Detection of nuclear antigens DNA in normal and pathologic human fluids by quantitative complement fixation.код для вставкиСкачать
Detection of Nuclear Antigens (DNA) in Normal and Pathologic Human Fluids by Quantitative Complement Fixation By EUGENE V. BARNGIT antibodies have been detected in the sera of many patients with collagen-vascular disease, particularly those with systemic lupus erythematosus.1 One such class of antinuclear antibodies, antibodies to deoxyribonucleic acid (antiDNA), are receiving increasing attention because they are commonly found in severely ill patients with systemic lupus erythematosus and rarely detected in other collagen-vascular disease^.^.^ Despite this interesting correlation, the exact pathogenic significance of such antinuclear antibodies is at present unknown. It has been suggested that such antinuclear antibodies may combine with nuclear antigens in the circulation of patients, and that the resultant antigen-antibody complexes contribute to a serum sickness-type illness that we describe as lupus glomerulo-nephritis? This hypothesis received great support from a report by Tan, Schur, Carr, and K ~ n k e lwho , ~ detected DNA antigens in the sera of some patients ill with systemic lupus erythematosus. This antigen was identified as DNA by a precipitation reaction with other human sera shown previously to contain precipitating antibody to DNA. By chemical tests, others6 have detected DNA in human sera, but these tests lack specificity and do not identify DNA as an antigen capable of fixing complement with lupus antibody. Studies in collaboration with Dr. John H. A Vaughanr have suggested that normal human sera may contain small amounts of antigenic nuclear materials, since rabbits immunized with whole human serum in complete Freunds’ adjuvant commonly develop antinuclear antibodies, and occasionally develop complement-fixing antibodies to DNA. The present report describes a more direct serologic attempt to measure nuclear antigens in normal and pathologic human sera and synovial fluids. Sera from 2 patients with systemic lupus erythematosus were shown to contain complement-fixing antibodies to native and single-strand DNA. These sera were used in quantitative complement fixation tests to detect and quantitate nuclear antigens (DNA) in normal and pathologic human sera and synovial fluids. Since sera from patients with systemic lupus erythematosus contain many antibodies directed against a variety of antigens in addition to DNA, their use in tests designed to measure DNA in “unknown” fluids may be criticized. Therefore, confirmation of measurements made utilizing lupus sera was attempted by: 1) haptene inhibition by purines and pyrimidines of reactions between human specimens and the lupus sera; 2) use of rabbit antibodies directed against DNA for the measurement of antigens in human fluids; and 3) extraction of human specimens with hot perchloric acid and meas- From the Department of Medicine, UCLA Medical School, University of California, Los Angeles, California 90024. Presented in part before the American Rheumatism Association, 18 June 1966. Supported in part by Research Grant N I H AM 10,000 and USPHS Training Grant AM 05068. EUGENE V . BARNETT, M.D.: Associate Professor of Medicine and Senior Znuestigator of the Arthritis Foundation. NTINUCLJUR ~ ARTHRITISAND RHEUMATISXI, VOL. 11, N O . 3 (JUNE 1968) 407 EUGENE V. BARNEm 408 Fig. 1.