Solid-phase immunoassay of digoxin by measuring time-resolved fluorescence.

1986 ◽  
Vol 32 (9) ◽  
pp. 1767-1769 ◽  
Author(s):  
P Helsingius ◽  
I Hemmilä ◽  
T Lövgren
Keyword(s):  
Time Resolution ◽  
Cardiac Glycoside ◽  
Solid Phase ◽  
Sample Volume ◽  
Time Resolved Fluorescence ◽  
Measuring Time ◽  
Time Resolved ◽  
Solid Phase Immunoassay

Abstract We describe a time-resolved fluoroimmunoassay for the cardiac glycoside digoxin. The assay depends on the competitive distribution of Eu3+-labeled anti-digoxin antibodies between solid-phase-bound digoxin and the digoxin in the sample or standard. After this immunoreaction, the bound fraction of the Eu3+-label is dissociated from the solid phase, converted into a highly fluorescent beta-diketone chelate, and measured in a fluorometer with time-resolution (1230 Arcus, LKB-Wallac). Sample volume is 20 microL, the incubation lasts 30 min, and the concentration range of the assay for digoxin is from 0.25 to 4.0 ng/mL (0.32-5.1 nmol of digoxin per liter). Correlation with an in-house RIA was good (r = 0.97, n = 43). The sensitivity was equivalent to that of most RIAs: 0.2 ng/mL (0.25 nmol/L).

scholarly journals Competitive solid-phase immunoassay of testosterone using time-resolved fluorescence

FEBS Letters ◽  
1984 ◽  
Vol 173 (1) ◽  
pp. 213-216 ◽  
Author(s):  
E. Bertoft ◽  
J.U. Eskola ◽  
V. Näntö ◽  
T. Lövgren
Keyword(s):  
Solid Phase ◽  
Time Resolved Fluorescence ◽  
Time Resolved ◽  
Solid Phase Immunoassay

Sensitive, rapid procedure for time-resolved immunofluorometry of lutropin.

1988 ◽  
Vol 34 (8) ◽  
pp. 1640-1644 ◽  
Author(s):  
M J Khosravi ◽  
R C Morton ◽  
E P Diamandis
Keyword(s):  
Monoclonal Antibody ◽  
Solid Phase ◽  
Detection System ◽  
Time Resolved Fluorescence ◽  
Time Resolved ◽  
Practical Procedure ◽  
Daily Monitoring ◽  
Fluorescence Measurements ◽  
Capture Antibody ◽  
Europium Chelate

Abstract In this new immunofluorometric method for quantification of lutropin in serum, the "sandwich" principle is combined with time-resolved fluorescence measurements, with the europium chelate 4,7-bis(chlorosulfophenyl)-1,10-phenanthroline-2,9-dicarboxylic acid (BCPDA) used as label. A monoclonal antibody to the alpha-subunit of lutropin is adsorbed onto the walls of white-opaque microtiter wells to form the solid-phase capture antibody, and a biotin-labeled soluble monoclonal antibody is used for antigen quantification. The detection system is completed with streptavidin, which has been linked to a protein bulking agent labeled with multiple BCPDA residues. In the presence of excess europium, the fluorescence of the final complex attached to captured lutropin molecules is measured on the dried solid phasse with an automated time-resolved fluorometer. The assay can be performed as a rapid (less than 60 min incubation) or regular (150 min incubation) procedure. The rapid assay is well-suited for routine daily monitoring of increasing or ovulatory lutropin concentrations; the regular assay, with its greater sensitivity (0.5 int. unit/L), is a practical procedure for lutropin measurements in hyposecretory states. The assay measures up to 240 int. units/L, and results compare well with those by a commercially available radioimmunoassay, an immunoradiometric assay, and another time-resolved immunofluorometric procedure.

Laser-excited immunofluorometric assay of prolactin, with use of antibodies coupled to lanthanide-labeled diethylenetriaminepentaacetic acid.

1986 ◽  
Vol 32 (7) ◽  
pp. 1323-1327 ◽  
Author(s):  
H Déchaud ◽  
R Bador ◽  
F Claustrat ◽  
C Desuzinges
Keyword(s):  
Plasma Sample ◽  
Solid Phase ◽  
Nitrogen Laser ◽  
Diethylenetriaminepentaacetic Acid ◽  
Time Resolved Fluorescence ◽  
Time Resolved ◽  
Step Procedure ◽  
Polystyrene Tube ◽  
Sandwich Type ◽  
One Step

Abstract We describe an immunofluorometric assay for prolactin based on lanthanide labeling of a monoclonal antibody and measuring time-resolved fluorescence. In this "sandwich"-type assay, the label (Eu3+) was bound to the second antibody by means of a simple, rapid method involving the anhydride of diethylenetriaminepentaacetic acid. To measure the photoluminescence of europium (or other lanthanides), we have developed a time-resolved fluorometer with a nitrogen laser as the pulsed excitation source. During the assay, the solid-phase antibody immobilized inside a polystyrene tube is incubated with the plasma sample and the second antibody in a one-step procedure. Results for 67 human plasmas correlated well (r = 0.98) with those by an immunoradiometric method.

