Pyrimidine-Specific Antibodies Which React With Deoxyribonucleic Acid (Dna)

Abstract

Progress in a program to synthesize antigens which might be used to elicit antibodies specific for nucleic acid components has been reported.1 In the foregoing paper, the properties and biochemical applications of an antiserum complementary to purin-6-oyl residues were outlined. It was demonstrated, using the complementfixation reaction, that this "anti-purinoyl" antibody cross-reacted with denatured DNA, but not with native DNA, from several species.This communication illustrates the principle of synthesis of polyfunctional antigens containing pyrimidine residues and describes conjugates which contain 5-acetyluracil and uracil residues, respectively, attached through the N1(N3) position, to serum albumins. Some of the characteristics of rabbit antibodies to these conjugates are also delineated. The interaction between "anti-pyrimidyl" antisera and DNA can be demonstrated by complement fixation; the reaction between antibody to the 5-acetyluracil conjugate and DNA has been shown in addition by passive cutaneous anaphylaxis in the guinea pig. Possible uses for these antisera in the investigation of the biological functions of nucleic acids are discussed.Materials and Methods.-Pyrimidine-protein conjugates: These substances were synthesized by adaptation of the elegant methods developed by Shaw and co-workers2-4 for synthesis of pyrimidines and (N1)-pyrimidyl amino acids, and for N-terminal amino acid analysis of proteins.Polyfunctional (5-acetyluracil-l-) bovine serum albumin (5-AcU-BSA): "Ethoxyacrylamide," mp 90-91', was synthesized from urethane, diketene, and triethylorthoformate by following the published2 3 procedure. To a cold solution of 2.3 gm BSA (0.03 mMole, Pentex Fraction P) in 50 ml of water plus 20 ml 0.1 N NaOH (pH 11.2), 750 mg "ethoxyacrylamide" (3.6 mMoles) was added in small portions. After agitation for 5 min, 10 ml tetrahydrofuran and another 20 ml of 0.1 N NaOH were added, and the mixture was stirred at 370 for an hour. Adjustment of the pH to 10.3 by addition of another 4 ml of NaOH was followed by further stirring for 18 hr at this temperature. The mixture was then dialyzed for 24 hr against running tap water. The clear, orange solution (pH 7.2) was treated dropwise with 1 N HC1 to bring the pH to 2.2. After standing for 10 min, the mixture was brought to pH 5.0 with the addition of 10% Na2CO3. The offwhite, precipitated conjugate was centrifuged in the cold, and was dissolved in 25 ml 0.15 M NaHCO3 to give a clear, rust-red solution. Addition of 27 ml 0.15 M HC1 to pH 4.9 caused reprecipitation of the conjugate. After centrifugation in the cold, the conjugate was dissolved in 30 ml 0.1 M phosphate buffer, pH 7.0. It was dialyzed against cold tap water for 24 hr, and then lyophilized to yield 2.2 gm of a light tan-colored product.The conjugate in phosphate buffer, pH 7.0, exhibited an absorption peak at 286 my, and at a concentration of 248 ,ug/ml had an O.D. of 1.38. On the assumption that an average 5-acetyl-

Antibodies to Native and Denatured Deoxyribonucleic Acid in Systemic Lupus Erythematosus*

“…These negative reactions with native DNA are expected for the antibodies elicited by immunization with purines and pyrimidines coupled to proteins (36)(37)(38)(39) or to synthetic polypeptides (40). The paradoxical finding of positive reactions of some anti-5-acetyl-uracyl antibodies with native DNA (37) remains unexplained.…”

Sensitization to Low Dose 5-Fluorouracil

“…At 10 wk, systemic administration of 5-FU was stopped. 30 wk after tumor implantation these five animals remained free of recurrence and, when sacrificed, showed no metastases.…”

“…other small molecules such as dinitrophenol). Furthermore, antibody formation has been described to nonfluorodinated pyrimidines, including uracil (30,31 --o specific to the sensitizing drug. Furthermore, the presence of this anti-5-FU binding activity correlated with the effectiveness of subsequent systemic 5-FU therapy on both local and metastatic tumor growth.…”

Review of American and English literature for the years 1963 and 1964 (Seventeenth Rheumatism Review)

“…DNA-reactive antibodies were experimentally induced. Purines (186,188), pyrimidines (1130,1240), nucleosides and nucleotides (350, 993) were coupled as haptens to protein or polypeptide carriers; rabbits immunized with the hapten-protein conjugates formed hapten-specific antibodies which reacted with DNA. Similar antibodies were elicited by the injection of DNA complexed with methylated bovine serum albumin (992).…”