Immunofluorometry of choriogonadotropin by time-resolved fluorescence spectroscopy, with a new europium chelate as label.

1987 ◽  
Vol 33 (11) ◽  
pp. 1994-1999 ◽  
Author(s):  
M J Khosravi ◽  
E P Diamandis
Keyword(s):  
Monoclonal Antibody ◽  
Dynamic Range ◽  
Solid Phase ◽  
High Dose ◽  
Nitrogen Laser ◽  
Time Resolved ◽  
Hook Effect ◽  
Step Procedure ◽  
Sandwich Type ◽  
Europium Chelate

Abstract We describe a new "sandwich"-type non-isotopic immunoassay for human choriogonadotropin (hCG) in serum. In the assay, hCG is captured by a beta-subunit-specific monoclonal antibody, which is immobilized in a white microtiter well. The sandwich is completed by adding a second biotinylated monoclonal antibody specific for the whole hCG molecule. The degree of binding of biotinylated antibody, which is proportional to the amount of hCG present in the sample, is quantified by adding streptavidin labeled with the europium chelate 4,7-bis(chlorosulfophenyl)-1,10-phenanthroline-2,9-dicarboxylic acid (BCPDA), in the presence of excess Eu3+. The fluorescent complex formed on the solid-phase [monoclonal antibody-hCG-monoclonal antibody-biotin-streptavidin-BCPDA-Eu3+] is measured by excitation at 337.1 nm with a nitrogen laser and monitoring the emission at 615 nm in a specially designed gated fluorometer working in a time-resolved mode. A two-step procedure is proposed for routine use to avoid the "high-dose hook effect" of the simpler and faster one-step procedure. The hCG assay described has a dynamic range of 1 to 500 int. units/L, and is precise and accurate. Results agree well with those obtained with a commercially available immunoradiometric and a time-resolved immunofluorometric procedure.

Time-resolved immunofluorometric assay of alpha-fetoprotein in serum and amniotic fluid, with a novel detection system.

1987 ◽  
Vol 33 (11) ◽  
pp. 2000-2003 ◽  
Author(s):  
M A Chan ◽  
A C Bellem ◽  
E P Diamandis
Keyword(s):  
Monoclonal Antibody ◽  
Amniotic Fluid ◽  
Dynamic Range ◽  
Solid Phase ◽  
Detection System ◽  
Alpha Fetoprotein ◽  
Nitrogen Laser ◽  
Time Resolved ◽  
Broad Dynamic Range ◽  
Sandwich Type

Abstract We describe a new "sandwich"-type nonisotopic immunoassay of alpha-fetoprotein (AFP) in serum and amniotic fluid. In the assay, AFP binds to a monoclonal antibody immobilized in a microtiter well and to a polyclonal soluble biotinylated antibody. A fluorescent product is created on the solid-phase after adding streptavidin labeled with a new Eu3+ chelate, 4,7-bis(chlorosulfophenyl)-1,10-phenanthroline-2,9-dicarboxylic acid (BCPDA), and excess Eu3+. The fluorescent complex, monoclonal antibody-AFP-polyclonal antibody-biotin-streptavidin-BCPDA-Eu3+, is quantified by nitrogen laser excitation at 337.1 nm, the emission at 615 nm being monitored in an especially designed gated fluorometer working in a time-resolved mode. This assay of AFP has a broad dynamic range (up to 1 mg/L), and is precise and accurate. The detection limit is approximately 0.1 microgram/L. Results agree well with those obtained by established techniques.

Sensitive time-resolved fluorescence immunoassay of somatotropin in serum

1990 ◽  
Vol 36 (3) ◽  
pp. 503-508 ◽  
Author(s):  
I Kahan ◽  
A Papanastasiou-Diamandi ◽  
G Ellis ◽  
S K Makela ◽  
J McLaurin ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Pediatric Patients ◽  
Solid Phase ◽  
Nitrogen Laser ◽  
Fluorescence Immunoassay ◽  
Time Resolved Fluorescence ◽  
Time Resolved ◽  
Sandwich Type ◽  
Europium Chelate ◽  
Growth Abnormalities

Abstract We describe a new "sandwich"-type non-isotopic immunoassay for human somatotropin (GH, growth hormone) in serum. In the assay, GH is captured by a monoclonal antibody immobilized in a white microtiter well and simultaneously reacted with a second biotinylated monoclonal antibody. The degree of binding of biotinylated antibody, which increases with increasing amount of GH in the sample, is quantified by adding streptavidin labeled with the europium chelate of 4.7 - bis(chlorosulfophenyl) - 1.10 - phenanthroline - 2.9 - dicarboxylic acid. The fluorescent complex on the solid phase is then measured by excitation at 337.1 nm (nitrogen laser) and monitoring the emission at 615 nm in a gated fluorometer/analyzer. The proposed procedure has short incubation times (less than 4 h protocol), uses only 25 microL of serum per microtiter well, and gives precise and accurate results. The method was clinically evaluated with samples obtained from pediatric patients undergoing investigation for growth abnormalities and from a patient with acromegaly.

