Streptavidin-polyvinylamine conjugates labeled with a europium chelate: applications in immunoassay, immunohistochemistry, and microarrays.
BACKGROUND
The favorable properties of lanthanide chelates compared with conventional fluorescent probes have attracted considerable interest. A Eu(3+) 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 approximately 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 microg/L PSA ( approximately 100 fg or approximately 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.
Scorilas
A
A
Department of Pathology and Laboratory Medicine, Mount Sinai Hospital, Toronto, Ontario, M5G 1X5 Canada.
Bjartell
A
A
Lilja
H
H
Moller
C
C
Diamandis
E P
EP
D000818:Animals; D002614:Chelating Agents / Q000737:chemistry; D002850:Chromatography, Gel; D005063:Europium / Q000737:chemistry; D005456:Fluorescent Dyes / Q000737:chemistry; D015200:Fluoroimmunoassay; D007074:Immunoglobulin G / Q000032:analysis*; D007150:Immunohistochemistry; D008297:Male; D051379:Mice; D010618:Phenanthrolines / Q000737:chemistry; D011145:Polyvinyls / Q000737:chemistry; D011467:Prostate / Q000737:chemistry; D017430:Prostate-Specific Antigen / Q000032:analysis*; D019809:Streptavidin / Q000737:chemistry
D002614:Chelating Agents; D005456:Fluorescent Dyes; D007074:Immunoglobulin G; D010618:Phenanthrolines; D011145:Polyvinyls; C054206:4,7-bis(chlorosulfophenyl)-1,10-phenanthroline-2,9-dicarboxylic acid; C008996:poly(vinylamine); D005063:Europium; D019809:Streptavidin; D017430:Prostate-Specific Antigen