Europium Chelates Research Articles

Development of a time-resolved immunofluorometric assay for quantitation of mucosal and systemic antibody responses

We developed a solid phase immunoassay that measured mucosal and systemic antibody responses from mice inoculated with either a staphylococcal enterotoxin B vaccine (SEBv) or noninfectious virus-like particles (VLP) of lentiviral origin. The assay used time-resolved fluorescence (TRF) with affinity-purified goat anti-mouse IgA and IgG conjugated to samarium and europium chelates, respectively. By employing these fluorogenic conjugates with different spectral emissions, IgA and IgG specific for SEB or VLP were readily detected in serum and saliva from mice inoculated intranasally. The TRF assay detected antigen-specific IgA in saliva 10 min after the addition of enhancement solution, while a conventional alkaline phosphatase-based assay for salivary IgA required 18 h after substrate addition. The TRF assay also provided a significantly higher signal-to-noise ratio and exhibited greater sensitivity. TRF assays detected both IgA and IgG in the same well, thereby reducing sample and reagent requirements.

Polyvinylamine-streptavidin complexes labeled with a europium chelator: a universal detection reagent for solid-phase time resolved fluorometric applications

Objectives: To describe the synthesis and characteristics of a new streptavidin-based universal detection reagent which is multiply labeled with the europium chelate of 4,7-bis(chlorosulfophenyl)-1,10-phenanthroline-2,9-dicarboxylic-acid (BCPDA). Methods and results: Polyvinylamine (PVA) was first labeled with biotin(b) and then with BCPDA to create (b) x-PVA-(BCPDA) y. By mixing controlled amounts of this complex with streptavidin (SA) and a fixed amount of Eu 3+, we were able to produce the conjugate (SA) z-(b) x-PVA(BCPDA) y-Eu 3+. This conjugate is reactive, highly fluorescent, and stable for at least 12 months. It was used to develop model solid-phase time-resolved fluoroimmunoassays for biotinylated mouse IgG and prostate spesific antigen (PSA). Detection limits achieved were around 1 to 2 ng/L (∼3×10 −18 moles/assay). Conclusions: A new universal detection reagent was synthesized, which can be used in combination with biotinylated reagents (e.g., antibodies, DNA probes, etc.) for the development of highly sensitive solid-phase time-resolved fluorescence-based assays.

Europium(III) and terbium(III) chelates of quinolonecarboxylic acid derivatives as labels for immunofluorimetric assay

Luminescence properties of Tb(III) and Eu(III) complexes of quinolonecarboxylic acid derivatives were studied. Optimal conditions of luminescence were determined, and the influence of surfactants and diethylenetriaminepentaacetic acid on the luminescence properties of the complexes was studied. It was demonstrated that species-specific immunoglobulins labeled with terbium ions can be determined with the detection limit of the lanthanide label 5 x 10-14 M.

Multiplex immunoassays on size-categorized individual beads using time-resolved fluorescence

Miniaturized multiplex immunoassays were studied on individual beads to detect analytes in the same reaction mixture. With this approach hands-on time, cost, and amount of reagents as well as waste produced in the assays were reduced. Particles were categorized according to size for immunoassays of prostate specific antigen (PSA) and acute myocardial infarction (AMI) markers. Additionally, free and dual PSA assays were carried out on a single bead. The analyte concentration was detected directly on the surface of the beads using stable, intrinsically fluorescent europium and terbium chelates, and time-resolved fluorometry. Less than 0.4ngml−1 PSA (corresponding to ca. 10amol) was detected in free, dual and multiplex PSA assays and 0.1 and 2.4ngml−1 of myoglobin (Mb) and creatine kinase MB (CK-MB), respectively, were monitored in the multiplex AMI marker assay. The effect of bead size and material on free PSA detection was also investigated. The beads were detected three times under different conditions; in liquid, after drying and after dissociating europium ions from the chelate into the DELFIA® fluorescence enhancement solution. The same detection sensitivity was found for the dissociative and non-dissociative methods indicating that the current labeling technology for the surface detection is comparable to the commercial DELFIA system.

Quantification of glycoalkaloids in tomato plants by time-resolved fluorescence using a europium chelator entrapped in liposomes.

