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.

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.

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.

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 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.

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.

Two sensitive time-resolved fluoroimmunoassays for cellular fibronectin

1995 ◽  
Vol 41 (9) ◽  
pp. 1283-1287 ◽  
Author(s):  
J Kropf ◽  
A M Gressner
Keyword(s):  
Human Plasma ◽  
Detection Method ◽  
Solid Phase ◽  
Sodium Dodecyl ◽  
Time Resolved Fluorescence ◽  
Time Resolved ◽  
Sandwich Type ◽  
Culture Supernatants ◽  
Cellular Fibronectin

Abstract Two sensitive sandwich-type immunoassays for determination of cellular fibronectin (cFN) in cell culture supernatants and in human plasma were developed. Both assays used a monoclonal antibody with specificity against the EDA sequence, which is characteristic for the cellular form of human and rat FN. Assay 1 involves binding of FN on gelatin-coated microwells followed by reaction with the anti-cFN antibody, whereas in assay 2 the anti-cFN antibody was immobilized first and detection was with an anti-FN antiserum. Time-resolved fluorescence spectrophotometry with measurement of an Eu3+ chelator after dissociation of the solid-phase complexes with urea/sodium dodecyl sulfate in the presence of excess Eu3+ was the detection method. The detection limit of the new assays was between 2.6 and 4.0 micrograms/L cFN. In serial dilution of human plasma samples, parallelism with the calibration curve was obtained over the whole measuring range (12-1000 micrograms/L) with assay 2, whereas assay 1 had deviations of the dilution curves at concentrations > or = 100 micrograms/L. The between-run CVs for assays 1 and 2 were 11.4% and 7.2%, respectively, at a concentration of 200 micrograms/L (median value of 18 experiments). Respective within-series CVs of 4.3% and 4.7% were obtained at the same concentration. The recovery of added cFN from human plasma was between 90% and 96%.

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.

Two new two-step immunoassays for free thyroxin evaluated: solid-phase radioimmunoassay and time-resolved fluoroimmunoassay

1990 ◽  
Vol 36 (7) ◽  
pp. 1355-1360 ◽  
Author(s):  
P Nuutila ◽  
P Koskinen ◽  
K Irjala ◽  
L Linko ◽  
H L Kaihola ◽  
...  
Keyword(s):  
Solid Phase ◽  
Equilibrium Dialysis ◽  
Late Pregnancy ◽  
Clinical State ◽  
Albumin Concentration ◽  
Time Resolved ◽  
One Step ◽  
Solid Phase Radioimmunoassay ◽  
Dialysis Method ◽  
Method R

Abstract We measured concentrations of free thyroxin (FT4) in serum by using two new two-step FT4 assays--a solid-phase two-step radioimmunoassay. Spectria, and a time-resolved fluoroimmunoassay. Delfia--and compared the results with those by a two-step FT4 assay (RIA-gnost), a one-step FT4 analog assay (Amerlex-M), and FT4 measured after equilibrium dialysis. The new FT4 assays classified 30 hypothyroid and 43 hyperthyroid patients (untreated) well. In 138 patients with nonthyroidal illness (NTI) and in late pregnancy (n = 36), fewer subnormal FT4 values were reported by Spectria (P less than 0.001), Delfia (P less than 0.001), and RIA-gnost (P less than 0.01) than by Amerlex-M. The results of the Spectria and Delfia methods correlated with the results of the dialysis method (r = 0.76) in NTI patients and pregnancy, and were in better agreement with the clinical state than was FT4 by Amerlex-M. The FT4 values by Amerlex-M, but not by other methods, correlated with albumin concentration. We conclude that these new two-step methods present good alternatives for FT4 analysis.

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.

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