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

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.

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

Clinical Chemistry ◽

10.1093/clinchem/46.9.1450 ◽

2000 ◽

Vol 46 (9) ◽

pp. 1450-1455 ◽

Cited By ~ 46

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.

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

Clinical Biochemistry ◽

10.1016/s0009-9120(00)00082-5 ◽

2000 ◽

Vol 33 (5) ◽

pp. 345-350 ◽

Cited By ~ 22

Author(s):

Andreas Scorilas ◽

Eleftherios P Diamandis

Keyword(s):

Solid Phase ◽

Time Resolved ◽

Phase Time ◽

Universal Detection ◽

Detection Reagent

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Detection of Oligonucleotide Hybridization on a Single Microparticle by Time-Resolved Fluorometry: Hybridization Assays on Polymer Particles Obtained by Direct Solid Phase Assembly of the Oligonucleotide Probes

Bioconjugate Chemistry ◽

10.1021/bc970033c ◽

1997 ◽

Vol 8 (3) ◽

pp. 378-384 ◽

Cited By ~ 23

Author(s):

Harri Hakala ◽

Pia Heinonen ◽

Antti Iitiä ◽

Harri Lönnberg

Keyword(s):

Solid Phase ◽

Oligonucleotide Probes ◽

Polymer Particles ◽

Time Resolved ◽

Oligonucleotide Hybridization ◽

Direct Solid ◽

Hybridization Assays

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Biotin binding to avidin. Oligosaccharide side chain not required for ligand association

Biochemical Journal ◽

10.1042/bj2480167 ◽

1987 ◽

Vol 248 (1) ◽

pp. 167-171 ◽

Cited By ~ 117

Author(s):

Y Hiller ◽

J M Gershoni ◽

E A Bayer ◽

M Wilchek

Keyword(s):

Concanavalin A ◽

Sugar Content ◽

Binding Activity ◽

Side Chain ◽

Affinity Column ◽

Binding Properties ◽

Commercial Preparation ◽

Oligosaccharide Moiety ◽

Biotin Binding

A commercially available, purified preparation of avidin was found to comprise two polypeptide bands (Mr 18,000 and Mr 15,500 respectively). Both bands bound biotin as assessed by biotin overlays of protein blots. The Mr 15,500 polypeptide was found to differ from the Mr 18,000 polypeptide only in its sugar content. When the commercial preparation was applied to a concanavalin A affinity column, the glycosylated forms were retarded as expected, and homotypic nonglycosylated avidin tetramers which failed to bind selectively to the column were collected in the effluent. The biotin-binding properties of the nonglycosylated avidin were equivalent to those obtained for the native (glycosylated) avidin molecule, indicating that the oligosaccharide moiety is not essential for the binding activity.

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Comparative analysis of the transferrin and lactoferrin binding proteins in the family Neisseriaceae

Canadian Journal of Microbiology ◽

10.1139/m89-063 ◽

1989 ◽

Vol 35 (3) ◽

pp. 409-415 ◽

Cited By ~ 77

Author(s):

Anthony B. Schryvers ◽

B. Craig Lee

Keyword(s):

Binding Proteins ◽

Solid Phase ◽

Bacterial Species ◽

Binding Activity ◽

Intact Cells ◽

Human Transferrin ◽

Affinity Isolation ◽

Lactoferrin Receptor ◽

Solid Phase Binding ◽

Branhamella Catarrhalis

Intact cells of several bacterial species were tested for their ability to bind human transferrin and lactoferrin by a solid-phase binding assay using horseradish peroxidase conjugated transferrin and lactoferrin. The ability to bind lactoferrin was detected in all isolates of Neisseria and Branhamella catarrhalis but not in isolates of Escherichia coli or Pseudomonas aeruginosa. Transferrin-binding activity was similarly detected in most isolates of Neisseria and Branhamella but not in E. coli or P. aeruginosa. The expression of transferrin- and lactoferrin-binding activity was induced by addition of ethylenediamine di-o-phenylacetic acid and reversed by excess FeCl3, indicating regulation by the level of available iron in the medium. The transferrin receptor was specific for human transferrin and the lactoferrin receptor had a high degree of specificity for human lactoferrin in all species tested. The transferrin- and lactoferrin-binding proteins were identified after affinity isolation using biotinylated human transferrin or lactoferrin and streptavidin–agarose. The lactoferrin-binding protein was identified as a 105-kilodalton protein in all species tested. Affinity isolation with biotinylated transferrin yielded two or more proteins in all species tested. A high molecular mass protein was observed in all isolates, and was of similar size (approximately 98 kilodaltons) in all species of Neisseria but was larger (105 kilodaltons) in B. catarrhalis.Key words: iron, Neisseria, transferrin, lactoferrin, receptor.

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

MRS Proceedings ◽

10.1557/proc-4-183 ◽

1981 ◽

Vol 4 ◽

Cited By ~ 3

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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The quantum yield of triplet formation and triplet lifetime in the solid phase by a time-resolved thermal lens method. Application to pyridazine

Chemical Physics Letters ◽

10.1016/0009-2614(88)80008-3 ◽

1988 ◽

Vol 145 (4) ◽

pp. 286-288 ◽

Cited By ~ 29

Author(s):

Masahide Terazima ◽

Tohru Azumi

Keyword(s):

Quantum Yield ◽

Thermal Lens ◽

Solid Phase ◽

Time Resolved ◽

Triplet Formation ◽

Triplet Lifetime

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Time-Resolved Reflectivity Study of Solid-Phase Epitaxial Regrowth in Relaxed and Strained Si1−xGex Epilayers

MRS Proceedings ◽

10.1557/proc-281-479 ◽

1992 ◽

Vol 281 ◽

Author(s):

T. E. Haynes ◽

C. Lee ◽

K. S. Jones

Keyword(s):

Composition Dependence ◽

Solid Phase ◽

Strain Relaxation ◽

Alloy Layer ◽

Atomic Fraction ◽

Bulk Alloy ◽

Strained Layers ◽

Time Resolved ◽

Epitaxial Regrowth ◽

Different Types

ABSTRACTThe rate of solid-phase epitaxial regrowth has been studied using time-resolved reflectivity in three different types of SiGe/Si epilayers amorphized by ion implantation. In two of these cases, the alloy epilayer contained either 12% or 20% Ge, and the amorphization depth was greater than the thickness (2000 Å) of the SiGe alloy layer. Time-resolved reflectivity measurements showed that the rate of regrowth was not constant in these two cases, but first decreased after passing the SiGe/Si interface, and then increased. The minimum regrowth rate occurred closer to the SiGe/Si interface in the epilayers with the larger Ge atomic fraction. In the third type of sample, the alloy epilayer thickness was ∼7μm, so that the initial epilayer (15% Ge) had the lattice constant of the bulk alloy. Furthermore, amorphization and regrowth occurred entirely within the relaxed alloy layer. In this case, the regrowth rate was constant. The composition dependence of the regrowth-rate transient in the strained layers is discussed in the context of a ‘critical-thickness’ model of strain relaxation.

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