A Simple Time-Resolved Fluoroimmunoassay of Total Thyroxine in Serum

1989 ◽  
Vol 26 (3) ◽  
pp. 238-243 ◽  
Author(s):  
Anastasia Papanastasiou-Diamandis ◽  
Vipin Bhayana ◽  
Eleftherios P Diamandis
Keyword(s):  
Dicarboxylic Acid ◽  
Binding Proteins ◽  
Solid Phase ◽  
Time Resolved Fluorescence ◽  
Competitive Immunoassay ◽  
Time Resolved ◽  
Serum Thyroxine ◽  
Total Thyroxine

We describe a non-isotopic heterogeneous competitive immunoassay of total thyroxine in serum. Thyroxine, released from its binding proteins by merthiolate (thimerosal), competes with immobilised thyroxine (thyroxine-bovine globulin conjugate) for binding to a monoclonal biotinylated antibody. The amount of biotinylated antibody bound, which is inversely related to the amount of thyroxine in the sample, is then quantified by adding streptavidin labelled with the europium chelator 4,7-bis(chlorosulphophenyl)-1,10-phenanthroline-2,9-dicarboxylic acid (BCPDA) in the presence of excess Eu3+. The complex formed (bovine globulin-thyroxine-antibody-biotin-streptavidin-BCPDA-Eu3+) is measured on the solid-phase by time-resolved fluorescence. The assay is simple to perform and its characteristics are similar to those of other currently used immunoassay techniques.

Ultrasensitive Thyrotropin Immunoassay Based on Enzymatically Amplified Time-Resolved Fluorescence with a Terbium Chelate

1992 ◽  
Vol 38 (4) ◽  
pp. 545-548 ◽  
Author(s):  
A Papanastasiou-Diamandi ◽  
T K Christopoulos ◽  
E P Diamandis
Keyword(s):  
Alkaline Phosphatase ◽  
Detection Limit ◽  
Solid Phase ◽  
Thyroid Stimulating Hormone ◽  
Fluorescence Immunoassay ◽  
Time Resolved Fluorescence ◽  
Time Resolved ◽  
Highly Sensitive ◽  
Assay Time ◽  
Highly Sensitive Detection

Abstract We describe an ultrasensitive, enzymatically amplified time-resolved fluorescence immunoassay of thyrotropin (thyroid-stimulating hormone) in serum with use of a terbium chelate as the detectable moiety. In this assay, thyrotropin is first simultaneously reacted with a solid-phase (microtiter well) monoclonal antibody and a soluble biotinylated monoclonal detection antibody. After washing, a streptavidin-alkaline phosphatase conjugate is added, followed by another washing. Alkaline phosphatase acts on the substrate 5-fluorosalicyl phosphate (FSAP) to produce 5-fluorosalicylic acid (FSA). FSA, but not FSAP, can then form with Tb3+ and EDTA a highly fluorescent ternary complex of long fluorescence lifetime. This complex is quantified with time-resolved fluorometry. The thyrotropin assay is highly sensitive (detection limit approximately 0.003 milli-int. unit/L when a total assay time of 85 min is used), precise, and accurate. The thyrotropin assay can also be completed in less than 30 min (detection limit 0.013 milli-int. unit/L), thus making this procedure a candidate technology for high-throughput automated analyzers.

Time-resolved fluorescence from biological systems: tryptophan and simple peptides

1980 ◽  
Vol 298 (1439) ◽  
pp. 321-334 ◽  
Keyword(s):  
Time Resolution ◽  
Single Photon ◽  
Photon Counting ◽  
Time Resolved Fluorescence ◽  
Time Resolved ◽  
Single Photon Counting ◽  
Fluorescence Measurements ◽  
Mode Locked Lasers ◽  
Electron Transfers ◽  
Sensitive Method

Four methods of applying mode-locked lasers to time-resolved fluorescence measurements in the subnanosecond region are compared. When time resolution below 100 ps is not required, the most precise and sensitive method is single-photon counting, and the application of this method to studies of time-resolved fluorescence of tryptophan in simple peptides is described. The dependence of lifetimes on pH and temperature are interpreted in terms of quenching by intramolecular proton and electron transfers.

scholarly journals Solid-Phase PCR with Hybridization and Time-resolved Fluorometry for Detection of HLA-B27

2001 ◽  
Vol 47 (3) ◽  
pp. 498-504 ◽  
Author(s):  
Minna Sjöroos ◽  
Jorma Ilonen ◽  
Timo Lövgren
Keyword(s):  
Solid Phase ◽  
Detection System ◽  
Pcr Primers ◽  
Dried Blood Spots ◽  
Microtiter Plate ◽  
Covalent Immobilization ◽  
Hla B27 ◽  
Time Resolved Fluorescence ◽  
Time Resolved ◽  
Consecutive Reactions

Abstract Background: Preactivated solid surfaces provide new possibilities for multiple consecutive reactions in a microtiter plate format. In this study, a combination of PCR and subsequent hybridization in the same microtiter well was applied for the detection of HLA-B27 alleles. Methods: A multiplex solid-phase PCR to amplify the HLA-B27 alleles together with β-actin as an amplification control gene was performed on the NucleoLinkTM (Nunc) surface. PCR was followed by hybridization and detection with time-resolved fluorescence. For the covalent capture of the PCR primers onto the solid support via a 1-(3-dimethylamino-propyl)-3-ethylcarbodiimide hydrochloride-mediated reaction, different 5′-end modifications of oligonucleotides were tested [amination, phosphorylation, and a poly(dT)10 linker]. Results: For covalent immobilization of the primers, amination of the 5′ end combined with use of the poly(dT)10 linker was superior. At least 19.5% of the primer added per well was attached via a stable bond. When the standard time-resolved, fluorescence-based HLA-B27 detection system was compared with the newly developed method in a sample series of 82 genomic DNAs and the corresponding dried-blood spots, all results were in full agreement. Conclusions: The new solid-phase PCR approach can be applied for multiple-target DNA detection. PCR followed by hybridization can be accomplished in a few hours using precoated strips and dried-blood spot PCR templates.

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