An enzyme-linked immunosorbent assay (ELISA) was developed to characterize spontaneously occurring, mono-and polyclonal anti-DNA antibodies. The assay consists of adsorbing single- (ss) and double-stranded (ds) DNA and various nucleoside-bovine serum albumin conjugates (e.g., A-, G-BSA, etc.) to microtiter wells and assesses the ability of various antibodies to bind to these immobilized antigens. The conformational and base specificity of two monoclonal antibodies (designated MRss-1 BWds-3) was examined in this manner. The exclusive binding of MRss-1 to ssDNA and guanosine-BSA (G-BSA) confirms our previous findings [Munns, T. W., Liszewski, M. K., Tellam, J. T., Ebling, F. M., & Hahn, B. H. (1982) Biochemistry 21, 2929-2936] that this antibody recognizes single-stranded nucleic acids by virtue of their guanine content. The extensive binding of BWds-3 to dsDNA, its limited binding to ssDNA, and complete absence of binding to nucleoside-BSA antigens implied a double-stranded conformational specificity. Further, competitive studies with naturally occurring and synthetic alternating copolymers indicated that BWds-3 preferentially recognized the native dsDNA antigens. ELlSA analysis of the spontaneously occurring, polyclonal anti-DNA antibodies from MRL/lpr and NZB/NZW-F1 mice revealed that the majority of anti-ssDNA antibodies bound to nucleoside-BSA conjugates. Anti-G antibodies were most prominent in both strains of mice, yet lesser and more variable quantities of anti-A, -C, -U, and -T antibodies were also detected. Preadsorption of serum with G-BSA/Sepharose resulted in the complete removal of anti-G antibodies and a 60% reduction in anti-ssDNA antibodies. Anti-ssDNA antibodies were completely removed by preadsorption of serum with a mixture of A-, G-, C-, and T-BSA/Sepharose. Anti-dsDNA antibodies accounted for approximately 30 and 60% of the total antibody population in the serums of MRL/lpr and NZB/NZW-F1 mice, respectively. Last, time-course studies with five individual MRL/lpr mice revealed that the appearance of all anti-DNA antibodies in their serum was coincident and occurred in all animals at the age of 10-11 weeks.