Supersensitive time-resolved immunofluorometric assay of free prostate-specific antigen with nanoparticle label technology.
BACKGROUND
The extreme specific activity of the long-lifetime fluorescent europium(III) chelate nanoparticles and the enhanced monovalent binding affinity of multivalent nanoparticle-antibody bioconjugates are attractive for noncompetitive immunoassay.


METHODS
We used a noncompetitive, two-step immunoassay design to measure free prostate-specific antigen (PSA). Europium(III) chelate nanoparticles (107 nm in diameter) were coated with a monoclonal anti-PSA antibody (intrinsic affinity, 6 x 10(9) L/mol). The nanoparticle-antibody bioconjugates had an average of 214 active binding sites per particle and a monovalent binding affinity of 7 x 10(10) L/mol. The assay was performed in a low-fluorescence microtitration well passively coated with an another monoclonal anti-PSA antibody (affinity, 2 x 10(10) L/mol), and the europium(III) fluorescence was measured directly from the bottom of the well by a standard time-resolved microtitration plate fluorometer.


RESULTS
The detection limit (mean + 2 SD) was 0.040 ng/L (7.3 x 10(5) molecules/mL), and the dynamic detection range covered four orders of magnitude in a 3-h total assay time. The imprecision (CV) over the whole assay range was 2-10%. The detection limit of the assay was limited by the fractional nonspecific binding of the bioconjugate to the solid phase (0.05%), which was higher than the nonspecific binding of the original antibody (<0.01%).


CONCLUSIONS
The sensitivity of the new assay is equal to that of the ambient-analyte, microspot immunoassay and will be improved by use of optimized, high binding-site density nanoparticle-antibody bioconjugates with reduced nonspecific binding and improved monovalent binding affinity.
Soukka
T
T
Department of Biotechnology, University of Turku, Tykistökatu 6, FIN-20520 Turku, Finland. tero.soukka@utu.fi
Paukkunen
J
J
Härmä
H
H
Lönnberg
S
S
Lindroos
H
H
Lövgren
T
T
D000085:Acetates; D000911:Antibodies, Monoclonal; D003102:Colloids; D005063:Europium; D015200:Fluoroimmunoassay / Q000379:methods; D017879:Isothiocyanates; D008863:Microspheres; D010316:Particle Size; D017430:Prostate-Specific Antigen / Q000032:analysis* / Q000276:immunology; D012680:Sensitivity and Specificity
D000085:Acetates; D000911:Antibodies, Monoclonal; D003102:Colloids; D017879:Isothiocyanates; C430267:N(1)-(4-isothiocyanatobenzyl)diethylenetriamine-N(1),N(2),N(3),N(4)-tetrakis(acetic acid); D005063:Europium; D017430:Prostate-Specific Antigen