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Streptavidin-polyvinylamine conjugates labeled with a europium chelate : Applications in immunoassay, immunohistochemistry, and microarrays

Scorilas, A. ; Bjartell, A. LU ; Lilja, H. LU orcid ; Moller, C. LU and Diamandis, E. P. (2000) In Clinical Chemistry 46(9). p.1450-1455
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... (More)

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. (C) 2000 American Association for Clinical Chemistry.

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author
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organization
publishing date
type
Contribution to journal
publication status
published
subject
in
Clinical Chemistry
volume
46
issue
9
pages
6 pages
publisher
American Association for Clinical Chemistry
external identifiers
  • scopus:0033811612
  • pmid:10973889
ISSN
0009-9147
DOI
10.1093/clinchem/46.9.1450
language
English
LU publication?
yes
id
474c6e66-9eb1-468e-bf8c-32b391a970c9
date added to LUP
2022-12-17 17:03:18
date last changed
2024-10-04 00:59:56
@article{474c6e66-9eb1-468e-bf8c-32b391a970c9,
  abstract     = {{<p>Background: The favorable properties of lanthanide chelates compared with conventional fluorescent probes have attracted considerable interest. A Eu<sup>3+</sup> 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. (C) 2000 American Association for Clinical Chemistry.</p>}},
  author       = {{Scorilas, A. and Bjartell, A. and Lilja, H. and Moller, C. and Diamandis, E. P.}},
  issn         = {{0009-9147}},
  language     = {{eng}},
  number       = {{9}},
  pages        = {{1450--1455}},
  publisher    = {{American Association for Clinical Chemistry}},
  series       = {{Clinical Chemistry}},
  title        = {{Streptavidin-polyvinylamine conjugates labeled with a europium chelate : Applications in immunoassay, immunohistochemistry, and microarrays}},
  url          = {{http://dx.doi.org/10.1093/clinchem/46.9.1450}},
  doi          = {{10.1093/clinchem/46.9.1450}},
  volume       = {{46}},
  year         = {{2000}},
}