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 double-stranded DNA ([32P]dsDNA) fragments were super-impossible, suggesting: 1) BV17-45/[32P]dsDNA binding is independent of dsDNA size using fragments greater than or equal to 192 base pairs in length, and 2) BV17-45 does not exhibit 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 (phi X174, 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 degrees C) were 7.4 X 10(6) M-1 s-1 and 9.2 X 10(-5) s-1, respectively, yielding a functional affinity of 8 X 10(10) 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.
D000906:Antibodies; D000911:Antibodies, Monoclonal; D000936:Antigen-Antibody Complex; D004247:DNA / Q000276:immunology*; D007700:Kinetics; D008970:Molecular Weight; D009690:Nucleic Acid Conformation; D010761:Phosphorus Radioisotopes; D010957:Plasmids; D011089:Polydeoxyribonucleotides / Q000276:immunology*
D000906:Antibodies; D000911:Antibodies, Monoclonal; D000936:Antigen-Antibody Complex; D010761:Phosphorus Radioisotopes; D011089:Polydeoxyribonucleotides; D004247:DNA