Direct solid-phase time-resolved immunofluorometric assay of cortisol in serum.

Abstract A dissociation-enhanced lanthanide fluoroimmunoassay of serum cortisol based on time-resolved fluorescence is described. The assay is a direct assay, where cortisol immobilized on the wall of a microtiter-strip well competes with cortisol in the sample for the europium-labeled polyclonal antibody. The amount of bound europium-labeled antibody is inversely proportional to the amount of cortisol in the sample. Separation is accomplished by washing the strip well. The assay is carried out in 2 h, at room temperature; it is easy to perform and gives accurate and reliable results. A chaotropic agent, trichloracetic acid, was very effective in releasing cortisol from binding proteins. This finding will have practical importance in the immunoassay field.

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Detection of Oligonucleotide Hybridization on a Single Microparticle by Time-Resolved Fluorometry: Hybridization Assays on Polymer Particles Obtained by Direct Solid Phase Assembly of the Oligonucleotide Probes

Bioconjugate Chemistry ◽

10.1021/bc970033c ◽

1997 ◽

Vol 8 (3) ◽

pp. 378-384 ◽

Cited By ~ 23

Author(s):

Harri Hakala ◽

Pia Heinonen ◽

Antti Iitiä ◽

Harri Lönnberg

Keyword(s):

Solid Phase ◽

Oligonucleotide Probes ◽

Polymer Particles ◽

Time Resolved ◽

Oligonucleotide Hybridization ◽

Direct Solid ◽

Hybridization Assays

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

Clinical Chemistry ◽

10.1093/clinchem/34.8.1640 ◽

1988 ◽

Vol 34 (8) ◽

pp. 1640-1644 ◽

Cited By ~ 5

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.

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

Clinical Chemistry ◽

10.1093/clinchem/32.7.1323 ◽

1986 ◽

Vol 32 (7) ◽

pp. 1323-1327 ◽

Cited By ~ 18

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.

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A Simple Time-Resolved Fluoroimmunoassay of Total Thyroxine in Serum

Annals of Clinical Biochemistry International Journal of Laboratory Medicine ◽

10.1177/000456328902600305 ◽

1989 ◽

Vol 26 (3) ◽

pp. 238-243 ◽

Cited By ~ 4

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.

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Ultra-trace level determination of advanced molecular tracers by combined solid-phase extraction (SPE)-time resolved fluorescence (TRF) for oil reservoir application

10.1021/scimeetings.0c02779 ◽

2020 ◽

Author(s):

Wei Wang

Keyword(s):

Solid Phase ◽

Oil Reservoir ◽

Phase Extraction ◽

Time Resolved Fluorescence ◽

Trace Level ◽

Time Resolved ◽

Molecular Tracers ◽

Ultra Trace ◽

Trace Level Determination

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

Clinical Chemistry ◽

10.1093/clinchem/38.4.545 ◽

1992 ◽

Vol 38 (4) ◽

pp. 545-548 ◽

Cited By ~ 24

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.

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Monitoring ovarian function by the simultaneous time-resolved fluorescence immunoassay of two urinary steroid metabolites

Clinical Chemistry ◽

10.1093/clinchem/44.7.1520 ◽

1998 ◽

Vol 44 (7) ◽

pp. 1520-1528 ◽

Cited By ~ 17

Author(s):

Geoff Barnard ◽

Fortune Kohen

Keyword(s):

Room Temperature ◽

Ovarian Function ◽

Early Morning ◽

Fluorescence Immunoassay ◽

Time Resolved Fluorescence ◽

Time Resolved ◽

Menstrual Cycles ◽

Lanthanide Chelates ◽

Coated Plate ◽

Urinary Steroid

Abstract We report the development of a novel time-resolved fluorescence immunoassay utilizing two different assay strategies for the simultaneous measurement of estrone-3-glucuronide (EG) and pregnanediol-3α-glucuronide (PG) in samples of early morning urine (EMU). The method for the measurement of EG involves the use of a labeled anti-idiotype as a surrogate antigen, whereas the other method (for the measurement of PG) is a regular competitive immunoassay using a labeled antigen. In addition, the procedure uses different lanthanide chelates as labels to monitor ovarian function in women. After washing the streptavidin-coated plate, we added 10 μL of undiluted urine or mixed standard to the coated wells, followed by the addition of 100 μL of assay buffer containing the labeled reactants (i.e., europium-labeled PG and samarium-labeled anti-idiotype recognizing the binding site of the antibody to EG). Subsequently, we added 100 μL of assay buffer containing the two biotinylated specific monoclonal anti-steroid glucuronide antibodies. After incubation for 1 h on a shaker at room temperature, we washed the plate and added 200 μL of enhancement solution to each well. We measured europium and samarium fluorescence, using a gated plate fluorometer with appropriate emission filters. The method demonstrates appropriate sensitivity and precision (all CVs, 5–8%) across the relevant working ranges for each analyte. The technique has been applied to serial EMUs collected from women with normal and stimulated menstrual cycles.

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