T. Soukka et al., Supersensitive time-resolved immunofluorometric assay of free prostate-specific antigen with nanoparticle label technology, CLIN CHEM, 47(7), 2001, pp. 1269-1278
Background: The extreme specific activity of the long-lifetime fluorescent
europium(III) chelate nanoparticles and the enhanced monovalent binding aff
inity of multivalent nanoparticle-antibody bioconjugates are attractive for
noncompetitive immunoassay.
Methods: We used a noncompetitive, two-step immunoassay design to measure f
ree prostate-specific antigen (PSA). Europium(III) chelate nanoparticles (1
07 nm in diameter) were coated with a monoclonal anti-PSA antibody (intrins
ic affinity, 6 x 10(9) L/mol). The nanoparticle-antibody bioconjugates had
an average of 214 active binding sites per particle and a monovalent bindin
g affinity of 7 x 10(10) L/mol. The assay was performed in a low-fluorescen
ce 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-resolv
ed microtitration plate fluorometer.
Results: The detection limit (mean + 2 SD) was 0.040 ng/L (7.3 x 10(5) mole
cules/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 w
as higher than the nonspecific binding of the original antibody (<0.01%).
Conclusions: The sensitivity of the new assay is equal to that of the ambie
nt-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. (C) 2001 Ame
rican Association for Clinical Chemistry.