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DNA antibodies detected by microhemagglutination test in the diagnosis of systemic lupus erythematosus
Journal o f Immunological Methods, 22 (1978) 149--153
149
© Elsevier/North-Holland Biomedical Press
D N A A N T I B O D I E S D E T E C T E D BY M I C R O H E M A G G L U T I N A T I O N IN T H E D I A G N O S I S O F S Y S T E M I C L U P U S E R Y T H E M A T O S U S
TEST
MIRTHA DEPIANTE-DEPAOLI, CLELIA M. RIERA, CARLOS YANTORNO *, ALBERTO RISEMBERG and ALBERTO STRUSSBERG Cdtedras de Qu(mica Cl(nica H e Inmunolog(a y Serologfa, Departamento de Bioqu{mica Cl(nica, Facultad de Ciencias Qu(micas, Universidad Nacional de Cdrdoba, C6rdoba, and Cdtedra de Cl(nica de las Enfermedades Infecciosas, Facultad de Ciencias Mddicas, Universidad Nacional de Cdrdoba, Cdrdoba, Argentina
(Received 17 January 1978, accepted 30 January 1978)
A method of tanned erythrocyte agglutination of wide applicability is described for the study of DNA antibodies. The practical aspects of the method have been explored in estimation of antibodies to DNA in human cases of systemic lupus erythematosus, discoid lupus erythematosus, rheumatoid arthritis, other collagenoses and not related diseases. This method was found to be of high sensitivity and specificity, detecting DNA antibodies only in sera of systemic lupus erythematosus. Results were compared with those obtained by immunofluorescence test using rat liver hepatocytes as source of nuclei.
INTRODUCTION In r e c e n t y e a r s c o n s i d e r a b l e d a t a h a v e b e e n a c c u m u l a t e d s u p p o r t i n g t h e p r e m i s e t h a t a n t i b o d i e s to native D N A are i m p o r t a n t in t h e p a t h o g e n e s i s o f s y s t e m i c l u p u s e r y t h e m a t o s u s (SLE) ( S e l i g m a n n , 1 9 6 3 ; Casals et al., 1 9 6 4 ; T a n et al., 1 9 6 6 ) a n d t h a t t h e levels c o r r e l a t e well w i t h a c t i v i t y , in p a r t i c u l a r w i t h active n e p h r i t i s ( R o t h f i e l d a n d Stollar, 1 9 6 7 ; S c h u r a n d S a n d s o n , 1 9 6 8 ; H u g h e s et al., 1 9 7 1 ) . A t t h e p r e s e n t t i m e t h e l a b o r a t o r y tests used m o s t w i d e l y in t h e d e t e c t i o n o f a n t i b o d i e s to D N A are i m m u n o p r e c i p i t a t i o n ( S e l i g m a n n , 1 9 5 7 a ) , c o m p l e m e n t f i x a t i o n ( S e l i g m a n n , 1 9 5 7 b ) , D N A - c o a t e d particle f l o c c u l a t i o n (Bozicevich et al., 1 9 6 0 ) , r a d i o l a b e l e d antigen b i n d i n g test (Pincus et al., 1 9 6 9 ) , passive h e m a g g l u t i n a t i o n ( J o k i n e n a n d J u l k u n e n , 1 9 6 5 ; I n a m i et al., 1 9 7 3 ) , etc. This c o m m u n i c a t i o n describes a sensitive a n d specific t a n n e d cell h e m a g g l u t i n a t i o n m e t h o d used t o d e t e c t a n t i b o d i e s t o native D N A . P r o t e i n conc e n t r a t i o n , i n c u b a t i o n t i m e a n d o t h e r variables o f t h e test h a v e b e e n studied.
* Died during the preparation of this manuscript.
150 M A T E R I A L S AND M E T H O D S S e ra
Fresh sera were obtained from 295 patients and 60 normal human blood donors. Sixty-seven cases of SLE, 9 of discoid lupus erythematosus (LE), 118 of rheumatoid arthritis (RA), 34 of other collagenoses and 67 of varying diseases such as idiopathic t r o m b o c y t o p e n i c purpura, cirrhosis, tuberculosis, hepatitis, etc. were diagnosed on the bases of clinical, laboratory, cytological and histopathological examinations. Sera samples were immediately frozen at --20°C until used.
