Utilization of kinetically enhanced monovalent binding affinity by immunoassays based on multivalent nanoparticle-antibody bioconjugates.

The monovalent binding affinity of high binding site density nanoparticle-antibody bioconjugates is shown to exceed the intrinsic affinity of the original, monoclonal antibody. The nanoparticle-antibody bioconjugates were prepared by covalent coupling of antibodies to long-lifetime fluorescent, europium(III) chelate nanoparticles, 107 nm in diameter. Experiments were carried out in standard microtitration wells to determine solid-phase association and dissociation rate constants, nonspecific binding, and affinity constants of the various binding site density nanoparticle-antibody bioconjugates and the conventionally labeled monoclonal antibody. The affinity constant for monovalent binding of a high binding site density bioconjugate (5.4 x 10(10) M(-1)) was 8-fold higher than the intrinsic affinity of the antibody (6.6 x 10(9) M(-1)). The separately measured association (2.5 x 10(6) M(-1) s(-1)) and dissociation (3.7 x 10(-5) s(-1)) rate constants of the bioconjugate were 2-fold higher and 4-fold lower, respectively, compared to the antibody. The dependence of the association rate constant of the density of the binding sites enhanced the kinetics and the affinity of the high binding site density bioconjugates. The nanoparticle labels with high specific activity, low nonspecific binding, and enhanced binding affinity of the nanoparticle-antibody bioconjugates contribute to the design of the next generation immunoassays with extreme sensitivity.