-Complement fixation with Serum E. 1:300 a n d S.S.DNA (calf thymus) diluted in normal human serum, and in phosphate buffered saline, pH 7.4 with and without DNAse treatment. protein (less than 0.5 per cent) was obtained from Worthington Biochemical Corporation and from Dr. Alexander Dounce of the Department of Biochemistry, University of Rochester School of MediMATERIAISAND METHODS cine and Dentistry. Preparations of calf thymus 250 sera from patients with systemic lupus ery- nucleoprotein and histone, as yell as DNA, nucleothematosus were kindly supplied by Dr. Carl M. protein, and histone extracted from human and Pearson. All these sera were shown to contain rabbit livers were also kindly supplied by Mr. Vinantinuclear antibodies against nuclear antigens in cent Agnello and Dr. Alexander Dounce.lzJ3 The immunofluorescence tests using human leukocyte DNA content of these preparations was estimated nuclei. These 250 sera were tested for precipitating by absorption at 260 mp. and in the Dephenylaantibody against single-strand DNA by double dif- mine test.14 T-4 phage DNA was kindly supplied fusion in 1 per cent Agarose (0.15 M Phosphate, by Dr. Verne Schumacker of the Department of pH 7.3) against single-strand calf thymus DNA at Chemistry, University of California at Los Angeles. Single-strand DNA was prepared by heating a concentration of 0.5 mg./ml. Fourteen sera were shown to have precipitating antibodies to DNA. Of preparations at 100 C for 10 minutes and then these 14, only 2 were shown to have complement- rapidly cooling in an ice bath. fixing antibodies to DNA in higher titer than the Specimens of normal and pathologic human sera anti-complementary activity of the sera. These 2 and synovial fluids were tested for their content sera were used as antibodies to detect nuclear an- of nuclear antigens in an initial dilution of 1:50 tigens in human fluids. or 1:lOO. Less dilute specimens were found to be Rabbits were immunized with single-strand calf anti-complementary in the quantitative complethymus DNA, complexed to methylated rabbit ment fixation test.7 The sera and synovial fluids serum albumin.8~9 The antisera produced reacted tested were generally separated from the clot withexclusively with single-strand and native calf thy- in 8 hours of aspiration. The Supernatants were mus DNA. Antibody was restricted to the gamma either tested directly following dilution or were M class of rabbit immunoglobulins as determined first centrifuged at 4,000 rpm. for 20 minutes. by DEAE chromatography,lo as previously re- Neither centrifugation nor hemolysis appeared to ported with antibody elicited by immunization affect the results obtained in the quantitative comwith DNA-bovine serum albumin complexes.11 plement fixation test. Inhibition tests utilizing nuThe rabbit antibodies had much greater activity cleic acid bases were performed.15 Known nuclear in the complement fixation test performed at 37 C. antigens as well as selected specimens of normal than 4 C. Calf thymus DNA without detectable pnthologic human sera and fluids were digested urement of the DNA content of the proteinfree material in the diphenylamine test. DETECTION OF ANTIGENS BY COMPLEMENT FIXATION Fig. 2.-Complement fixation with Serum V. 1:200and S.S. and native 409 T-4phage DNA. with DNAse prior to testing in some of the complement fixation tests.7 Not all specimens were digested with DNAse or tested in inhibition tests utilizing nucleic acid bases because of the limited quantities of sera with complement-fixing antibodies to DNA. Plasma was treated with perchloric acid16 and the extracts tested with the diphenylamine reagent for deoxyribose content.14 DNA was indeed DNA and not some contaminant, complement fixation tests were repeated, utilizing single-strand DNA, previously digested with DNAse as well as DNA preparation which had been similarly incubated without the addition of DNAse. Complement fixation was entirely abolished RESULTS when single-strand DNA was digested with Serum from Patient V when diluted 1: DNAse prior to testing, whether the DNA 100 gave positive complement fixation with was added to undiluted human serum or single-strand calf thymus DNA and calf diluted in buffer containing calcium and thymus nucleoprotein; less complement fix- magnesium (Fig. 1). Identical quantities ation with native calf thymus DNA; and of single-strand calf thymus DNA diluted insignscant complement fixation with RNA in buffer or in normal human serum for 6 and histone.'T The serum from Patient V hours at 37 C. gave identical complement when diluted 1:300 gave highly