocytes are lysed, the ligand is released and reacts with europium present in the lysis solution to produce a highly fluorescent and stable chelate. The fluorescence signal can be measured by time-resolved fluorometry and correlates directly with the number of adherent cells. In this study, we have optimized both the TDA-labeling and adhesion assay conditions in isolated human neutrophils. Furthermore, we have compared the assay with a traditional microscopic counting method. This time-resolved fluorometric assay provides a rapid, reproducible and convenient method for the routine analysis of leukocyte adhesion.

Cell Adhesion↗

PubMed1999

Terbium and rhodamine as labels in a homogeneous time-resolved fluorometric energy transfer assay of the beta subunit of human chorionic gonadotropin in serum.

BACKGROUND: Fluorescence resonance energy transfer (FRET) is a powerful tool in analytical chemistry. The aim of the present work was to use FRET to design a homogeneous immunoassay. METHODS: We used a highly fluorescent terbium (Tb3+) chelate (donor) and the organic fluorochrome rhodamine (acceptor) combined with time-resolved detection of the acceptor emission in homogeneous assay format for the measurement of the beta subunit of human chorionic gonadotropin (betahCG) in serum. We used two antibodies labeled with Tb3+ and rhodamine, respectively, recognizing different epitopes on betahCG. The close proximity between the labels in the immunocomplex permitted energy transfer between the pulse-excited Tb3+ donor (decay time >1 ms) and the acceptor rhodamine (decay time of 3.0 ns). The prolonged emission of donor-excited acceptor (energy transfer) was measured after the short-lived background and acceptor emissions had decayed. The emission of donor-excited rhodamine was measured at a wavelength of where the emission of unbound donor is minimal. RESULTS: The energy transfer signal was directly proportional to the betahCG concentration in the sample. The limit of detection was 0.43 microgram/L, and the assay was linear up to 200 microgram/L. Total assay imprecision in the range 10-185 microgram/L was between 7.5% and 2.8%. CONCLUSIONS: Although less sensitive than heterogeneous, dissociation-enhanced europium-based separation assays, the presented assay format has advantages such as speed and simplicity, which make the assay format ideal for assays requiring a high throughput.

Antibodies, Monoclonal↗

PubMed1998

Homogeneous time-resolved IL-2-IL-2R alpha assay using fluorescence resonance energy transfer.

A homogeneous receptor-ligand assay based on fluorescence resonance energy transfer is described. In the assay, recombinant human interleukin 2 (IL-2) and a monoclonal antibody against the human IL-2 receptor alpha chain were labelled with a highly fluorescent europium chelate and Cy5, respectively. As a result of a successful receptor-ligand complex formation, these labels are brought into close proximity, which will thereby allow an energy transfer to occur from the donor (europium) to the acceptor (Cy5), upon excitation of the donor. Utilization of specific non-neutralizing antibodies made it possible to use crude hIL-2R alpha membranes prepared from recombinant baculovirus-infected Sf9 insect cells. The specific energy transfer was measured at the emission wavelength of Cy5 using a time-resolved fluorometer. The data presented, demonstrate that this assay design can be utilized for saturation as well as competitive binding experiments in addition to regular receptor titrations. As a rapid simple homogeneous assay it is particularily suitable for high throughput screening analyses.

Animals↗

PubMed1997

Solid phase IL-2-IL-2R alpha assay with time resolved fluorometry.

A time-resolved fluorometric, solid phase, receptor ligand interaction assay is described. The assay consists of wells coated with anti-human IL-2 receptor alpha (hIL-2R alpha) monoclonal antibodies (mAb), europium labelled hIL2 (Eu-IL-2) and human recombinant IL-2 receptor alpha subunits expressed in the baculovirus expression vector system (BEVS). In the assay hIL-2R alpha-Eu-IL-2 complexes bind to the solid phase mAb. Receptor bound Eu is dissociated into an enhancer solution where it forms highly fluorescent complexes. The fluorescence is measured in a time-resolved fluorometer. The Kd value calculated from the saturation curve is in good agreement with previously reported values for the low affinity type of IL-2R, making the described assay a simple and nonradioactive alternative for measurement of soluble hIL-2R alpha in biological systems. Furthermore this assay format provides convenient separation of bound ligand from unbound and is therefore suitable for high throughput screenings.

Europium↗

PubMed1996

Time-resolved fluorometric assay for natural killer activity using target cells labelled with a fluorescence enhancing ligand.