Antigenic preparations A highly polymerized commercial DNA (Koch-Light Laboratories, U.K.) was used as antigen. This was allowed to dissolve overnight with McIlvaine's buffer, pH 4.9, in a 37°C water bath.
Immunologic assays The formolinized cell hemagglutination method was performed as described by Jokinen and Julkunen (1965). One volume of washed human erythrocytes (0 *) at 50% suspension was treated with 4 vol of formaldehyde 10% and kept at 4°C during at least 15 days. At the m o m e n t of use the cells were resuspended at 10% in the coupling solution containing native DNA (0.1 mg/ml). The passive hemagglutination method using DNA coupled to tanned erythrocytes was essentially performed as described by Boyden (1951). Washed human erythrocytes (0+) at 8% suspension were treated for 1 h at 4°C with an equal volume of 1 : 20,000 tannic acid solution. Equal volumes of 4% tanned erythrocytes were mixed with different concentrations of native DNA in order to obtain the optimal antigen dilution. The sensitized erythrocytes were incubated 1 h at 4°C, washed thrice with phosphatebuffered saline (PBS) and made up to a 2% suspension in diluent (PBS containing 1/60 normal rabbit serum). The sera to be tested were diluted in microtiter plates, 1:3, 1:9, 1:27, etc. in diluent. To each well containing 50 gl serum dilution, 25 gl of cell suspension were added. The results were read after 60 min and the end-point was taken as the highest serum dilution giving a smooth mat of cells covering the b o t t o m of the well. Fluorescent antibody technique was employed as described previously (Yantorno et al., 1974). RESULTS
Samples of serum from patients with SLE, discoid LE, RA, other collagenoses and diseases not related with collagen were studied searching for DNA antibodies. Table I shows results of sera from some groups assayed by hemagglutina-
151 TABLE 1 P A S S I V E H E M A G G L U T I N A T I O N WITH F O R M O L I N I Z E D R E D C E L L S I n c i d e n c e o f D N A a n t i b o d i e s in SLE a n d o t h e r diseases. T h e f o r m o l i n i z e d cells were c o a t e d w i t h n a t i v e D N A at a 0.1 m g / m l p r o t e i n c o n c e n t r a t i o n . Disease
R a t i o p o s i t i v e / t o t a l n u m b e r of sera
SLE RA O t h e r diseases
15/18 3/31 0/23
tion using formolinized e r y t h r o c y t e s coated with native DNA. The ratio of positive/total is high in the patients suffering of SLE, whereas the incidence is quite lower in RA. The titer observed in SLE ranged from 1:9 to 1:81 while in RA it was up to 1 : 9. DNA antibodies were not det ect ed in ot her diseases. As can be seen, with this t echni que it is n o t possible to have a clearcut distinction b e t w e e n SLE and RA, since antibodies t o DNA were det ect ed in b o t h diseases. In order to solve this problem the passive hemagglutination test was perf o r m e d as described by B oyde n (1951). Previously, the optimal conditions of the reactions were established. Different concentrations o f the antigen coating tanned h u m a n e r y t h r o c y t e s were c o n f r o n t e d with sera from SLE and RA patients. Results are presented in Table 2. The optimal c o n c e n t r a t i o n of t h e DNA was 1 mg/ml, since with t hat dilution of the antigen the highest titer was obtained in sera f r om SLE. No reactivity was observed in sera from R.A. Besides, dilution prepared at 1:30, 1:60 and 1:100, variations o f incubation time and temperatures were tested. It was observed t hat dilution at 1:60 