Purine-specific antibodies which react with deoxvribonucleic acid (DNA).

Substances that have the properties of antibodies and react with DNA from various sources are frequently found in the sera of patients with systemic lupus erythematosus." 2 They appear to be formed spontaneously, perhaps as autoantibodies, but the stimulus for their production is not known. There have been many attempts to induce the formation of antibodies with specificity towards DNA,3-5 but those which react with purified DNA have been conclusively demonstrated in only one instance.6 In this example, the antigen, a partially denatured DNA from T4 bacteriophage, was unusual in that it contained a glucosylated pyrimidine, and the antibody produced was found to be specific towards this antigenic component.' DNA from sources other than T-even phages did not react with this antibody. Although antibodies specific for a variety of haptens have been obtained following the classical technic of Landsteiner,8 no record has been found of the use of nucleic acid components for such studies. Accordingly, purines and pyrimidines were covalently linked to protein carriers to determine whether they might stimulate the formation of antibodies. Bovine serum albumin conjugates containing the [6-purinyl]-sulfonyl, orotic acid, and [6-purinyl]-f3-alanyl moieties were prepared, but antibodies to these antigens gave equivocal results when examined for purine or pyrimidine specificity.9 This report describes the coupling of 6-trichloromethylpurine'0 to serum albumin, and the use of the resulting conjugate as an antigen to induce the formation of antibodies which exhibit a purine specificity and react with DNA. Materials and Methods.-Purinoyl-protein conjugates: These substances were synthesized by a method which was derived from a previously described technic1' for the preparation of aroylprotein conjugates. A 'chilled solution containing 750 mg bovine serum albumin (BSA; Pentex Fraction V) and 170 mg 6-trichloromethylpurine in 70 ml of 4% tetrahydrofuran-water was stirred at room temperature for 3 hr. The pH was maintained throughout at 10-10.5 by the addition of 0.1 N NaOH and the solution was dialyzed overnight against running tap water. Upon acidification to pH 4.5 with 0.1 N HCl, the conjugated protein precipitated. It was redissolved in 25 ml of 0.15 M NaHCO3, dialyzed against running water, and lyophilized. The product (Pur-BSA) thus obtained dissolved readily in water or buffer solutions and migrated in agar gel electrophoresis on a microscope slide as a single component toward the anode at pH 8.2 more rapidly than did unconjugated BSA. From spectrophotometric measurements, Pur-BSA was estimated to contain approximately 24 purine groups per mole of BSA (molecular weight, 67,000).12 In the same way, the conjugate (Pur-HSA) with human serum albumin (HSA, Pentex Fraction V) was prepared. It contained about 27 moles of purine per mole. Spectral properties of these derivatives determined in a Cary spectrophotometer in 1 cm cells are given in Table 1. Immunochemical procedures: Rabbits were immunized by injection, into the foot-pads,'3 of Pur-BSA or Pur-HSA in complete Freund's adjuvant mixture weekly for three weeks. The animals were bled three times by cardiac puncture, beginning seven days after the last injection, and were exsanguinated 14 days after the final injection. The bleedings from individual rabbits were pooled.