A time-resolved fluorometric assay for the measurement of natural killer cell activity against target cells labelled with the acetoxymethyl ester of the fluorescence enhancing ligand 2,2':6',2"-terpyridine-6,6"-dicarboxylic acid (TDA) is described. The hydrophobic esterified form of TDA (bis(acetoxymethyl) 2,2':6',2"-terpyridine-6,6"-dicarboxylate, BATDA) diffuses readily through the cell membrane of viable cells. BATDA is hydrolysed by intracellular esterases resulting in accumulation of membrane impermeable TDA inside the target cells. After incubation of labelled K-562 cells with effector cells the TDA released from lysed cells into the supernatant is chelated with Eu3+. The natural killer cell activity is then quantified by measuring the intense fluorescence of the EuTDA chelates formed. Target cells are rapidly labelled when incubated with BATDA, TDA is released from target cells faster than 51Cr, the spontaneous release permits a short-term release assay to be set up and the detection of EuTDA is fast (5 min/96 well plate). Furthermore, this non-radioactive method permits the use of complex culture media since, in contrast to methods based on prompt fluorometry, the problem with autofluorescence can be avoided by the use of time-resolved fluorometry.

2,2'-Dipyridyl↗

PubMed1996

One-step all-in-one dry reagent immunoassays with fluorescent europium chelate label and time-resolved fluorometry.

The availability of an intrinsically fluorescent, inert, and stable Eu chelate label made it feasible to design one-step all-in-one immunoassays with time-resolved fluorometry for detection. Both competitive and noncompetitive immunoassays are performed in microtitration wells containing all assay-specific components in a stable dry form. Only the sample and one assay buffer common for all analytes need to be added. Model assays for human chorionic gonadotropin (hCG), alpha-fetoprotein (AFP), and progesterone all reached equilibrium in 15 min or less without compromising the performance characteristics of the measurements, all of which perform at least equivalent to state-of-the-art assays. The detection limits for hCG, AFP, and progesterone were 0.3 IU/L, 0.1 microgram/L, and 0.5 nmol/L, respectively. The assay ranges for hCG and AFP were linear to 5000 IU/L and 1200 micrograms/L, respectively. The immunoassay format can be readily implemented in a fully automated random-access immunoassay system with optimal performance characteristics and no handling of analyte-specific assay components.

Binding, Competitive↗

PubMed1994

Simultaneous measurement of NK cell cytotoxicity against two target cell lines labelled with fluorescent lanthanide chelates.

We describe a cytotoxicity assay which permits the simultaneous measurement of natural killer cell activity against two different cell lines. The target cell lines are labelled either with a fluorescent europium chelate or with a fluorescent terbium chelate and cell death is quantified by measuring the chelate release. K-562, Molt4 and Daudi cell lines have been used as targets. The release of the two chelates from the target cells can be detected with the help of time resolved fluorometry. As the measurements are made after background fluorescence has decayed no additional steps are needed to correct for the background from the medium. The assay procedure used for measurement of cytotoxicity against two target cell lines is very similar to the widely used 51Cr release assay.

Cell Line↗

PubMed1994

Simultaneous measurement of natural killer cell cytotoxicity against each of three different target cell lines.

A time-resolved fluorometric assay for the simultaneous measurement of natural killer cell activity against three different lanthanide diethylenetriaminopentaacetate (LaDTPA) labelled target cell lines is described. The target cell line K-562 was labelled with SmDTPA, the cell line Molt with TbDTPA and the cell line Raji with EuDTPA. After co-incubation of the three target cell lines with effector cells the fluorescence of the lanthanides released from the lysed target cells was measured in an enhancer solution in which they formed highly fluorescent complexes. It was possible to differentiate the specific release from the three target cell lines because the emission lines of the europium, samarium and terbium complexes formed in the enhancer solution are well separated from each other. The autofluorescence from culture media supplemented with serum was avoided by the use of time-resolved fluorometry. The results show that applying fluorometry based on the combination of spectral and temporal resolution to natural killer cell assays, makes possible the simultaneous determination of lysis in up to three target cell lines in complex culture medium.

Culture Media↗

PubMed1993

Fluorescent europium chelates as target cell markers in the assessment of natural killer cell cytotoxicity.