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.

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.

Europium and samarium as labels in time-resolved immunofluorometric assay of follitropin.

1987 ◽  
Vol 33 (1) ◽  
pp. 48-51 ◽  
Author(s):  
R Bador ◽  
H Déchaud ◽  
F Claustrat ◽  
C Desuzinges
Keyword(s):  
Solid Phase ◽  
Lanthanide Ions ◽  
Nitrogen Laser ◽  
Plasma Samples ◽  
Diethylenetriaminepentaacetic Acid ◽  
Time Resolved ◽  
Step Procedure ◽  
One Step ◽  
The Difference ◽  
Incorporation Ratio

Abstract This time-resolved immunofluorometric assay for human follitropin involves use of europium- or samarium-labeled monoclonal antibodies, with an average incorporation ratio of 3 mol of Eu3+ or Sm3+ per mole of antibody. These lanthanide ions are bound to the antibody molecules by means of the anhydride of diethylenetriaminepentaacetic acid. The solid-phase antibody is immobilized inside polystyrene tubes in which plasma samples were assayed in a one-step procedure. After incubation, the fluorescence intensity of Eu3+ or Sm3+ label is measured by time-resolved fluorometry, with a nitrogen laser as the pulsed excitation source. The sensitivity of the assay is largely better with Eu3+ than with Sm3+ because of the difference in their intrinsic luminescence properties. Results obtained with the proposed methods correlated well with those by an immunoradiometric method.

Time-resolved fluoroimmunoassay of human choriogonadotropin.

1983 ◽  
Vol 29 (1) ◽  
pp. 60-64 ◽  
Author(s):  
K Pettersson ◽  
H Siitari ◽  
I Hemmilä ◽  
E Soini ◽  
T Lövgren ◽  
...  
Keyword(s):  
Dynamic Range ◽  
Solid Phase ◽  
Cross Reactivity ◽  
Sandwich Assay ◽  
Time Resolved ◽  
Alpha Subunits ◽  
Human Choriogonadotropin ◽  
Step Procedure ◽  
One Step ◽  
The One

Abstract We describe time-resolved fluoroimmunoassay for human choriogonadotropin involving monoclonal antibodies directed against the beta- and alpha-subunits. The latter antibody was labeled with europium, which was measured by counting for 1 s after the immunoreaction was completed. In the solid-phase sandwich assay, both a one-step and two-step procedure were used; the respective measuring ranges were 0.7-135 and 0.7-350 int. units/L, the latter covering a 500-fold dynamic range. The CV within the assay range was between 4 and 8%, depending on the dose. Cross reactivity with lutropin in the one- and two-step procedures was 1.6% and 1.0%, respectively.

Determination of Fetal Hemoglobin by Time-Resolved Immunofluorometric Assay

1992 ◽  
Vol 38 (10) ◽  
pp. 2013-2018 ◽  
Author(s):  
U Turpeinen ◽  
U H Stenman
Keyword(s):  
High Performance ◽  
Fetal Hemoglobin ◽  
Gamma Chain ◽  
Time Resolved ◽  
Assay Procedure ◽  
Analytical Recovery ◽  
Sandwich Type ◽  
Capture Antibody ◽  
Europium Chelate

Abstract We have developed a "sandwich"-type time-resolved immunofluorometric assay (IFMA) for fetal hemoglobin (HbF) in hemolysates from adults and newborns, amniotic fluid, and plasma, based on a polyclonal and a monoclonal antibody against human fetal hemoglobin. Microtiter wells are coated with polyclonal capture antibody, and the gamma-chain-specific monoclonal tracer antibody is labeled with a europium chelate. In a simple and fast assay procedure, prediluted hemolysates are incubated in the microtiter wells first with capture antibody for 1 h and, after washing, for 1 h with tracer antibody. The wells are washed and the fluorescence of europium is measured. The mean analytical recovery is 102% and results by IFMA agreed well with values obtained by high-performance liquid chromatography. The analytical range of IFMA is large and well suited for clinical purposes. The detection limit of the assay is 0.2 microgram/L and the measuring range extends to 500 micrograms/L.

scholarly journals Streptavidin-Polyvinylamine Conjugates Labeled with a Europium Chelate: Applications in Immunoassay, Immunohistochemistry, and Microarrays

2000 ◽  
Vol 46 (9) ◽  
pp. 1450-1455 ◽  
Author(s):  
Andreas Scorilas ◽  
Anders Bjartell ◽  
Hans Lilja ◽  
Christina Moller ◽  
Eleftherios P. Diamandis
Keyword(s):  
Solid Phase ◽  
Specific Antigen ◽  
Microarray Experiment ◽  
Immunohistochemical Localization ◽  
Macromolecular Complex ◽  
Serum Samples ◽  
Time Resolved ◽  
Europium Chelate ◽  
Highly Correlated ◽  
Detection Reagent