reproduc- fixation curves. These data suggest that ible complement-fixationcurves with single- there were no significant inhibitors or DNA strand calf thymus DNA.17 On multiple de- enzymes in the normal human serum that terminations, 50 per cent complement fixa- would be active in vitro during that period. tion was obtained with 0.03 to 0.45 pg. DNAse was shown to destroy the antigenic single-strand calf thymus DNA/ml. In all activity. experiments where human specimens were Serum from Patient V was shown to give tested for nuclear antigen content, a simul- complement fixation with single-strand hutaneous complement fixation curve was ob- man, and T-4 phage DNA as well as calf tained with single-strand calf thymus DNA. thymus DNA. In every case the percent To confirm that the antigen in the prepa- complement fixation was greater with ration labeled single-strand calf thymus single-strand than native DNA (Fig. 2). EUGENE V. BARNE'IT 410 Fig. 3.-Complement fixation with Serum V. 1:300 and S.S. T-4 phage and S.S. DNA (calf thymus). Fig. 4.-corn lement fixation with Serum E. 1:ZOO and Serum V. 1:300, vs. S.S. DNA (calf thpymus) . Single-strand calf thymus and T-4 phage DNA (Fig. 3 ) gave the greatest percent complement fixation per microgram of antigen. Serum V at a dilution of 1:300 in a complement fixation test utilizing 0.05 pg./ml. of single-strand calf thymus DNA, was completely inhibited by 300 millimicromoles per ml. of cytidylic acid. Insufficient quantities of serum V were available for further inhibition studies. Serum from the second patient with systemic lupus erythematosus, Patient E, when diluted 1:200 gave similar complement fixation curves with single-strand DNA as obtained with the serum from Patient V diluted 1:300 (Fig. 4). The sera from Patient E diluted 1:200 and Patient V, 1:300 411 DETECTION OF ANTIGENS BY COMPLEMENT FIXATION Table 1.-Complement Fixation by Serum E. 1:300 and Synovial Fluid Specimens Treated with DNAse Specimen No. JF-1 JF-3 JF-5 JF-8 JF-9 Dh&%O& Result R. A. Anti-complementary Destroyed by DNAse Enhanced by DNAse Destrayed by DNAse Destroyed by DNAse Gout Infection Gout R. A. Table 2.-Complement Fixation Inhibition of Serum E. 1:300 and Specimens of Synovial Fluid, JF-3, JF-9 and Abnormal Sera, Ab-14, Ab-21 and SingleStrand Calf Thymus DNA by 300 m,um./ml. of Cytidylic Acid Specimen No. JF-3 JF-9 Ab-14 Ab-21 ss-calf thymus - DNA % Inhibition DissnOSis Gout R. A. S. L. E. S. L. E. 60 5 25 0 - 50 Table 3.-Nuclear Antigen Content of Synovial Fluid Specimens Disease NumberTtsted RA SLE Gout Reiter’s Infection gave no complement fixation with specimens of histone or RNA but did detect calf thymus nucleoprotein. At these dilutions neither sera fixed complement in the presence of DNA or nucleoprotein that had been previously digested with DNAse. The complement fixation of serum E in a dilution of 1:300 in the presence of 0.05 pg./ml. single-strand calf thymus DNA was inhibited up to 76 per cent by 300 millimicromoles per ml. of cytidylic acid. Synovial fluid specimens from two patients with gout were tested in dilutions from 1:100 to 1:2700. Significant complement fixation was obtained with both specimens.17 Complement fixation was abolished with Specimens No. JF-3, JF-8, and JF-9, but not with Specimens JF-1 and JF-5,when the specimens were digested with DNAse prior to testing (Table 1).The addition of ( “SS calf thymus DNA” ) 74.3, 31.8, 7.6 37.8 42.6, 0.4 2.9 <0.2 300 mpm./ml. of cytidylic acid markedly inhibited complement fixation by serum E, at dilution of 1:300 and JF-3 and abnormal serum, Ab-14 (Table 2). JF-3 was from a patient with gout and serum Ab-14 was a serum from a patient with systemic lupus erythematosus. Complement fixation with JF-9 (patient with rheumatoid arthritis) and with abnormal serum Ab-21 (systemic lupus erythematosus) was not inhibited with 300 mpm./ml. of cytidylic acid. Eight synovial fluids were tested for the content of nuclear antigen. In 7 of these, nuclear antigen was detected in high concentration (Table 3). There was no correlation between the synovial leukocyte count and the quantity of antigen detected. Twenty-seven sera from patients with various diseases were tested for their content of nuclear antigen (Table 4). Seven of these patients gave complement fixation 412 EUGENE V. BARNETT Table I.