Ilkka Hemmilä

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).

Time-Resolution in Fluorometry Technologies, Labels, and Applications in Bioanalytical Assays

Referee: C. Morgan, School of Environmental and Life Sciences. University of Salford, U.K.

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.

Determination of hormones by time-resolved fluoroimmunoassay

Immunoassays based on europium labels and time-resolved fluorescence as the detection method, have been developed. The specific activity of the label is several orders of magnitude higher than that of radioactive labels. Consequently, the technique provides great potential, especially in the determination of analytes which require high sensitivity. Both competitive and immunometric assays which use labelled antibodies have been worked out. In competitive assays the antigen is immobilized on a solid phase with a protein carrier. The antigen in the standard or sample then competes with the labelled antibody in solution. Separation is done simply by washing the wells in the microtitre strip where the assays are performed. Model systems are described for the measurement of testosterone and cortisol. Immunometric assays of human thyrotropin (hTSH) and luteotropin (LH) were performed with monoclonal antibodies, by either a one-step (hTSH) or two-step (LH) incubation procedure. These assays, which exploit the specific activity of the label, give a very high sensitivity and good reproducibility. The standard curves are linear and the dynamic range is at least 1000-fold. Because of the properties of the europium label and the simple assay design, the immunoassays based on time-resolved fluorescence are expected to gain wide application both in research and in routine determinations.

Time-resolved fluorometry: an overview of the labels and core technologies for drug screening applications

Time-resolved fluorometry (TRF) with lanthanide chelate labels is a well-established technology in diagnostics. Recently, TRF has attracted great interest as a tool for application in a range of assay formats in drug screening. The advent of highly fluorescent lanthanide chelates has made it possible to develop a wide variety of highly sensitive TRF-based assay technologies, both heterogeneous and homogeneous. In addition, the ability of TRF technology to combine temporal resolution with high spatial resolution opens up the possibility of assay configurations with features such as miniaturization, multilabel or multiparametric analysis and greatly improved sample throughput.

The synthesis and use of activated N-benzyl derivatives of diethylenetriaminetetraacetic acids: Alternative reagents for labeling of antibodies with metal ions

A series of bifunctional chelating agents--substituted benzyl derivatives of diethylenetriaminetetra acids--was synthesized. These agents were used to label antibodies with a lanthanide, Eu3+. The stabilities of their antibody conjugates were evaluated under different conditions and the dissociation rates of Eu3+ were measured at pH 3.2, which is used for fluorescence enhancement in time-resolved fluoroimmunoassays. The synthesized complexing agents were also compared to other reagents used for metal labelings, to diethylenetriaminepentaacetic acid-dianhydride, and to p-isothiocyanatophenyl-EDTA. The asymmetric p-isothiocyanatobenzyl derivative of diethylenetriaminetetraacetic acid-Eu3+ showed reasonably good stability at neutral pH but released Eu3+ rapidly in the acidic fluorescence enhancement solution. This makes it an optimal choice for chelate labeling in dissociation-based time-resolved fluoroimmunoassays.

Co-fluorescence enhancement system based on pivaloyltrifluoroacetone and yttrium for the simultaneous detection of europium, terbium, samarium and dysprosium

Abstract A sensitive co-fluorescence enhancement system was developed for the fluorimetric detection of europium, terbium, samarium and dysporium ions. The fluorescence intensities of the ternary chelates of Eu 3+ (and Tb 3+ , Sm 3+ or Dy 3+ ) with pivaloyltrifluoroacetone and 1,10-phenanthroline in a solution containing Triton X-100 and ethanol were greatly enhanced by the addition of excess of Y 3+ , Lu 3+ , Gd 3+ or La 3+ . Y 3+ enhanced the fluorescence intensities of Eu 3+ , Tb 3+ , Sm 3+ and Dy 3+ chelates 130-, 1078-, 61- and 102-fold, respectively. The limits of detection of Eu 3+ , Tb 3+ , Sm 3+ and Dy 3+ in the developed co-fluorescence enhancement solution were 0.035, 0.34, 7.9 and 46 pM, respectively, when measured with a sensitive time-resolved fluorimeter. The present enhancement system is well suited for the fluorimetric determination of lanthanide ions used, e.g., as labels in time-resolved fluorimetric immunoassays. As compared with a direct enhancement system, the co-fluorescence enhancement solution benefits from higher fluorescence intensities and prolonged decay times. Therefore, it is particularly well suited for a multi-label immunoassay, where several analytes are to be measured simultaneously from a single sample by employing three or four chelate labels.

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.

Homogeneous Time-Resolved Fluorescence Quenching Assay (LANCE) for Caspase-3

In addition to kinases and G protein—coupled receptors, proteases are one of the main targets in modern drug discovery. Caspases and viral proteases, for instance, are potential targets for new drugs. To satisfy the current need for fast and sensitive high-throughput screening for inhibitors, new homogeneous protease assays are needed. We used a caspase-3 assay as a model to develop a homogeneous time-resolved fluorescence quenching assay technology. The assay utilizes a peptide labeled with both a luminescent europium chelate and a quencher. Cleavage of the peptide by caspase-3 separates the quencher from the chelate and thus recovers europium fluorescence. The sensitivity of the assay was 1 pg/μl for active caspase-3 and 200 pM for the substrate. We evaluated the assay for high-throughput usage by screening 9600 small-molecule compounds. We also evaluated this format for absorption/distribution/metabolism/excretion assays with cell lysates. Additionally, the assay was compared to a commercial fluorescence caspase-3 assay.

Novel detection strategies for drug discovery.

The Human Genome Project is expected to increase the number of potential drug targets from the current figure of 500 to approximately 3,000-4,000. This increased number of targets, and increasing knowledge of signaling-pathway networks and their complexities, sets new demands for efficiency on HTS assay technologies. Assessment of the total efficacy of a given drug candidate requires not only the classical assays, but also a wide variety of assays related to signaling cascades and cellular functions. Discrete functional assays traditionally involved Ca(2+) flux, kinases and cAMP, but today extend to the whole signaling network, from ligand binding to expression. This review discusses emerging novel non-radioisotopic assays, such as ligand-stimulated GTP-binding, the inositol triphosphate assay, cellular receptor trafficking, and protein-protein interactions.