scholarly journals Polyvinylamine-streptavidin complexes labeled with a europium chelator: a universal detection reagent for solid-phase time resolved fluorometric applications

2000 ◽  
Vol 33 (5) ◽  
pp. 345-350 ◽  
Author(s):  
Andreas Scorilas ◽  
Eleftherios P Diamandis
Keyword(s):  
Solid Phase ◽  
Time Resolved ◽  
Phase Time ◽  
Universal Detection ◽  
Detection Reagent

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.

Novel application of streptavidin-hapten derivatives as protein-tracer conjugate in competitive-type immunoassays involving biotinylated detection probes

1991 ◽  
Vol 37 (1) ◽  
pp. 58-63 ◽  
Author(s):  
M J Khosravi ◽  
R C Morton
Keyword(s):  
No Reduction ◽  
Solid Phase ◽  
Binding Activity ◽  
Fluorescence Signal ◽  
Antibody Immobilization ◽  
Aggregate Formation ◽  
Cross Link ◽  
Time Resolved ◽  
Biotin Binding ◽  
Detection Reagent

Abstract To investigate the use of streptavidin-hapten derivatives as potential protein-tracer conjugates for competitive-type immunoassays, we labeled streptavidin with cortisol and compared biotin-binding activity of the conjugates with that of unlabeled streptavidin. In this model system, streptavidin labeled with one to approximately 17 cortisol molecules retained its capability to cross-link a biotinylated protein on microtiter wells to a biotin-based general detection reagent developed for time-resolved fluorometry. Compared with unlabeled streptavidin, there was no reduction in the binding activity of the conjugate carrying as many as 2.6 cortisol molecules per molecule of streptavidin. Conjugation ratios greater than 4.4 showed a slight decrease in binding activity, presumably because of the aggregate formation evident at these labeling ratios. As expected, the conjugates were also capable of linking a solid-phase-bound anti-cortisol monoclonal antibody to the biotinylated detection reagent. The fluorescence signal generated increased almost linearly with increasing conjugation ratios from about three to nine cortisol molecules per molecule of streptavidin. At greater ratios, the assay response plateaued. The calibration curves obtained were typical for competitive-type immunoassays when the conjugates were incorporated in a cortisol assay based on a second-antibody immobilization approach.

Direct Time-Resolved Fluorescence Immunoassay of Progesterone in Serum Involving the Biotin-Streptavidin System and the Immobilized-Antibody Approach

1992 ◽  
Vol 38 (5) ◽  
pp. 725-730 ◽  
Author(s):  
S E Kakabakos ◽  
M J Khosravi
Keyword(s):  
Dynamic Range ◽  
Fluorescence Immunoassay ◽  
Time Resolved Fluorescence ◽  
Igg Antibody ◽  
Time Resolved ◽  
Typical Performance ◽  
Dilution Experiments ◽  
Universal Detection ◽  
Derivatives Of ◽  
Detection Reagent

Abstract We developed a direct competitive-type immunoassay for progesterone in serum that combines the advantages of the biotin-streptavidin system with the antibody-immunobilization approach. We synthesized biotinylated progesterone derivatives of five different proteins and, after initial evaluation of the conjugates, selected biotinylated bovine IgG-progesterone as the most suitable tracer. Progesterone released from binding proteins with danazol competes with the biotinylated tracer conjugate for binding to a limited amount of a mouse anti-progesterone monoclonal antibody in microtitration wells coated with a goat anti-mouse IgG antibody. The binding of the biotinylated tracer is then monitored by reaction with a streptavidin-based universal detection reagent developed for time-resolved fluorometry. The assay demonstrated typical performance characteristics with respect to the dynamic range, detection limit, and precision. Recovery averaged 99.3% (SD 8.3%) and dilution experiments showed good linearity. Measurements correlated well with those from three commercially available direct immunoassays for progesterone.

Solid-phase time-resolved immunofluorometric assay of progesterone by EU-labelled protein-A

1986 ◽  
Vol 25 ◽  
pp. 54
Author(s):  
A. Ius ◽  
L. Ferrara ◽  
G. Meroni ◽  
M.A. Bacigalupo
Keyword(s):  
Solid Phase ◽  
Protein A ◽  
Time Resolved ◽  
Phase Time

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

Direct solid-phase time-resolved fluoroimmunoassay of 17 alpha-hydroxyprogesterone in serum and dried blood spots on filter paper

1990 ◽  
Vol 36 (9) ◽  
pp. 1667-1672 ◽  
Author(s):  
R R Gonzalez ◽  
O Mäentausta ◽  
J Solyom ◽  
R Vihko
Keyword(s):  
Filter Paper ◽  
Specific Activity ◽  
Solid Phase ◽  
High Specific Activity ◽  
Dried Blood Spots ◽  
Serum Samples ◽  
Time Resolved ◽  
Blood Spots ◽  
Phase Time ◽  
Direct Solid

Abstract We describe a direct, solid-phase time-resolved fluoroimmunoassay (TRFIA) for measuring 17 alpha-hydroxyprogesterone (17OHP) in serum and blood spots on filter paper. We used 17OHP-3-carboxymethyloxime (17OHP3CMO) coupled to polylysine as the label, which enabled incorporation of up to 34 atoms of europium per molecule of 17OHP, for a very high specific activity. The assay is based on competition between labeled 17OHP3CMO and 17OHP in blood specimens for polyclonal rabbit anti-17OHP antibodies. The antibody-label complex is separated by binding to anti-rabbit antibodies coated onto microtiter strips. The assay buffer contains danazol to displace 17OHP from steroid-binding proteins in serum. For serum samples, the assay is accomplished in 1 h of incubation at room temperature. The blood spot assay with filter paper discs involves incubation overnight at 4 degrees C. Results for both types of specimens from the same subjects correlated well. The lowest measurable concentrations of 17OHP (nmol/L) were 0.10 (3 SD) and 0.75 (3 SD) for serum and dried blood on filter paper, respectively. Intra- and interassay CVs were about 5-15% for both types of samples.

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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