In the control of tomato transgenic cultivars it is important to determine the glycoalkaloid content, which requires the screening of many samples. We have developed a simple method for the extraction and determination of glycoalkaloids in the leaf and fruit extracts of tomatoes using a europium chelator entrapped in phosphatidylcholine-cholesterol containing liposomes. The concentration values were quantified from liposome lysis effected by glycoalkaloid action. The fluorescent signal is linear between 1 and 10 micrograms of tomatine, which makes the method useful for the analysis of tomato samples.

Quantitative Reverse Transcription-PCR Assay with an Internal Standard for the Detection of Prostate-specific Antigen mRNA

Circulating prostate cells can be detected with a reverse transcription-PCR (RT-PCR) assay for prostate-specific antigen (PSA) mRNA. We have developed a new quantitative RT-PCR method for measuring PSA mRNA. The method uses a PSA-like internal standard (IS) mRNA that is added into the sample at the beginning of the RNA extraction and coamplified by RT-PCR with the PSA in the sample. After PCR amplification, the IS and PSA products are selectively detected by hybridization in a microtitration plate using probes labeled with fluorescent europium chelates. The method was validated with PSA and IS mRNAs and PSA-expressing cells to obtain a detection limit of 50 PSA mRNA copies (i.e., signal 2 times the mean of zero signal), linearity up to 10(6) copies, and detection of a single PSA-expressing cell. In preliminary evaluations, 60% (n = 10) of the prostate cancer patients with skeletal metastases gave results above the detection limit (500 PSA mRNA copies in 5 mL of blood). The total number of PSA copies ranged from 900 +/- 200 to 44 100 +/- 4900 (mean +/- SD) in the samples, corresponding to approximately 1-100 PSA-expressing cells in 5 mL of blood. In the controls (n = 34), none of the healthy females and 2 of 19 healthy males had detectable PSA mRNA [700 +/- 100 and 2000 +/- 900 (mean +/- SD) PSA mRNA copies in 5 mL of blood for the 2 males]. The assay provides sensitive and quantitative detection of PSA mRNA expression from blood samples and can be used to establish the clinically significant number of PSA mRNA copies in prostate cancer.

Light emission from sources located within metallodielectric planar microcavities

A simple, rigorous electromagnetic formula is derived for predicting the electromagnetic power provided by sources located in transparent or dissipative planar microcavities. With this simple approach, we compare numerically and experimentally the electromagnetic power that escapes the microcavity when the source is located in a metallodielectric or in an all-dielectric resonant planar structure. Although a strong light-extraction coefficient might be expected for metallodielectric microcavities, we show that these attractive structures suffer from metal absorption even when thin metallic layers are used. Experiments implemented with europium chelates located in metallodielectric or in all-dielectric microcavities confirm this result.

Time-resolved fluorescence imaging (TRFI) for direct immunofluorescence of PSA and alpha-1-antichymotrypsin in prostatic tissue sections.

We have developed a direct immunofluorescence technique utilising chelates of the lanthanide ions europium and terbium conjugated to monoclonal IgGs (Mabs) against prostate-specific antigen (PSA) and alpha-1-antichymotrypsin (ACT) for the detection and quantification on the same tissue section. Strong signals without disturbance from tissue autofluorescence were demonstrated in paraffin sections of ten benign and six malignant prostate tissue specimens. The signal intensity increased linearly with the amount of labelled Mab until epitope saturation began. The highest concentrations of bound IgG in tissue sections were 27.3 fmol/pixel for ACT and 7.2 for PSA. Time-resolved fluorescence imaging (TRFI) offers an attractive method for histochemical studies based on specific and quantitative detection of fluorescent lanthanide chelates.

Synthesis of novel europium-labeled estradiol derivatives for time-resolved fluoroimmunoassays.

The O-(5-carboxypentyl)-, O-(4-aminobutyl)-, O-(6-aminohexyl)oximes of 2- and 4-formylestradiol as well as the 4-carboxyethylthioether derivative of estradiol were synthesized. These estradiol derivatives were characterized using IR-, 1H-, and 13C NMR spectroscopy. All synthesized estradiol derivatives were labeled with europium chelates. These "tracers" were purified and tested in a competitive time-resolved fluoroimmunoassay with monoclonal antibody (SSI 57-2) raised against the 6-O-(carboxymethyl)oxime-bovine serum albumin derivative of estradiol. All the tested europium-labeled estradiol-4-derivatives were bound by the antibody, whereas tracers linked via position 2 were not recognized by this antibody. It was observed that tracers conjugated via C-4 gave more sensitive standard curves than tracers conjugated via C-6. Especially, the estradiol-4-thioether derivative was found to be highly useful in time-resolved fluoroimmunoassays of estradiol while using this antibody.