-Nuclear Antigen Content of Abnormal Human Sera Disease Number Tested RA SLE Nephritis SLE without Nephritis Systemic Mastocytosis Uremia with Chronic Glomerulonephritis 2 1 2 RA SLE 4 5 5 1 1 2 1 + + Nephritis SLE without Nephritis Vasculitis, Local Rheumatic Heart Psoriasis with RA Gout Scleroderma Units/ml. ( “SS calf thymus DNA”) 15.0, 2.3 8.6 16.0, 13.5 1 4.9 1 9.0 less than 0.2 1 Table 5.-Nuclear Antigen Content of Normal Human Sera source R.K. G. D. G. w. D. A. D. C. D. G . 8 others Pool of “Normal” Serum greater than that achieved with 0.2 pg./ml. of single-strand calf thymus DNA. Although it is particularly unlikely that these specimens contain single-strand calf thymus DNA, the nuclear antigen content of these specimens is expressed in units equal to u./ml. of single-strand calf thymus DNA that would be capable of achieving the percent complement fixation obtained by the specimen with either serum E diluted 1:300 or serum V diluted 1:200. Twenty of these specimens gave no complement fixation when diluted 150 or 1:100, and thus have less than the equivalent of 0.2 u./ml. of single-strand calf thymus DNA in the undiluted specimen. The diagnoses of the 27 patients studied are listed. There was no obvious correlation between the diagnoses and nuclear antigen content, nor was there UllitS/d. (equivalent to SS calf thymus DNA) 3.50 2.60 1.94 0.80 0.28 0.26 <0.26 - not measurable 1.50 between the clinical severity or peripheral white count of these patients. The sera of 14 normal people were promptly tested following phlebotomy and separation of the serum from the clot (Table 5). Nuclear antigen was detected in 6 of these. In only one specimen did the quantity of antigen detected approach that found in 7 of the 27 pathologic sera. The serum from rabbit no. 44 fixed complement with native DNA of calf thymus origin at 4 C., but to a greater degree with greater reproducibility at 37 C. (Fig. 5 ) . It gave comparable complement fixation with single-strand DNA of human liver origin, but less complement fixation with rat liver nucleoprotein and native DNA of human liver origin. Greatest complement fixation was achieved with single-strand calf thy- DETECTION OF ANTIGENS BY COMPLEMENT FIXATION 413 % C' fix. Fig. 5.-Complement fixation with Rabbit Antibody #44 1:400 and Native DNA (calf thymus) tested at 37" C. ALF THYMUS 1.0 0.1 0.01 u p S.S. 0.001 DNA Fig. 6.-Complement fixation with Rabbit Antibody #44 1:400 and single-strand DNA (calf thymus) tested at 37O C. and T-4phage. mus DNA and least complement fixation with single-strand DNA from T-4 phage (Fig. 6), and no complement fixation with RNA. In haptene inhibition tests (Table 6) serum from rabbit no. 44 was best inhibited by cytidylic acid of the ribonucleotides used and by cytosine of the nucleic acid bases. Seventeen serum specimens were available for repeat testing with rabbit antibody no. 44. Table 7 compares results of complement fixation with human antibody E in a dilution of 1:300 at 4 C. and rabbit antibody no. 44 at 37 c . Of 17 specimens measured utilizing human antibody E, 5 had detectable amounts of antigen. Seven of 17 tested with rabbit antibody had detectable amounts of antigen. In not a single case did both human and rabbit antibodies give a positive reaction with the same specimen. The plasma was separated from two units of outdated blood obtained from the UCLA Blood Bank. The plasma was treated with EUGENE V. BARNETT 414 Table 6.-Complement Fixation Inhibition b y Haptenes of Rabbit Antibody # 44 (1:400) at 37" C. with 0.006 pg./ml. Single-Strand Calf Thymus D N A ~ Millimicromoles/ml. of Haptene % Inhibition Haptene Cytidylic acid Adenylic acid Guanylic acid Cytosine Adenine Guanine Thymine 100 100 100 1000 1000 1000 1000 84 43 0 45 0 0 0 Antigen Content of Abnormal Sera as Detected by Human and Rabbit Antibodies Table 7.