A time-resolved fluorometric assay for the detection of natural killer cell activity against target cells labelled with the fluorescent chelate europium-6,6"-bis[N,N-bis(carboxymethyl)-aminomethyl]-4'-phenyl-2,2',6', 2"-terpyridine (EuCAPT) has been developed. In the assay released EuCAPT from lysed K-562 cells is measured in the supernatant after co-incubation of the target cells with effector cells. Thus, the performance of the assay is essentially similar to the previously described EuDTPA assay and the widely used 51Cr assay. EuCAPT is released from target cells lysed by effector cells faster than 51CrO4(2-) but somewhat slower than EuDTPA. In contrast to methods based on prompt fluorometry the autofluorescence from culture medium supplemented with serum can be avoided by the use of time-resolved fluorometry. The result shows that fluorescent europium chelates provide an alternative to radioactive markers currently used for the assessment of in vitro cellular cytotoxicity.

Chelating Agents↗

PubMed1992

Simultaneous quadruple-label fluorometric immunoassay of thyroid-stimulating hormone, 17 alpha-hydroxyprogesterone, immunoreactive trypsin, and creatine kinase MM isoenzyme in dried blood spots.

We describe a quadruple-label fluorometric immunoassay for simultaneously measuring four analytes: thyroid-stimulating hormone (TSH), 17 alpha-hydroxyprogesterone (17 alpha-OHP), immunoreactive trypsin (IRT), and creatine kinase MM (CK-MM). The assay is based on immunoreagents labeled with four different lanthanide ions (Eu3+, Tb3+, Sm3+, and Dy3+), on dissociative fluorescence enhancement applying the principle of co-fluorescence, and on time-resolved fluorometry. The monoclonal anti-alpha-TSH and anti-IRT antibodies and the polyclonal anti-CK-MM antibody were labeled with Eu3+, Sm3+, and Dy3+, respectively; 17 alpha-OHP was labeled with Tb3+. The assay was performed in microtitration strip wells coated with a mixture of monoclonal antibodies against beta-TSH, IRT, and CK-MM and a polyclonal goat anti-rabbit IgG for capture of the rabbit anti-17 alpha-OHP antibodies. After completion of the immunoreactions, the bound fractions of the lanthanides were dissociated into the co-fluorescence enhancement solution, creating highly fluorescent chelates. The four lanthanide-specific signals were subsequently measured in a time-resolved fluorometer. The detection limits of the assay were 0.1 mIU/L for TSH, 2 nmol/L for 17 alpha-OHP, 2 micrograms/L for IRT, and 4 U/L for CK-MM.

17-alpha-Hydroxyprogesterone↗

PubMed1988

Determination of cytotoxic T lymphocyte activity by time-resolved fluorometry using europium-labelled concanavalin A-stimulated cells as targets.

Time-resolved fluorometry was used to detect the cytolysis of concanavalin A-stimulated blast cells by employing cells stimulated in a mixed lymphocyte culture as effectors. The target cells were labelled with europium diethylenetriaminopentaacetate (EuDTPA) chelates. The results obtained showed that this method, which has been successfully applied to the measurement of natural killer cell activity, was also applicable to the more specific cytotoxic T lymphocyte reaction. The specific release was higher with EuDTPA-labelled target cells than with 51chromium-labelled target cells. As with 51chromium, EuDTPA can be used to distinguish between the cytolysis of specific and non-specific target cells.

Chromium Radioisotopes↗

PubMed1986

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

The significance, specificity and high sensitivity of a new method to determine the natural killer cell cytolysis of europium diethylenetriaminepentaacetate (EuDTPA)-labelled target cells has been confirmed. The targets used in this release assay were the NK sensitive cell line K-562 and the resistant cell line Raji. The released EuDTPA was detected by a method based on time-resolved fluorometry. The specific EuDTPA release was higher than specific 51chromium (51Cr) release. Competitive assays, where half of the target cells were labelled with EuDTPA and the other half with 51Cr and the use of double labelled target cells showed that the results were identical with those of single labelled cells. The reliability of the EuDTPA release assay was further confirmed by performing experiments using NK cells from a patient whose complete lack of NK activity had earlier been demonstrated with the 51Cr release assay. Furthermore, our studies show that the amount of incorporated EuDTPA was directly proportional to the concentration of marker used. Due to the proportional incorporation of EuDTPA the labelling conditions can be chosen to obtain a sensitivity which allows even single cells to be detected.

Cells, Cultured↗

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↗

PubMed1985