Molecule by Molecule Direct and Quantitative Counting of Antibody−Protein Complexes in Solution

We have used two-color fluorescence coincidence detection to directly count individual protein-antibody complexes of protein G or herpes simplex virus labeled with one or more red- and blue-excited antibodies. This allowed quantitative measurement of the concentration of the protein-antibody complexes over 3 orders of magnitude down to the femtomolar level. Single molecule measurements in diluted serum are also possible. The sample preparation is simple, takes place in solution, and requires no separation. Both the antibody affinity and complex dissociation rate are important in determining the sensitivity of the method. At present, the sensitivity limit of 50 fM is determined by the encounter rate of the labeled analyte with the probe volume. This method can be used to detect and quantitate proteins and to measure the stoichiometry, equilibrium constant, and dissociation rate of protein-protein complexes at low concentrations.