and 1 h o f incubation at 4°C were the optimal conditions. The tech n iq u e described above was used to study sera from 5 groups of patients. In the results presented in Table 3 it can be seen t hat antibodies to native DNA were det ect ed only in sera from patients with SLE. Thirty-nine o f the 67 sera exhibited titers ranging from 1:3 to 1:729. TABLE 2 T I T E R E X P R E S S E D AS T H E R E C I P R O C A L O F H I G H E S T D I L U T I O N S H O W I N G AGGLUTINATION OF TANNED ERYTHROCYTES SENSITIZED WITH NATIVE DNA Disease
SLE1 SLE2 RA1 RA2
T i t e r of D N A a n t i b o d i e s . D N A c o n c e n t r a t i o n 1 mg/ml
0.1 m g / m l
0.01 m g / m l
27 9 <3 <3
9 3 <3 <3
<3 <3 <3 <3
152 TABLE 3 PASSIVE HEMAGGLUTINATION WITH TANNED RED CELLS Incidence of DNA antibodies in SLE and other diseases. The tanned cells were coated with native DNA at a 1 mg/ml protein concentration. Disease
Ratio positive/total number of sera
SLE Discoid LE RA Other collagenoses Other diseases Normal human sera
39/67 0/9 0/118 0/34 0/67 0/60
F u r t h e r m o r e , t h e s a m e sera were studied b y i m m u n o f l u o r e s c e n c e t e s t l o o k i n g f o r p e r i p h e r a l staining p a t t e r n s t h a t a c c o r d i n g to G o n z a l e z and R o t h f i e l d ( 1 9 6 6 ) and L u c i a n o and R o t h f i e l d ( 1 9 7 3 ) are d u e t o D N A antib o d i e s . T h e results s h o w a v e r y close c o r r e l a t i o n b e t w e e n t h e p r e s e n c e o f p e r i p h e r a l figures a n d a n t i b o d y t i t e r in h e m a g g l u t i n a t i o n test. DISCUSSION T h e p r e s e n c e o f a n t i b o d i e s t o native D N A in the sera o f p a t i e n t s w i t h SLE is well stablished a n d t h e i r d e t e c t i o n is i m p o r t a n t b e c a u s e t h e y are t h e principal f a c t o r s r e s p o n s i b l e in t h e p a t h o g e n e s i s o f S L E (Tan et al., 1966). D i f f e r e n t p r o c e d u r e s a p p l i e d t o t h e s t u d y o f these a n t i b o d i e s had t h e disa d v a n t a g e s o f l o w sensitivity or s p e c i f i c i t y . S e l i g m a n n ( 1 9 5 8 ) used t a n n e d s h e e p red cells in h e m a g g l u t i n a t i o n test f o r t h e d e t e c t i o n o f D N A a n t i b o d i e s . T h e results w e r e n o t p e r f e c t l y r e p r o d u c ible a n d this was a t t r i b u t e d to the d e f i c i e n t a d s o r p t i o n of the D N A t o t h e t a n n e d s h e e p e r y t h r o c y t e s . Besides, t h e y o b s e r v e d p r o z o n e p h e n o m e n a at l o w titers. O t h e r a u t h o r s ( I n a m i et al., 1 9 7 3 ) , using f o r m o l i n i z e d and t a n n e d h u m a n red cells c o a t e d with D N A , f o u n d in 51 S L E sera 22% o f D N A a n t i b o d i e s . H e m a g g l u t i n a t i o n was n o t o b s e r v e d in m o s t o f the negative sera. In the p r e s e n t w o r k , f o l l o w i n g t h e t e c h n i q u e p r o p o s e d b y J o k i n e n and J u l k u n e n ( 1 9 6 5 ) t o c o a t f o r m o l i n i z e d red cells b y D N A , the p e r c e n t a g e of p o s i t i v i t y in the 18 S L E sera s t u d i e d was 83%. In 3 o u t of 31 sera f r o m R A a t i t e r o f 1:9 was o b s e r v e d . T h e p r e p a r a t i o n of f o r m o l i n i z e d cells is t i m e cons u m i n g a n d t h e results are n o t t o o clear to read. In c o n t r a s t , w h e n t h e h e m a g g l u t i n a t i o n test p r o p o s e d b y B o y d e n ( 1 9 5 1 ) was used t o d e t e c t D N A a n t i b o d i e s , a high specificity and elevated sensitivity was o b s e r v e d . As was s h o w n in 67 SLE sera s t u d i e d t h e p e r c e n t a g e of positivity was 56%. It is i n t e r e s t i n g to n o t e t h a t in 2 8 8 sera f r o m n o r m a l h u m a n a n d n o n - S L E p a t i e n t s n o a n t i b o d i e s to D N A were o b s e r v e d w i t h this tech-
