Determination of the Composition and Functional Activity of the Conjugates of Colloidal Gold and Antibodies
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A.N. Bach Institute of Biochemistry, Russian Academy of Sciences, Russia
Dmitriy V. Sotnikov   

A.N. Bach Institute of Biochemistry, Federal Research Centre “Fundamentals of Biotechnology”, Russian Academy of Sciences, Leninsky prospect 33, Moscow, Russia
Publication date: 2017-10-27
Eurasian J Anal Chem 2016;11(3):169–179
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 anti-species 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.
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