Measuring an antibody affinity distribution molecule by molecule.
Single molecule fluorescence microscopy was used to observe the binding and unbinding of hapten decorated quantum dots to individual surface immobilized antibodies. The fluorescence time history from an individual antibody site can be used to calculate its binding affinity. While quantum dot blinking occurs during these measurements, we describe a simple empirical method to correct the apparent/observed affinity to account for the blinking contribution. The combination of many single molecule affinity measurements from different antibodies yields not only the average affinity, it directly measures the full shape and character of the surface affinity distribution function.
Temirov
Jamshid P
JP
Center for Integrated Nanotechnologies and Bioscience Division, Los Alamos National Laboratories, Los Alamos, New Mexico 87545, USA.
Bradbury
Andrew R M
AR
Werner
James H
JH
D000818:Animals; D055910:Antibodies, Immobilized / Q000276:immunology; D000915:Antibody Affinity; D000918:Antibody Specificity; D051379:Mice; D008856:Microscopy, Fluorescence; D045663:Quantum Dots; D012984:Software; D013499:Surface Properties; D013997:Time Factors
D055910:Antibodies, Immobilized