153 nique. M o r e o v e r , this is a very simple m e t h o d and it avoids t h e f o r m o l treatm e n t o f red cells t h a t in m a n y cases causes a u t o a g g l u t i n a t i o n with the cons e q u e n c e o f false positive reactions. In view o f the d a t a p r e s e n t e d here, it is possible to c o n s i d e r the t a n n e d h u m a n e r y t h r o c y t e s agglutination t e c h n i q u e as a useful t o o l t o o b t a i n D N A specific reactions. F u r t h e r m o r e , the close c o r r e l a t i o n with i m m u n o f l u o r e s c e n c e m a k e s this t e c h n i q u e very useful in clinical centers w h e r e the i m m u n o f l u o r e s c e n c e m i c r o s c o p e is n o t always available. REFERENCES Boyden, S.V., 1951, J. Exp. Med. 93,107. Bozieevich, J., J.P. Nasou and D.E. Kayhoe, 1960, Proc. Soc. Exp. Biol. Med. 103,636. Casals, S.P., G.J. Friou and L.L. Myers, 1964, Arthr. and Rheum. 7,379. Gonzalez, E.N. and N.F. Rothfield, 1966, New Engl. J. Med. 274, 1333. Hughes, G.R.V., A.S. Cohen and C.L. Christian, 1971, Ann. Rheum. Dis. 30,259. Inami, Y.H., R.M. Nakamura and E.M. Tan, 1973, J. Immunol. Methods 3,287. Jokinen, E.J. and H. Julkunen, 1965, Ann. Rheum. Dis. 24,477. Luciano, A. and N.F. Rothfield, 1973, Ann. Rheum. Dis. 32,337. Pincus, T., P.H. Schur and J.A. Rose, 1969, New Engl. J. Med. 281,701. Rothfield, N.F. and B.D. Stollar, 1967, J. Clin. Invest. 46, 1785. Schur, P.H. and J. Sandson, 1968, New Engl. J. Med. 278,533. Seligmann, M., 1957a, C.R. Acad. Sci. 245,243. Seligmann, M., 1957b, C.R. Acad. Sci. 245, 1472. Seligmann, M., 1958, Rev. Fran(;. Etud. Clin. Biol. 111,558. Seligmann, M., 1963, Arthr. and Rheum. 6,542. Tan, E.M., P.H. Schur, R.I. Garr and H.G. Kunkel, 1966, J. Clin. Invest. 45, 1732. Yantorno, C., C.M. Riera, A. Risemberg and A. Strussberg, 1974, J. Immunol. Methods 5, 253.
149
© Elsevier/North-Holland Biomedical Press
D N A A N T I B O D I E S D E T E C T E D BY M I C R O H E M A G G L U T I N A T I O N IN T H E D I A G N O S I S O F S Y S T E M I C L U P U S E R Y T H E M A T O S U S
TEST
MIRTHA DEPIANTE-DEPAOLI, CLELIA M. RIERA, CARLOS YANTORNO *, ALBERTO RISEMBERG and ALBERTO STRUSSBERG Cdtedras de Qu(mica Cl(nica H e Inmunolog(a y Serologfa, Departamento de Bioqu{mica Cl(nica, Facultad de Ciencias Qu(micas, Universidad Nacional de Cdrdoba, C6rdoba, and Cdtedra de Cl(nica de las Enfermedades Infecciosas, Facultad de Ciencias Mddicas, Universidad Nacional de Cdrdoba, Cdrdoba, Argentina
(Received 17 January 1978, accepted 30 January 1978)
A method of tanned erythrocyte agglutination of wide applicability is described for the study of DNA antibodies. The practical aspects of the method have been explored in estimation of antibodies to DNA in human cases of systemic lupus erythematosus, discoid lupus erythematosus, rheumatoid arthritis, other collagenoses and not related diseases. This method was found to be of high sensitivity and specificity, detecting DNA antibodies only in sera of systemic lupus erythematosus. Results were compared with those obtained by immunofluorescence test using rat liver hepatocytes as source of nuclei.
