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I Hemmilä

Publications and source records attributed to I Hemmilä.

At least 19 recordsLinked to original sources
PubMed2005

Progress in lanthanides as luminescent probes.

Lanthanides have recently found applications in different fields of biomolecular and medical research. Luminescent lanthanide chelates have created interest mainly due to their unique luminescent properties, such as their long Stokes' shift and exceptional decay times allowing efficient temporal discrimination of background interferences in the assays, such as immunoassays. Recently, new organometallic complexes have been developed giving opportunities to novel applications, in heterogeneous and homogeneous immunoassays, DNA hybridization assays, high-throughput screening as well as in imaging. In addition, encapsulating the chelates into suitable matrix in beads enables the use of new members of lanthanides extending the emission wavelength to micrometer range and decays from a few microseconds to milliseconds. As the luminescence is derived from complicated intra-chelate energy transfer, it also gives novel opportunities to exploit these levels in different types of energy transfer based applications. This review gives a short overview of recent development of lanthanide chelate-labels and discusses in more details of energy levels and their exploitation in new assay formats.

Animals↗
PubMed2000

Development of a ubiquitin transfer assay for high throughput screening by fluorescence resonance energy transfer.

An assay based on fluorescence resonance energy transfer (FRET) has been developed to screen for ubiquitination inhibitors. The assay measures the transfer of ubiquitin from Ubc4 to HECT protein Rsc 1083. Secondary reagents (streptavidin and antibody to glutathione-S-transferase [GST]), pre-labeled with fluorophores (europium chelate, Eu(3+), and allophycocyanin [APC]), are noncovalently attached via tags (biotin and GST) to the reactants (ubiquitin and Rsc). When Rsc is ubiquitinated, Eu(3+) and APC are brought into close proximity, permitting energy transfer between the two fluorescent labels. FRET was measured as time-resolved fluorescence at the emission wavelength of APC, almost entirely free of nonspecific fluorescence from Eu(3+) and APC. The FRET assay generated a lower ratio of signal to background (8 vs. 31) than an assay for the same ubiquitination step that was developed as a dissociation-enhanced lanthanide fluoroimmunoassay (DELFIA). However, compared to the DELFIA method, use of FRET resulted in higher precision (4% vs. 11% intraplate coefficient of variation). Quenching of fluorescence was minimal when compounds were screened at 10 microg/ml using FRET. Employing a quick and simple homogeneous method, the FRET assay for ubiquitin transfer is ideally suited for high throughput screening.

Dimethyl Sulfoxide↗
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↗
PubMed1994

Use of fluorescent europium chelates as labels in microscopy allows glutaraldehyde fixation and permanent mounting and leads to reduced autofluorescence and good long-term stability.

In the present study europium chelates were introduced as alternative fluorescent labels for microscopy and their effect on enhanced autofluorescence caused by the glutaraldehyde fixative was investigated. Glutaraldehyde fixation was used to stabilize the cells for a permanent mount after the immunocytochemical reaction. The europium signal in time-resolved fluorescence microscopy was shown to be free of autofluorescence when strong cross-linking fixation with glutaraldehyde was used and the signal-to-background ratio obtained was 2,400 or better. It was also shown that the europium signal was stable in daylight and at room temperature. Fluorescent europium chelate used in this experiment provides excellent contrast and long-term stability for the samples with glutaraldehyde fixation and permanent mounting.

Antibodies, Monoclonal↗
PubMed1993

Europium-labelled recombinant protein G. A fast and sensitive universal immunoreagent for time-resolved immunofluorometry.

Recombinant protein G was labelled with europium by conjugating the protein with Eu3+ chelate of a p-isothiocyanatobenzyl derivative of diethylenetriaminetetraacetic acid, a bifunctional chelating agent specifically optimized for labelling of immunoreagents with lanthanide ions. The labelling produced a universal reagent for time-resolved fluorometric immunoassays based on the principle of dissociative fluorescence enhancement (DELFIA). The optimum labelling level of about eight chelates per protein yielded a highly sensitive and stable reagent which retained its affinity for IgG and exhibited low non-specific binding to coated solid surfaces. The reagent was evaluated in an immunoassay of anti-tetanus antibodies in human serum samples and the results were compared to those obtained with Eu-labelled polyclonal and Eu-labelled monoclonal anti-human IgG antibodies. The detection limit of the assay was 0.003 mU/ml (0.3 microU per assay well). After a 100-fold dilution of the samples, the assay range extended from 0.3 mU/ml to 100,000 mU/ml with a linear range of five log orders. The incubation with Eu-labelled protein G reached equilibrium after a 15 min incubation. The rapid kinetics, the low non-specific background and the high specific binding suggest that Eu-protein G can serve as a universal label for immunoassays based on IgG binding to solid surfaces.

Antibodies, Bacterial↗
PubMed1993

Di- and tetracarboxylate derivatives of pyridines, bipyridines and terpyridines as luminogenic reagents for time-resolved fluorometric determination of terbium and dysprosium.

