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.

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.

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.

Measurement of estrone-3-glucuronide in urine by rapid, homogeneous time-resolved fluoroimmunoassay.

1989 ◽  
Vol 35 (4) ◽  
pp. 555-559 ◽  
Author(s):  
G Barnard ◽  
F Kohen ◽  
H Mikola ◽  
T Lövgren
Keyword(s):  
Monoclonal Antibodies ◽  
Liquid Phase ◽  
Clinical Utility ◽  
Ovarian Function ◽  
Fluorescence Immunoassay ◽  
Time Resolved Fluorescence ◽  
Time Resolved ◽  
Europium Chelate ◽  
Covalently Linked ◽  
Sensitivity Specificity

Abstract We describe a liquid-phase nonseparation time-resolved fluorescence immunoassay for measuring estrone-3-glucuronide in undiluted urine. The sensitivity, specificity, and accuracy are similar to those for a conventional separation fluoroimmunoassay or radioimmunoassay, but the speed, convenience, precision, reliability, and clinical utility of the new method are more advantageous. The labeled antigen, a fluorescent europium chelate covalently linked to estrone-3-glucuronide, is incubated for 10 min with a limited concentration of polyclonal or monoclonal antibodies to estrone-3-glucuronyl-6-bovine serum albumin and 10 microL of standard or sample (undiluted urine) in microtiter wells. The fluorescence emanating from the antibody-free label, which is proportional to the concentration of estrone-3-glucuronide in the standard or sample, is then measured in a time-resolved fluorometer. The method is useful for monitoring ovarian function in women.

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.

Multiple labeling and time-resolvable fluorophores

1991 ◽  
Vol 37 (9) ◽  
pp. 1486-1491 ◽  
Author(s):  
E P Diamandis
Keyword(s):  
Dicarboxylic Acid ◽  
Detection Limits ◽  
Alpha Fetoprotein ◽  
Macromolecular Complex ◽  
Fluorescence Immunoassay ◽  
Time Resolved Fluorescence ◽  
Time Resolved ◽  
Highly Sensitive ◽  
Europium Chelate ◽  
New Time

Abstract A new time-resolved fluorescence immunoassay system involving use of the europium chelate of 4,7-bis(chlorosulfophenyl)-1,10-phenanthroline-2,9-dicarboxylic acid as label is reviewed. This stable chelate by itself is not very fluorescent but, used in multiple labeling strategies, improves the achievable detection limits. By using multiple labeling, streptavidin tailing, and Eu3+ activation, one can create a very stable, easy-to-use reagent that is suitable for devising highly sensitive immunoassays and other biotechnological assays. This reagent, a streptavidin-based macromolecular complex, is able to detect approximately 300,000 molecules (approximately 0.5 amol) of alpha-fetoprotein in a model noncompetitive immunoassay.

A sensitive solid-phase time-resolved fluorescence immunoassay apparatus

2009 ◽  
Author(s):  
Yun-lei Wang ◽  
Ke-fei Song ◽  
Wei-lai Zhang ◽  
Jun-ling Li
Keyword(s):  
Solid Phase ◽  
Fluorescence Immunoassay ◽  
Time Resolved Fluorescence ◽  
Time Resolved ◽  
Phase Time

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%.

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