Time-Resolved Fluorometric Detection of DNA Using a Tetradentate β-Diketonate Europium Chelate as a Label

A tetradentate β-diketonate europium chelate, 4,4′-bis(1″,1″,1″,2″,2″,3″,3″-heptafluoro-4″,6″-hexanedion-6″-yl)-chlorosulfo-o-terphenyl(BHHCT)-Eu3+, was labeled to a streptavidin-bovine serum albumin conjugate for time-resolved fluorometric detection of DNA by hybridization with a biotinylated probe DNA in a microtiter well. The method gave a detection limit of 23 pg/well (solid-phase measurement) and 4 pg/well (solution-phase measurement) for specific DNA.

Highly Sensitive Detection of Bensulfuron-methyl by Time-Resolved Fluoroimmunoassay Using a Tetradentate β-Diketonate Europium Chelate as a Label

A tetradentate β-diketonate europium fluorescent chelate, 4,4′-bis(1″,1″,1″,2″,2″,3″,3″-heptafluoro-4″,6″-hexanedion-6″-yl)-chlorosulfo-o-terphenyl (BHHCT)-Eu3+, was used as a label for highly sensitive time-resolved fluoroimmunoassay of bensulfuron-methyl (BSM). A BSM-BSA (BSA=bovine serum albumin) conjugate was labeled with BHHCT-Eu3+ and the labeled BSM-BSA conjugate was used for competitive immunoassay. The assay was carried out in a 96-well microtiter plate coated with the anti-BSM monoclonal antibody. The method gives the detection limit of 24 pg/ml, which is one to two orders of magnitude lower than those of the conventional HPLC and other methods. The RSD of the present method is less than 10%, and the recovery is in the range of 90 - 110% for BSM water samples.

Highly sensitive quantitation of methamphetamine by time-resolved fluoroimmunoassay using a new europium chelate as a label.

A simple and highly sensitive time-resolved fluoroimmunoassay of methamphetamine (MA) using a new fluorescent europium chelate (BHHCT-Eu3+) as a label is described. Two variations of competitive immunoassay were attempted. In the first (one-step) assay, microtiter plates coated with anti-MA were used, and the new label was bound to a conjugate of bovine serum albumin and N-(4-aminobutyl)-MA (MA-BSA). In the second (two-step) assay, instead of the labeled MA-BSA, biotinylated MA-BSA and BHHCT-Eu3+-labeled streptavidin-BSA were used. The lowest measurable concentrations of MA for the one-step and the two-step methods were 1 ng/mL (25 pg/assay) and 1 pg/mL (25 fg/assay), respectively. These were 10 to 1000 times superior to the detection limits of MA in any other immunoassay. Intra-assay coefficient of variation was approximately 2-8% at eight different concentrations (n = 4). Analysis of 34 urine samples with the new method and conventional gas chromatography showed a good correlation (r = 0.954). The high detectability of the present assay also enabled segmental hair analysis with a few centimeters of a hair.

Time-resolved fluorescence in immunocytochemical detection of prostate-specific antigen in prostatic tissue sections.

Chelates with fluorescent lanthanides such as europium and terbium are widely used in immunofluorometric assays, e.g. for the measurement of different molecular forms of prostate-specific antigen (PSA) in serum for detection and monitoring of prostate cancer. These chelates have also been introduced as non-radioactive labels in immunocytochemistry and in situ hybridization. In the present study, sections of non-malignant prostate were investigated using monoclonal IgGs against PSA. Detection of specific immunostaining employing time-resolved fluorescence with europium-labeled streptavidin was compared with conventional detection by streptavidin conjugated to horse-radish peroxidase. The high PSA concentration in the tissue produced high intensity, specific time-resolved fluorescence signals in the epithelial cells of the prostate gland without disturbance from non-specific tissue autofluorescense. This allowed short exposure times to be used which resulted in insignificant photobleaching. Two of the three europium-chelates evaluated yielded high signal intensities. Counterstaining was found to be optimal with Gill No. 1-Haematoxylin solution and Merckoglas was the best mounting medium for the europium chelates tested. In conclusion, time-resolved fluorescence imaging is an attractive alternative to conventional detection of streptavidin conjugated to horse-radish peroxidase, as it provides linear, high intensity, specific signals subsequent to the decay of non-specific tissue autofluorescence.

