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-a ntibody bioconjugates is shown to exceed the intrinsic affinity of the orig inal, monoclonal antibody. The nanoparticle-antibody bioconjugates were pre pared by covalent coupling of antibodies to long-lifetime fluorescent, euro pium(III) chelate nanoparticles, 107 nn in diameter. Experiments were carri ed out in standard microtitration wells to determine solid-phase associatio n and dissociation rate constants, nonspecific binding, and affinity consta nts 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 1 0(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 bin ding 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 imm unoassays with extreme sensitivity.