Abstract Background: The favorable properties of lanthanide chelates compared with conventional fluorescent probes have attracted considerable interest. A Eu3+ chelator, 4,7-bis(chlorosulfophenyl)-1,10-phenanthroline-2,9-dicarboxylic acid (BCPDA), has been synthesized previously. Methods: We here describe immunoassay, immunohistochemistry, and microarray applications of a new streptavidin-based universal polyvinylamine (PVA) detection reagent that is multiply labeled with the europium chelate of BCPDA. Solid-phase time-resolved immunofluorometric assays for biotinylated mouse IgG and prostate-specific antigen (PSA) were developed using the new conjugate as a detection reagent. The new conjugate was also used for the immunohistochemical localization of PSA expression in paraffin-embedded prostatic tissues. A model microarray with spotted biotinylated antibody as target was also performed. Results: Approximately 50–100 BCPDA moieties were covalently bound to PVA, which was then linked to streptavidin via biotin interaction. The macromolecular complex successfully recognized and bound biotinylated detection reagents, e.g., antibodies. The new reagent enabled measurement of solid phase-immobilized biotinylated mouse IgG with a detection limit of ∼1 pg/assay and demonstrated excellent linearity. In an ELISA-type sandwich PSA assay that included two PSA monoclonal antibodies using the new conjugate as detection reagent, we detected 0.001 μg/L PSA (∼100 fg or ∼3 amol/assay). Serum samples analyzed for PSA by this method and a commercial assay gave highly correlated results. The new reagent enabled excellent immunohistochemical localization of PSA expression in prostate tissues. Using the new reagent in a model microarray experiment with biotinylated mouse IgG as target, we demonstrated excellent spatial resolution of 5- to 10-nL microspots. Conclusions: The new detection reagent may find important applications in biotechnology.

New approach to competitive lanthanide immunoassay: time-resolved fluoroimmunoassay of progesterone with labeled analyte.

1988 ◽  
Vol 34 (3) ◽  
pp. 501-504 ◽  
Author(s):  
H Déchaud ◽  
R Bador ◽  
F Claustrat ◽  
C Desuzinges ◽  
R Mallein
Keyword(s):  
Trichloroacetic Acid ◽  
Solid Phase ◽  
Chelating Agent ◽  
Nitrogen Laser ◽  
Plasma Samples ◽  
Time Resolved ◽  
New Approach ◽  
Protein Conjugate ◽  
Preliminary Extraction ◽  
Approach Time

Abstract This solid-phase competitive europium immunoassay of progesterone in plasma relies on antigen labeling. With this new approach, time-resolved fluoroimmunoassay can attain sensitivity and precision similar to that of conventional radioimmunoassay. The europium-labeling involves coupling diethylenetriaminepentaacetic acid (chelating agent for Eu3+) to a progesterone-protein conjugate. The solid-phase antibody is immobilized inside polystyrene tubes in which plasma samples (50 microL) are assayed directly, without preliminary extraction. After incubation in the presence of trichloroacetic acid, the tubes are washed and the fluorescence intensity of europium is measured by time-wavelength-resolved fluorometry, with a nitrogen laser as the pulsed excitation source. Progesterone values obtained by this procedure agreed well with those obtained by radioimmunoassay (r = 0.98, n = 97). The detection limit was equivalent to that of most RIAs (0.2 micrograms/L).

Multianalyte Immunoassay Based on Spatially Distinct Fluorescent Areas Quantified by Laser-Excited Solid-Phase Time-Resolved Fluorometry

1992 ◽  
Vol 38 (3) ◽  
pp. 338-342 ◽  
Author(s):  
S E Kakabakos ◽  
T K Christopoulos ◽  
E P Diamandis
Keyword(s):  
Infectious Disease ◽  
Solid Phase ◽  
Specific Area ◽  
Time Resolved ◽  
Specific Antibodies ◽  
Highly Sensitive ◽  
Europium Chelate ◽  
Simultaneous Immunoassay ◽  
Diverse Groups ◽  
Multianalyte Immunoassay

Abstract We describe a new multianalyte immunoassay principle and apply it to the simultaneous immunoassay of lutropin, follitropin, choriogonadotropin, and prolactin in serum. The method is based on the coating of distinct areas of polystyrene with analyte-specific antibodies. These antibodies react with the analyte and immobilize it in a specific area while another biotinylated antibody also reacts with the analyte to form a sandwich. After addition of streptavidin labeled with the fluorescent europium chelate of 4,7-bis(chlorosulfophenyl)-1,10-phenanthroline-2,9-dicarboxylic acid, fluorescent areas are formed, the intensity of which is related to the amount of each analyte present in the sample. The fluorescent areas are quantified on the dry solid phase with laser-excited time-resolved fluorometric measurements. The assays developed are highly sensitive, precise, and accurate. We believe that this system shows potential for multianalyte immunoassay of diverse groups of compounds in disciplines such as endocrinology, infectious disease, hematology, and oncology.

Sign in / Sign up

Export Citation Format

Share Document