Surface-immobilized antibody-antigen binding studies by single molecule fluorescence imaging

摘要

In this work we report Single Molecule (SM) microscopy studies of binding events between individual surface-immobilized antibodies and fluorescent antigens using Total Internal Reflection (TIR) microscopy. Specific binding events of single biotin-conjugated 'quantum dots to anti-biotin antibodies, immobilized on an amine-coated cover glass using a heterobifunctional photo-reactive cross-linker were observed. The methodology of calculating the binding affinity (dissociation constant) from the time series of 2-D images is described.