INTRODUCTION In r e c e n t y e a r s c o n s i d e r a b l e d a t a h a v e b e e n a c c u m u l a t e d s u p p o r t i n g t h e p r e m i s e t h a t a n t i b o d i e s to native D N A are i m p o r t a n t in t h e p a t h o g e n e s i s o f s y s t e m i c l u p u s e r y t h e m a t o s u s (SLE) ( S e l i g m a n n , 1 9 6 3 ; Casals et al., 1 9 6 4 ; T a n et al., 1 9 6 6 ) a n d t h a t t h e levels c o r r e l a t e well w i t h a c t i v i t y , in p a r t i c u l a r w i t h active n e p h r i t i s ( R o t h f i e l d a n d Stollar, 1 9 6 7 ; S c h u r a n d S a n d s o n , 1 9 6 8 ; H u g h e s et al., 1 9 7 1 ) . A t t h e p r e s e n t t i m e t h e l a b o r a t o r y tests used m o s t w i d e l y in t h e d e t e c t i o n o f a n t i b o d i e s to D N A are i m m u n o p r e c i p i t a t i o n ( S e l i g m a n n , 1 9 5 7 a ) , c o m p l e m e n t f i x a t i o n ( S e l i g m a n n , 1 9 5 7 b ) , D N A - c o a t e d particle f l o c c u l a t i o n (Bozicevich et al., 1 9 6 0 ) , r a d i o l a b e l e d antigen b i n d i n g test (Pincus et al., 1 9 6 9 ) , passive h e m a g g l u t i n a t i o n ( J o k i n e n a n d J u l k u n e n , 1 9 6 5 ; I n a m i et al., 1 9 7 3 ) , etc. This c o m m u n i c a t i o n describes a sensitive a n d specific t a n n e d cell h e m a g g l u t i n a t i o n m e t h o d used t o d e t e c t a n t i b o d i e s t o native D N A . P r o t e i n conc e n t r a t i o n , i n c u b a t i o n t i m e a n d o t h e r variables o f t h e test h a v e b e e n studied.
* Died during the preparation of this manuscript.
150 M A T E R I A L S AND M E T H O D S S e ra
Fresh sera were obtained from 295 patients and 60 normal human blood donors. Sixty-seven cases of SLE, 9 of discoid lupus erythematosus (LE), 118 of rheumatoid arthritis (RA), 34 of other collagenoses and 67 of varying diseases such as idiopathic t r o m b o c y t o p e n i c purpura, cirrhosis, tuberculosis, hepatitis, etc. were diagnosed on the bases of clinical, laboratory, cytological and histopathological examinations. Sera samples were immediately frozen at --20°C until used.
Antigenic preparations A highly polymerized commercial DNA (Koch-Light Laboratories, U.K.) was used as antigen. This was allowed to dissolve overnight with McIlvaine's buffer, pH 4.9, in a 37°C water bath.
Immunologic assays The formolinized cell hemagglutination method was performed as described by Jokinen and Julkunen (1965). One volume of washed human erythrocytes (0 *) at 50% suspension was treated with 4 vol of formaldehyde 10% and kept at 4°C during at least 15 days. At the m o m e n t of use the cells were resuspended at 10% in the coupling solution containing native DNA (0.1 mg/ml). The passive hemagglutination method using DNA coupled to tanned erythrocytes was essentially performed as described by Boyden (1951). Washed human erythrocytes (0+) at 8% suspension were treated for 1 h at 4°C with an equal volume of 1 : 20,000 tannic acid solution. Equal volumes of 4% tanned erythrocytes were mixed with different concentrations of native DNA in order to obtain the optimal antigen dilution. The sensitized erythrocytes were incubated 1 h at 4°C, washed thrice with phosphatebuffered saline (PBS) and made up to a 2% suspension in diluent (PBS containing 1/60 normal rabbit serum). The sera to be tested were diluted in microtiter plates, 1:3, 1:9, 1:27, etc. in diluent. To each well containing 50 gl serum dilution, 25 gl of cell suspension were added. The results were read after 60 min and the end-point was taken as the highest serum dilution giving a smooth mat of cells covering the b o t t o m of the well. Fluorescent antibody technique was employed as described previously (Yantorno et al., 1974). RESULTS
