Determination of the composition and functional activity of the conjugates of colloidal gold and antibodies

Abstract

This article describes a method for estimating the composition of conjugates of gold
nanoparticles with antibodies and its binding capacity for the antigen. The conjugates
are separated in first order from the unbound antibodies by centrifugation after
synthesis, and then the concentration of unbound antibodies in the supernatant is
analysed by an enzyme immunoassay (ELISA). Conclusions regarding the composition of
these conjugates are made on the basis of the difference between the concentrations of
the added and the unbound antibodies. This approach protects against the influence of
nanoparticles on the label, and the high precision of the immunosorbent assay can
reliably detect even small changes in the concentration of antibodies caused by the
immobilisation. The amount of antigen binding to the obtained conjugate is registered,
and thus the stored reactivity of immobilised antibodies is assessed in the same system.
The developed method was applied to characterise colloidal gold conjugates with antispecies antibodies (sheep antibodies to human immunoglobulin). It is shown that in the
course of the interaction between the immobilized sheep antibodies and free human
immunoglobulins, not more that 12% of the binding sites of the sheep antibodies are
able to bind the human immunoglobulins.