Kaj Blomberg

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.

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.

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.

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.

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.

Solid phase IL-2–IL-2Rα 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.