A one-step fluorescent detection method for lipid fingerprints; Eu(TTA) 3·2TOPO

This paper describes the development of a one-step fluorescent lipid reagent that involves europium chelates whose generic formula can be represented as europium(organic ligand) 3·2(synergic reagent). An optimized formulation is presented and a comparison with physical developer was conducted which showed the new technique to be very good when fresh fingerprints were recovered but inferior to physical developer on aged prints (over a week). In addition, the compatibility of the technique with sequential methods such as ninhydrin and DFO is reported. The experimentation, results and chemical formulation of this study were presented to delegates of the International Symposium on Fingerprint Detection and Identification held in Israel in June 1995 [1]. Prior to that a patent application had been made [2].

Evaluation of solution structures of highly luminescent europium(III) chelates by using laser induced excitation of the [formula omitted] transition

Solution structures of 13 Eu(III) chelates were examined by using laser induced excitation of the 7 F 0 → 5 D 0 transition. Remarkable variations in the 7 F 0 → 5 D 0 excitation spectra of 2,2′,2″,2″−{[aryl]bis(methylenenitrilo}tetrakis(acetic acid) complexes of Eu(III) are observed depending on the denticity of the ligand and the number and character of the coordinated nitrogen atoms. The evaluation of the structures is made on the basis of the energy of the 7 F 0 → 5 D o excitation transition of Eu(III) because the 7 F 0 → 5 D 0 transition energy is dependent on the number and type of coordinating atoms in the first coordination sphere of Eu(III). Additional information about the structures is obtained by measuring the excited-state lifetimes of the Eu(III) chelates. The 7 F 0 → 5 D 0 transition energy shifts always an equal amount to lower energies due to the coordination of a certain group or atom. The energies of the 7 F 0 → 5 D 0 excitation transitions are also used to calculate these ‘nephelauxetic’ shift parameters for coordinated nitrogen heteroatoms iin the 2,2′,2″,2″−{[4-phenylethynyl)pyridine-2,6-diyl]bis (methylenenurido)}tetrakis(acetic acid) ( 3). 2,2′,2″,2″-[(2,2′-bipyridine-6,6′-diyl)bis-(methylenenitrilo)] tetrakis (acetic acid) ( 4). 2,2′,2″,2″-[(2,2′:6,2″-terpyridine-6,6″-terpyridine-6.6″-diyl)bis(methylenenitrilo)]tetrakis(acetic acid) ( 5), 2,2′,2″,2″- (pyrazole-1,3-diyl)-bis(pyridine)-2,2′-diyl]bis(methylenenitrilo)]tetrakis(acetic acid) ( 7). 2,2′,2″,2″-([thizole-2,4-diyl)bis(pyridine)- 2,2′-diyl]-bis(methylenenitrilo(]tetrakis(acetic acid) ( 8) and 2,2′,2″,2″-([2,2,′-pyridine-2,6-diyl)bis(thiazole)-4,4′-diyl]bis-(methylenenitrilo)]-tetrakis(acetic acid) ( 9) complexes. The variation in the shift parameters of the nitrogen heteroatoms is suggested to be due to the different distances between the nitrogen heteroatoms and Eu(III) ions.

Indirect time-resolved fluorescence detection of both non-fluorescent and fluorescent compounds separated by high-performance liquid chromatography

Indirect time-resolved fluorescence (ITRF) detection of non-fluorescent phenylthiohydantoin (PTH)-Ala and fluorescent dansyl (DNS)-Ala separated by high-performance liquid chromatography (HPLC) was carried out. Addition of europium (Eu 3+) chelates to the eluent makes it possible to detect both non-fluorescent and fluorescent compounds simultaneously. This is because the fluorescence lifetime of Eu 3+ chelates is extremely long, whereas that of normal fluorescent compounds is short. The HPLC conditions with the Eu 3+ chelate system were studied.

Sensitive bioaffinity assays with individual microparticles and time-resolved fluorometry

Future immunoassays and nucleic acid hybridization assays will be performed in miniaturized formats that utilize microchips or microparticles. This will require a sensitive detection technology that allows spatial resolution. By using fluorescent europium chelates and time-resolved microfluorometry, one can detect 11,000 europium molecules on individual microparticles. In a miniaturized noncompetitive immunoassay of prostate-specific antigen (PSA), we quantitatively detected 5 ng/L (0.05 amol per particle) of the analyte on an individual microparticle with excellent precision over the whole measurement range (CV <10%). Using a hybridization assay, we also could detect the deltaF508 mutation for cystic fibrosis on individual microparticles. Consequently, fluorescent lanthanide chelate labels and time-resolved microfluorometry qualify as the next generation of technology in this field.

