Antibody affinity. VIII. Measurement of affinity of anti-lactose antibody by fluorescence quenching with a DNP-containing ligand.

The method of fluorescence quenching by bound ligand for the measurement of antibody affinity has been extended to anti-lactose antibodies. The basis for this extension is the use of a lactosyl ligand covalently linked to the 2,4-dinitrophenyl (DNP) group. This group serves as a sensor of the bound state by causing fluorescence quenching similar to that observed with anti-DNP antibody. The synthesis and characterization of this new ligand, N-(Nalpha-acetyl,Nepsilon-DNP-L-lysyl)-rho-aminophenyl-beta-lactoside, are described. The affinity of its interaction with three rabbit IgG and one equine IgM antilactose antibody preparations has been measured. The Qmax values range from 24 to 47% and the calculated association constants from 2.5 times 10-5 M-1 to 7.2 times 10-5 M-1. For one IgG preparation, measurement of the association constant by equilibrium dialysis with the tritiated ligand gave a value of 6.75 times 10-5 M-1 compared to the value of 7.2 times 10-5 M-1 obtained by fluorescence quenching. It is evident that with monoclonal anti-lactose antibody the accurate determination of Qmax, which is possible in this system, provides another structurally dependent clonal characterization of the variable region of the antibody. This optical probe along with others can be used for the identification of individual clones of antibody-producing cells through time and inheritance.