A group of compounds composed of pyridine, 2,2'-bipyridine and 2,2':6',2''-terpyridine as the energy absorbing, triplet sensitizing moieties and two or four carboxylic acids as the chelating groups were synthesized and their ability to enhance terbium ion luminescence was elucidated. The dicarboxylic acid derivatives form highly luminescent Tb3+ chelates with luminescence quantum yields approaching 50%. Additional groups, such as methoxy groups or additional pyridine rings in the aromatic structure can be utilized to shift the excitation maxima towards longer wavelengths in order to produce more practicable excitation for practical use. The ligands giving the highest Tb3+ emissions were further evaluated in a two-step fluorescence enhancement system constructed for specific binding assays based on the simultaneous use of three or four ions as the labels (Eu3+, Tb3+, Sm3+ and Dy3+). In the two-step procedure, the first step comprises dissociative fluorescence enhancement based on an acidic solution of an aromatic beta-diketone used as the luminogenic ligand, whereafter Eu3+ and Sm3+ are quantitated. In the second step, the lanthanide ions form new chelates with the added ligands. These ligands are able to transfer the excitation energy they have absorbed with high efficiency to the chelated ions and produce high ion luminescence for Tb3+ and Dy3+.

2,2'-Dipyridyl↗
PubMed1993

Expression of the tumour-associated antigen CA-242 in transitional cell bladder tumours: a comparison with CA-50.

The tissue expression of carbohydrate antigen CA-242 was analysed in formalin-fixed biopsy specimens from 147 transitional cell bladder tumours. The staining was related to established prognostic factors and survival during a mean follow-up of over 12 years and the staining results were also compared to expression of CA-50 antigen. Forty-one percent (60/147) of the tumours were negative for CA-242 and 59% (87/147) were positive. Normal bladder mucosa was positive for CA-242 and the umbrella cells in particular showed intense positive staining. In tumours, the umbrella cells were usually positive (when present) and in tumour tissue, positive cells appeared either as individual positive cells or in groups. None of the tumours was entirely positive for CA-242. The tissue expression of CA-242 could not be significantly related to TNM classification, papillary status, WHO grade or quantitative variables (DNA ploidy, S phase fraction, mitotic frequency, nuclear factors). The tissue expression of CA-242 was significantly weaker than the expression of CA-50. The expression of CA-242 was related to favourable prognosis in survival analysis (P = 0.04). The results show that the expression of the novel tumour marker antigen CA-242 as determined in paraffin-embedded material is a weak prognostic factor as compared with established prognostic factors in transitional cell bladder tumours.

Aged↗
PubMed1992

Europium-labelled streptavidin as a highly sensitive universal label. Indirect time-resolved immunofluorometry of FSH and TSH.

Labelling of streptavidin with a fluorogenic europium ion was optimized with the aim of obtaining a universal, stable and highly sensitive non-isotopic label for time-resolved fluorometric immunoassays (TR-FIA) based on dissociative fluorescence enhancement (DELFIA). Even the conjugation of all the free amino groups of streptavidin with Eu chelates had only a minor effect on the binding capacity of the protein or its affinity. The labelled streptavidin was evaluated in indirect time-resolved immunofluorometric assays of human follicle and thyroid stimulating hormones (FSH and TSH). The interassay imprecision was below 3% within the concentration range from 2.5 to 94 U/l for the FSH samples and below 5% in the range from 2.4 to 35 mIU/l for the TSH samples. The detection limits of the assays for FSH and TSH were 0.05-0.10 U/l and 0.01-0.025 mIU/l, respectively, when a CV of 15% was regarded as the acceptable upper limit of imprecision. The results obtained by the indirect assays correlated closely with those obtained by corresponding direct sandwich assays. The model assays demonstrated the utility of Eu-labelled streptavidin as a universal reagent for immunoassays requiring a wide dynamic range and high sensitivity.

Bacterial Proteins↗
PubMed1992

Time-resolved fluorescence imaging of europium chelate label in immunohistochemistry and in situ hybridization.

Fluorescent lanthanide chelates with long decay times allow the suppression of the fast decaying autofluorescence in biological specimens. This property makes lanthanide chelates attractive as labels for fluorescence microscopy. As a consequence of the suppression of the background fluorescence the sensitivity can be increased. We modified a standard epifluorescence microscope for time-resolved fluorescence imaging by adding a pulsed light source and a chopper in the narrow aperture plane. A cooled CCD-camera was used for detection and the images were digitally processed. A fluorescent europium chelate was conjugated to antisera and to streptavidin. These conjugates were used for the localization of tumor associated antigen C242 in the malignant mucosa of human colon, for the localization of type II collagen mRNA in developing human cartilaginary growth plates, and for the detection of HPV type specific gene sequences in the squamous epithelium of human cervix. The specific slowly decaying fluorescence of the europium label could be effectively separated from the fast decaying background fluorescence. It was possible to use the europium label at the cell and tissue level and the autofluorescence was effectively suppressed in in situ hybridization and immunohistochemical reactions in both frozen and formaldehyde-fixed, wax-embedded specimens.