-Nuclear Human h t i b o d y E 1:300 (4OC.) Serum No. Ab-1 Ab-2 AB-3 Ab-5 Ab-6 Ab-7 Ab-8 Ab-10 Ab-14 Ab-15 Ab-17 Ab-19 Ab-20 Ab-21 Ab-22 Ab-35Ab-36 Rabbit A n t i i y #44 (37OC.) SerUm Dilution* C' Fix. 1:loo 1:loo N.D. <30% <30% N.D.f 1:loo 1:loo 1:loo 1:100 1:100 1:loo 1:loo 1:loo 1:loo 1:loo 1:lOO 1:loo 1:1350 1:1350 <a% 3.0pLS <30% <30% <30% 9.op <30% <30% <m <30% 16.0~ <30% 8.6~ 13.5~ Serum Dilution 1:25 1:200 1:50 1:50 1:25 1:50 1:50 1:50 1:100 1:25 1:225 1:s 1:25 1:loo 1:50 1:50 1:50 + 1:400 C' Fix. <30% 0.46~ 0.28~ 0.09rr <30% <30% 0.08~ 0.13:~ <30% 0.07~ <m <30% 0.05~ <30% <30% <30% <30% 'Dilution of abnormal serum yielded complement fixation with either human or rabbit antibodies. Less dilute specimens were anti-complementary. fN. D. not tested. $Units equal pg. of S. S. calf thymus DNA that would yield percent complement fixation obtained with the human or rabbit antibodies. - perchloric acid and the protein-free mate- treated with hot perchloric acid and the rial measured in the dinhenylamine test. supernatants tested in both diphenylamine The values obtained ranged from 0.15 to tests and comolement fixation tests, the 0.35 micrograms per ml. on specimens that amount of DNA detected in both tests had been concentrated by pressure dialysis. equaled the amount of DNA added to sera In aliquots of outdated aged whole blood or plasma. and the hemolyzed plasma treated with hot DISCUSSION perchloric acid, the DNA in the proteinfree material measured 0.53 micrograms A sensitive serologic technique has been per ml. in the diphenylamine tests. When applied for the detection and quantitation known amounts of calf thymus DNA were of nuclear antigens in serum and synovial added to normal and abnormal plasma or fluid specimens. The antigens detected in sera and these mixtures subsequently these specimens are most likely DNA or DETECTION OF ANTIGENS BY COMPLEMENT FIXATION DNA-containing material, in that the antisera in the dilutions used in these tests detected only native, single-strand DNA or nucleo-protein. The antigens in both the control specimens of DNA and the human specimens were destroyed by DNAse in nearly every case tested. The only exception was JF-5 from the case of infectious arthritis. Inhibition of complement fixation was achieved with both the control DNA antigen and with 2 of 4 of the human specimens tested by the addition of 300 mpm./ ml. nucleotides. This sensitive technique has confirmed observations obtained by the use of chemical tests which suggested that DNA was present in human sera. Considering the different techniques, Tan, Schur, Carr, and Kunkel suggested that normal sera may contain up to 3 micrograms per ml. of DNA.16 The present report, using different antisera and different techniques, would support this estimate. Furthermore, the experiments utilizing perchloric acid treatment of plasma in testing for DNA in the diphenylamine tests suggest that no more than a microgram of DNA per ml. of serum could be accounted for by in uitro destruction of blood cells. This latter figure is probably a gross overestimation, since the material tested was the concentrated extract of plasma from 250 cc. of whole, aged, bank blood. As suggested by earlier work,16 DNA measurements of human serum obtained by the usual techniques without gross hemolysis measure quantities of DNA present in uiuo. The detection of small amounts of nuclear antigens in normal sera by the serologic test is in accord with suggestions made as a result of previous work, where it was shown that immunization of rabbits with normal human serum or freshly separated plasma may result in antibodies to DNA.? Much greater quantities in a range of 8-12 mg. per cent of DNA have been detected by chemical tests6 The much lower amounts of nuclear 415 material detected in the present study suggest that: 1) a significant proportion of nuclear