Dual-label time-resolved immunofluorometric assay for simultaneous determination of pregnancy-associated plasma protein A and free β-subunit of human chorionic gonadotrophin

Using time-resolved fluorometry, a simple one-step dual-label immunometric assay has been developed, which allows simultaneous determination of pregnancy-associated plasma protein A (PAPP-A) and free β-subunit of human chorionic gonadotrophin (β-hCG) in first-trimester maternal serum samples. Two monoclonal antibodies were biotinylated and immobilized onto the surface of streptavidin-coated microtitration plates, and used to capture PAPP-A and β-hCG, respectively. Europium (Eu) and Samarium (Sm) chelates were conjugated to two additional monoclonal antibodies acting as detection antibodies for PAPP-A and β-hCG. The assay was performed using a 4-h one-step format. The within-run precision with buffer-based calibrators was below 8% over the working range of PAPP-A (40–10 000 mIU/l) and β-hCG (7.3–525 μg/l) and no hook effect was observed. The intra- and inter-assay coefficients of variation were below 7.1% for serum samples. PAPP-A and β-hCG concentrations measured by the dual assay in 39 first-trimester serum samples correlated excellently with those obtained by DELFIA single-label PAPP-A ( r=0.997) and the β-hCG part ( r=0.993) of the DELFIA AFP/βhCG dual-label assay.

Simple triple-label detection of seven cystic fibrosis mutations by time-resolved fluorometry

We describe a simple hybridization assay performed in microtitration wells with use of DNA probes labeled with three different lanthanide chelates for detection of seven mutations that cause cystic fibrosis. The assay is based on DNA amplification of four fragments containing the mutations (delta F508, G1717-->A, G542X, R553X, 3905 insertion T, W1282X, and N1303K) by PCR, followed by hybridization with short, allele-specific oligonucleotide probes labeled with europium, terbium, or samarium chelates. Because the technology makes it possible to hybridize three DNA probes simultaneously in one reaction, all 14 mutation-related alleles were detected in a total of five reaction wells. Blood spot specimens, obtained from children with cystic fibrosis, their parents, and their siblings, have been assayed, and for all the probes the positive signal-to-noise ratios are > 10. Solution hybridization utilizing triple-label time-resolved fluorometry combined with PCR is a suitable procedure for large-scale screening and automation.

Thiol-Reactive, Luminescent Europium Chelates: Luminescence Probes for Resonance Energy Transfer Distance Measurements in Biomolecules

Lanthanide chelates have recently been shown to be extremely promising luminescence probes for distance measurements in biomolecules using luminescence resonance energy transfer measurements [P. R. Selvin, T.M. Rana, and J.E. Hearst (1994)J. Am. Chem. Soc.116, 6029–6030; P.R. Selvin, and J.E. Hearst (1994)Proc. Natl. Acad. Sci. USA91, 10024–10028]. In this work we describe simple procedures for preparing highly fluorescent thiol-reactive europium chelates. These new compounds contain a uv-absorbing coumarin group which sensitizes europium emission, diethylenetriaminepentaacetic acid or triethylenetetraaminehexaacetic acid groups which provide europium chelating function, and a pyridyl disulfide group which allows specific modification of thiol groups. These reagents can be used to label proteins at Cys residues or synthetic oligonucleotides which contain thiol groups. Modification can be reversed easily by treatment with a reducing agent (dithiothreitol). Luminescence energy transfer between these new chelates and CY5 fluorochrome attached to the opposite ends of 15-bp double-stranded DNA was measured to test their usefulness for distance measurements in macromolecules. The distance measured between the chelate (donor) and CY5 (acceptor) was in the range expected for the length of 15-bp DNA. The stability of europium chelates and their conjugates with a protein, the precision of distance measurements using these chelates, possible errors due to intramolecular energy transfer, and the modulation of theR0value with deuterium oxide were tested. The results obtained fully confirmed the great potential of these new probes for sensitive, simple, and precise distance measurements in biomolecules using luminescence resonance energy transfer.