Samples of serum from patients with SLE, discoid LE, RA, other collagenoses and diseases not related with collagen were studied searching for DNA antibodies. Table I shows results of sera from some groups assayed by hemagglutina-
151 TABLE 1 P A S S I V E H E M A G G L U T I N A T I O N WITH F O R M O L I N I Z E D R E D C E L L S I n c i d e n c e o f D N A a n t i b o d i e s in SLE a n d o t h e r diseases. T h e f o r m o l i n i z e d cells were c o a t e d w i t h n a t i v e D N A at a 0.1 m g / m l p r o t e i n c o n c e n t r a t i o n . Disease
R a t i o p o s i t i v e / t o t a l n u m b e r of sera
SLE RA O t h e r diseases
15/18 3/31 0/23
tion using formolinized e r y t h r o c y t e s coated with native DNA. The ratio of positive/total is high in the patients suffering of SLE, whereas the incidence is quite lower in RA. The titer observed in SLE ranged from 1:9 to 1:81 while in RA it was up to 1 : 9. DNA antibodies were not det ect ed in ot her diseases. As can be seen, with this t echni que it is n o t possible to have a clearcut distinction b e t w e e n SLE and RA, since antibodies t o DNA were det ect ed in b o t h diseases. In order to solve this problem the passive hemagglutination test was perf o r m e d as described by B oyde n (1951). Previously, the optimal conditions of the reactions were established. Different concentrations o f the antigen coating tanned h u m a n e r y t h r o c y t e s were c o n f r o n t e d with sera from SLE and RA patients. Results are presented in Table 2. The optimal c o n c e n t r a t i o n of t h e DNA was 1 mg/ml, since with t hat dilution of the antigen the highest titer was obtained in sera f r om SLE. No reactivity was observed in sera from R.A. Besides, dilution prepared at 1:30, 1:60 and 1:100, variations o f incubation time and temperatures were tested. It was observed t hat dilution at 1:60 and 1 h o f incubation at 4°C were the optimal conditions. The tech n iq u e described above was used to study sera from 5 groups of patients. In the results presented in Table 3 it can be seen t hat antibodies to native DNA were det ect ed only in sera from patients with SLE. Thirty-nine o f the 67 sera exhibited titers ranging from 1:3 to 1:729. TABLE 2 T I T E R E X P R E S S E D AS T H E R E C I P R O C A L O F H I G H E S T D I L U T I O N S H O W I N G AGGLUTINATION OF TANNED ERYTHROCYTES SENSITIZED WITH NATIVE DNA Disease
SLE1 SLE2 RA1 RA2
T i t e r of D N A a n t i b o d i e s . D N A c o n c e n t r a t i o n 1 mg/ml
0.1 m g / m l
0.01 m g / m l
27 9 <3 <3
9 3 <3 <3
<3 <3 <3 <3
152 TABLE 3 PASSIVE HEMAGGLUTINATION WITH TANNED RED CELLS Incidence of DNA antibodies in SLE and other diseases. The tanned cells were coated with native DNA at a 1 mg/ml protein concentration. Disease
Ratio positive/total number of sera
SLE Discoid LE RA Other collagenoses Other diseases Normal human sera
39/67 0/9 0/118 0/34 0/67 0/60
F u r t h e r m o r e , t h e s a m e sera were studied b y i m m u n o f l u o r e s c e n c e t e s t l o o k i n g f o r p e r i p h e r a l staining p a t t e r n s t h a t a c c o r d i n g to G o n z a l e z and R o t h f i e l d ( 1 9 6 6 ) and L u c i a n o and R o t h f i e l d ( 1 9 7 3 ) are d u e t o D N A antib o d i e s . T h e results s h o w a v e r y close c o r r e l a t i o n b e t w e e n t h e p r e s e n c e o f p e r i p h e r a l figures a n d a n t i b o d y t i t e r in h e m a g g l u t i n a t i o n test. DISCUSSION T h e p r e s e n c e o f a n t i b o d i e s t o native D N A in the sera o f p a t i e n t s w i t h SLE is well stablished a n d t h e i r d e t e c t i o n is i m p o r t a n t b e c a u s e t h e y are t h e principal f a c t o r s r e s p o n s i b l e in t h e p a t h o g e n e s i s o f S L E (Tan et al., 1966). D i f f e r e n t p r o c e d u r e s a p p l