material present in human specimens is not detectable as antigen by the antisera used in the present study, possibly because the nuclear material is in part bound to serum proteins or antibodies; and/or 2) the chemical tests detected materials other than DNA in human specimens. Using precipitin tests for DNA, Tan, Schur, Carr and Kunkel detected nuclear antigens in human sera. Since they utilized other human antisera as reagents, the difference in quantities detected may well be explained by differences in specificity of the human antibodies used, as well as by differences in sensitivities of the tests employed. Indeed, the inhibition of the antisera used by cytidylic acid points out the marked differences in specscity of different human anti-DNA antibodies, since Stollar studied human DNA antibodies that were not inhibited by cytidylic acid.15 It is of particular interest that the human antibody which was capable of reacting with single-strand DNA of T-4 phage origin detected antigens in entirely digerent human sera as were detected by the rabbit antibody which showed little or no reaction with single-strand T-4 phage DNA. It thus appears that the antigens in human fluids may be as heterogenous as the antinuclear antibodies previously described. Since no elevation of serum DNA was detected in 5 of 6 S.L.E. patients with neph ritis, it is unlikely that DNA-anti-DNA complexes with DNA in antigen excess played a role in these 5 patients’ disease at the particular time in their disease when the specimens were tested. This in no way excludes the possible pathogenetic role for antigen-antibody complexes in lupus nephritis. Indeed, Tan, Schur, Carr and KunkeP6 have described marked fluctuations in the DNA content of the serum of individual patients. Their study suggests that the appearance of DNA in the serum of some, EUGENE V. BAR"IT 416 but not a majority, of patients with systemic lupus may signal the onset of glomerulonephritis. These observations should perhaps encourage us to search for antigenantibody complexes other than DNA-antiDNA as instrumental in the etiology of lupus glomerulo-nephriti~.~ In the present study nuclear antigen or DNA content of synovial fluids was markedly elevated regardless of the diagnosis. No studies were made to test the possibility that synovial fluid leukocytes in vitro when lyzed might release large amounts of DNA. However this appears unlikely since so little DNA was released from the leukocytes of 500 ml. of whole blood kept at refrigeration or at room temperature for long periods. The approach described in the present study shows promise for the detection of nuclear antigens, both DNA as well as other antigens in fractions of normal and abnormal sera. Such approaches may shed light on the possible serum binding by various plasma proteins of the nuclear material in various disease states. It is possible that abnormalities in transport for nuclear antigens due to differences in serum binding of nuclear antigens may be instrumental in the induction of auto-antibodies to nuclear material as seen in systemic lupus erythematosus and other collagen-vascular diseases. Furthermore, this approach shows promise for the detection of nuclear antigens in diseases of lower animals. One such situation is Aleutian Disease of Mink. We have detected nuclear antigens in serum from mink with Aleutian Disease but not in normal mink serum nor in sera from 15 other species.l'J* This nuclear antigenemia may have some relationship to the viremia postulated in Aleutian disease. ACKNOWLEDGMENTS The author gratefully acknowledges the technical assistance of Miss Margaret Alspaugh and Miss Dorothy Corey. SUMMARY A sensitive serologic technique is described which shows promise for the quantitation of nuclear antigens, particularly DNA in serum and synovial fluid specimens. The nuclear antigen or DNA content of synovial fluids tested was markedly elevated regardless of the diagnoses. Six of 14 normal human sera had small but detectable amounts of