Laser-excited time-resolved solid-phase fluoroimmunoassays with the new europium chelate 4,7-bis(chlorosulfophenyl)-1,10-phenanthroline-2,9-dicarboxylic acid as label

1988 ◽  
Vol 60 (10) ◽  
pp. 1069-1074 ◽  
Author(s):  
Esther. Reichstein ◽  
Yehezkel. Shami ◽  
Mohabir. Ramjeesingh ◽  
Eleftherios P. Diamandis
Keyword(s):  
Dicarboxylic Acid ◽  
Solid Phase ◽  
Time Resolved ◽  
Europium Chelate

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.

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.

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.

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.

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.

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.

Solute Segregation and Dynamics of Solid-Phase Crystallization In in and Sb-Implanted Silicon

1981 ◽  
Vol 4 ◽  
Author(s):  
J. Narayan ◽  
G. L. Olson ◽  
O. W. Holland
Keyword(s):  
Laser Power ◽  
Solid Phase ◽  
Solute Segregation ◽  
Dislocation Loops ◽  
Solid Phase Crystallization ◽  
Time Resolved ◽  
Plan View ◽  
Ion Channeling ◽  
Retrograde Solubility ◽  
Transmission Electron

ABSTRACTTime-resolved-reflectivity measurements have been combined with transmission electron microscopy (cross-section and plan-view), Rutherford backscattering and ion channeling techniques to study the details of laser induced solid phase epitaxial growth in In+ and Sb+ implanted silicon in the temperature range from 725 to 1500 °K. The details of microstructures including the formation of polycrystals, precipitates, and dislocations have been correlated with the dynamics of crystallization. There were limits to the dopant concentrations which could be incorporated into substitutional lattice sites; these concentrations exceeded retrograde solubility limits by factors up to 70 in the case of the Si-In system. The coarsening of dislocation loops and the formation of a/2<110>, 90° dislocations in the underlying dislocation-loop bands are described as a function of laser power.

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