i e d t o t h e s t u d y o f these a n t i b o d i e s had t h e disa d v a n t a g e s o f l o w sensitivity or s p e c i f i c i t y . S e l i g m a n n ( 1 9 5 8 ) used t a n n e d s h e e p red cells in h e m a g g l u t i n a t i o n test f o r t h e d e t e c t i o n o f D N A a n t i b o d i e s . T h e results w e r e n o t p e r f e c t l y r e p r o d u c ible a n d this was a t t r i b u t e d to the d e f i c i e n t a d s o r p t i o n of the D N A t o t h e t a n n e d s h e e p e r y t h r o c y t e s . Besides, t h e y o b s e r v e d p r o z o n e p h e n o m e n a at l o w titers. O t h e r a u t h o r s ( I n a m i et al., 1 9 7 3 ) , using f o r m o l i n i z e d and t a n n e d h u m a n red cells c o a t e d with D N A , f o u n d in 51 S L E sera 22% o f D N A a n t i b o d i e s . H e m a g g l u t i n a t i o n was n o t o b s e r v e d in m o s t o f the negative sera. In the p r e s e n t w o r k , f o l l o w i n g t h e t e c h n i q u e p r o p o s e d b y J o k i n e n and J u l k u n e n ( 1 9 6 5 ) t o c o a t f o r m o l i n i z e d red cells b y D N A , the p e r c e n t a g e of p o s i t i v i t y in the 18 S L E sera s t u d i e d was 83%. In 3 o u t of 31 sera f r o m R A a t i t e r o f 1:9 was o b s e r v e d . T h e p r e p a r a t i o n of f o r m o l i n i z e d cells is t i m e cons u m i n g a n d t h e results are n o t t o o clear to read. In c o n t r a s t , w h e n t h e h e m a g g l u t i n a t i o n test p r o p o s e d b y B o y d e n ( 1 9 5 1 ) was used t o d e t e c t D N A a n t i b o d i e s , a high specificity and elevated sensitivity was o b s e r v e d . As was s h o w n in 67 SLE sera s t u d i e d t h e p e r c e n t a g e of positivity was 56%. It is i n t e r e s t i n g to n o t e t h a t in 2 8 8 sera f r o m n o r m a l h u m a n a n d n o n - S L E p a t i e n t s n o a n t i b o d i e s to D N A were o b s e r v e d w i t h this tech-
153 nique. M o r e o v e r , this is a very simple m e t h o d and it avoids t h e f o r m o l treatm e n t o f red cells t h a t in m a n y cases causes a u t o a g g l u t i n a t i o n with the cons e q u e n c e o f false positive reactions. In view o f the d a t a p r e s e n t e d here, it is possible to c o n s i d e r the t a n n e d h u m a n e r y t h r o c y t e s agglutination t e c h n i q u e as a useful t o o l t o o b t a i n D N A specific reactions. F u r t h e r m o r e , the close c o r r e l a t i o n with i m m u n o f l u o r e s c e n c e m a k e s this t e c h n i q u e very useful in clinical centers w h e r e the i m m u n o f l u o r e s c e n c e m i c r o s c o p e is n o t always available. REFERENCES Boyden, S.V., 1951, J. Exp. Med. 93,107. Bozieevich, J., J.P. Nasou and D.E. Kayhoe, 1960, Proc. Soc. Exp. Biol. Med. 103,636. Casals, S.P., G.J. Friou and L.L. Myers, 1964, Arthr. and Rheum. 7,379. Gonzalez, E.N. and N.F. Rothfield, 1966, New Engl. J. Med. 274, 1333. Hughes, G.R.V., A.S. Cohen and C.L. Christian, 1971, Ann. Rheum. Dis. 30,259. Inami, Y.H., R.M. Nakamura and E.M. Tan, 1973, J. Immunol. Methods 3,287. Jokinen, E.J. and H. Julkunen, 1965, Ann. Rheum. Dis. 24,477. Luciano, A. and N.F. Rothfield, 1973, Ann. Rheum. Dis. 32,337. Pincus, T., P.H. Schur and J.A. Rose, 1969, New Engl. J. Med. 281,701. Rothfield, N.F. and B.D. Stollar, 1967, J. Clin. Invest. 46, 1785. Schur, P.H. and J. Sandson, 1968, New Engl. J. Med. 278,533. Seligmann, M., 1957a, C.R. Acad. Sci. 245,243. Seligmann, M., 1957b, C.R. Acad. Sci. 245, 1472. Seligmann, M., 1958, Rev. Fran(;. Etud. Clin. Biol. 111,558. Seligmann, M., 1963, Arthr. and Rheum. 6,542. Tan, E.M., P.H. Schur, R.I. Garr and H.G. Kunkel, 1966, J. Clin. Invest. 45, 1732. Yantorno, C., C.M. Riera, A. Risemberg and A. Strussberg, 1974, J. Immunol. Methods 5, 253.