nuclear antigen. Seven of 27 athologic human sera had markedly increased amounts of nuclear antigen (DNA). Adough antigen was detected in human sera by rabbit antibody to DNA, in no case in this study did the human and rabbit antibodies give a positive result with the same human serum. There was no correlation between clinical severity, diagnosis, or peripheral white count and the level of nuclear antigens detected. SUMMARIO IN INTERLINGUA Es describite un sensibile technica serologic de promissa pro le quantification de antigenos nucleari, particularmente de ADN in sero e liquid0 synovial. Le antigeno nucleari o le contento de ADN del liquidos synovial testate esseva marcatemente elevate sin reguardo a1 diagnose del casos individual. Sex de 14 normal seros human habeva micre sed detegibile quantitates de antigeno nucleari. Septe de 27 pathologic seros human habeva marcatemente augmentate de antigeno nucleari (ADN). Ben que antigeno esseva detegite in seros human per anticorpore de conilio anti ADN, in nulle del casos studiate esseva notate que le anticorpores de conilio o human rendeva resultatos positive con le mesme sero human. Esseva trovate nulle correlation inter le severitate clinic, le diagnose, o le peripheric numeration leucocytic e le nivello del antigenos nucleari detegite. REFERENCES 1. Barnett, E. V.: Antinuclear antibodies and 2. Seligman, M.: DNA antibodies. Arthritis nuclear antigens. A review. Calif. Med. 104:463, Rheum. 7:542, 1963. 1966. 3. Tan, E. M.: An immunologic precipitin sys- DETECTION OF ANTIGENS BY COMPLEMENT FIXATION tem between soluble nucleoprotein and antibody in systemic lupus erythematosus. J. Clin. Invest. 46:735, 1967. 4. Kunkel, H. G.: Current comment: Mechanisms of renal injury in systemic lupus erythematosus. Arthritis Rheum. 9:725, 1966. 5. Tan, E. M., Schur, P. H., and Kunkel, H. G.: DNA in the serum of patients with systemic lupus erythematosus (SLE). J. Clin. Invest. 44: 1104, 1965. 6. Gray, J. D.: The relationship between rheumatoid “factor” and serum deoxyribonucleic acid. J. Immunol. 92:827, 1964. 7. Barnett, E. V., and Vaughan, J. H.: Antinuclear antibodies in rabbit antisera. J. Exp. Med. 123:733, 1966. 8. Plescia, 0.J., Braun, W., and Palczuk, N. C.: Production of antibodies to denatured deoxyribonucleic acid (DNA). Nat. Acad. Sci. (Wash.) 52: 279,1964. 9. Stollar. B. D., and Sandberg, A. L.: Comparisons of antibodies reacting with DNA. I. Systemic lupus erythematosus sera and rabbit antibodies induced by DNA-methylated bovine serum albumin complexes. J. Immun. 96:755, May, 1966. 10. Ishizaka, K., Ishizaka, T., Lee, H. L., and Fudenberg, H.: Immunochemical properties of human yA isohemagglutinin. I. Comparison with yG and yM globulin antibodies. J. Immun. 95:197, 1965. 417 11. Sandberg, A. L., and Stollar, B. D.: A 19s anamestic response to DNA-methylated bovine serum albumin complexes. Immunology 11:547, 1966. 12. Dounce, A. L.: In Chargaff, E. and Davidson, J. N. (Eds.):The Nucleic Acids. New York, Academic Press, 1955. 13. Kay, E. R. M., Simons, N. S., and Dounce, A. L.: An improved preparation of sodium deoxyribonucleate. J. Amer. Chem. SOC.74:1724, 1952. 14. Ciles, K. W., and Myers, A.: An improved diphenylamine method for the estimation of deoxyribonucleic acid. Nature (Lond.) 206:93, 1965. 15. Stollar, D., and Levine, L.:Antibodies to denatured deoxyribonucleic acid in lupus erythematoms serum. Evidence for purine determinants in DNA. Arch. Biochem. 101:417, 1963. 16. Tan, E. M., Schur, P. H., Carr, R. I., and Kunkel, H. G.: Deoxyribonucleic acid (DNA) and antibodies to DNA in the serum of patients with systemic lupus erythematosus. J. Clin. Invest. 45: 1732,1966. 17. Barnett, E. V.: The role of nuclear antigens and antinuclear antibodies in inflammation. J. Clin. Pharmacol. (in press). 18. Barnett, E. V., Williams, Jr., R. C., and Kenyon, A. J.: Nuclear antigens and antinuclear antibodies in mink sera. Presented at the IV Congreso Panamericano De Rheumatologia. Mexico City, Mexico, Oct. 23, 1967.