Specificity and kinetics defining the interaction between a murine monoclonal autoantibody and DNA

Interactions between a murine monoclonal anti-DNA autoantibody (BV17-45) and DNA were examined by direct binding and competitive radioimmunoassays. Binding isotherms constructed by titration of purified BV17-45 with a series of distinct ³²P-labeled doublestranded DNA ([³²P]dsDNA) fragments were superimposable, suggesting: (1) BV17-45/[³²P]dsDNA binding is independent of dsDNA size using fragments ≥192 base pairs in length, and (2) BV17-45 does not inhibit stringent sequence specificity. Single-stranded DNA-specific monoclonal antibody BV04-01 did not react with [³²P]dsDNA, confirming its duplex character. In competition experiments, BV17-45 cross-reacted with phage (ΦX174, M13) RF and virion DNAs at picomolar concentrations. Selectivity for B-form DNA was suggested by the ability of poly(dA) · poly(dT), but not other helical duplex forms, to block BV17-45/[³²P]dsDNA binding. Among the four deoxyribohomopolymers, only deoxyadenylic acid polymers completely inhibited BV17-45/[³²P]dsDNA complex formation. [³²P]dsDNA binding was relatively insensitive to ionic strength, suggesting minimal contribution of electrostatic forces to the binding free energy. Measured BV17-45/[³²P]dsDNA association and dissociation rate constants (4°C) were 7.4 x 10⁶ m^-1 s^-1 and 9.2 x 10^-5 s^-1, respectively, yielding a functional affinity of 8 x 10¹⁰ M^-1. Results are discussed in terms of the relative contribution of B-DNA structural and substructural determinants to the mechanism of BV17-45 recognition.