Alkaline Phosphatase↗
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↗
PubMed1991

Co-fluorescence of europium and samarium in time-resolved fluorimetric immunoassays.

In the presence of an excess of Y3+, the fluorescence intensities of Eu3+ and Sm3+, chelated with benzoyltrifluoroacetone (BTA) or thenoyltrifluoroacetone (TTA) in an aqueous solution containing 1,10-phenanthroline, were increased by factors ranging from 209- to 811-fold. This co-fluorescence phenomenon was used in a highly sensitive time-resolved fluorimetric detection of the lanthanides, Eu3+ and Sm3+. The detection limits of Eu3+ in the BTA- and TTA-based solutions were 4 and 15 fmol dm-3, respectively. The detection limits of Sm3+ were 0.11 and 0.12 pmol dm-3, respectively. The co-fluorescence enhancement systems were also applied in the double-label time-resolved fluorimetric immunoassay of luteinizing hormone and follicle stimulating hormone using specific antibodies labelled either with Eu3+ or Sm3+. The co-fluorescence enhancement solution was superior as compared with the commercial 'direct' fluorescence enhancement solution based on the acidic solution of beta-naphthoyltrifluoroacetone, trioctylphosphine oxide and Triton X-100, in respect to the signal level obtained and the sensitivity. It is suited to time-resolved fluorimetric immunoassays in which particularly high detection sensitivities are required, and it can also be used in double-label assays employing Eu3+ and Sm3+ chelate labels.

Europium↗
PubMed1991

Magnetic field dependence of longitudinal relaxation rates of solutions of various protein-gadolinium3+ chelate conjugates.

Bovine serum albumin (BSA) and porcine serum fibrinogen (FIB) were multiply labeled with gadolinium3+ by using three different ligands: DTPA dianhydride, isothio-cyanato-benzyl-DTPA (ITCB-DTPA), in which none of the five coordinating carboxylates is employed for macromolecular linkage, and isothiocyanatobenzyl-TRITA, a macrocyclic ligand. The nuclear magnetic resonance dispersion (NMRD) profiles of the protein-(Gd chelate) conjugates were characteristic to each chelate involved, possessing, as expected, greater longitudinal relaxivities than the corresponding Gd chelates alone and exhibiting prominent peaks at the proton Larmor frequency range of 10 to 40 MHz. Particularly favorable relaxation enhancement was measured in the solutions of BSA-(ITCB-DTPA-Gd) at this field range. When the number of chelates conjugated with protein increased, up to 163 Gd chelates per one fibrinogen, a progressive decrease in relaxivity was observed. This study demonstrates the relaxation properties of novel macromolecular contrast agents designed for magnetic resonance imaging.

Animals↗
PubMed1990

Proton relaxation enhancement of albumin, immunoglobulin G, and fibrinogen labeled with Gd-DTPA.

Bovine serum albumin, immunoglobulin G, and fibrinogen were labeled with Gd-DTPA using a bifunctional chelating agent DTPA anhydride. The protein-(Gd-DTPA) conjugates had 1.4- to 2.0-fold greater longitudinal relaxivities at 0.02 and 0.44 T than the relaxivity of plain Gd-DTPA. The increase of longitudinal relaxivity was not directly related to the size of carrier protein. Up to 50 Gd-DTPA chelates per protein, longitudinal relaxivity of the conjugates was proportional to the concentration of Gd and independent of the Gd/protein ratio.

Animals↗
PubMed1990

Time-resolved fluorescence in biospecific assays.

Fluorescent lanthanide chelates and their detection using time-resolved fluorometry for in vitro as well as for microscopic bio-specific assays are reviewed, with reference to other fluorescent dyes and labelling materials. The minimum detectable dose of labelled components and the theoretical ultimate sensitivities of immunoassays and DNA-assays are discussed and compared with experimental results. It is concluded that the time-resolved fluorometry provides a high sensitivity labelling method in immunoassays, and a sensitivity comparable to phosphorus-32 in DNA hybridization assays. It is also concluded that lanthanide chelates are a potential alternative for organic fluorescent dyes in microscopy and that they are most useful in multiparameter assays. The combination of organic fluorescent dyes and lanthanide chelates provides the highest separation efficiency between two assay parameters.

DNA Probes↗
PubMed1989

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.

Animals↗
PubMed1989

Simultaneous quantification of two plant viruses by double-label time-resolved immunofluorometric assay.

For simultaneous and sensitive detection of two antigens in one sample, monoclonal antibody (MAb) to potato virus M (PVM) was labelled with a lanthanide Eu3+ and MAb to potato virus X (PVX) with another lanthanide Sm3+. A mixture of the labelled MAbs was used as a tracer. After performing the immunoreactions, the fluorescence of the dissociated lanthanides was measured at different wave-lengths in a time-resolved fluorometer to quantificate the PVX and PVM amount in a sample. Double-label time-resolved fluoroimmunoassay (TR-FIA) detected 1 ng/ml of each virus and was therefore more sensitive for simultaneous detection of PVX and PVM than reported for a single virus detection with double antibody sandwich ELISA (DAS-ELISA).

Antibodies, Monoclonal↗