Micro-hemagglutination tests for detection of native and single-strand DNA antibodies and circulating DNA antigen

Journal of Immunological ,Methods 3 (1973) 287 -300. O North-llol!and Publishing Company

MICRO-HEMAGGLUTINATION TESTS FOR DETECTION OF NATIVE AND SINGLE-STRAND DNA ANTIBODIES AND CIRCULATING DNA ANTIGEN* Y.11. INAMI, R.M. NAKAMURA, anti E.M. TAN** Department o] Patholo,~v. California Colh'ge o]MedichTe, Unirersit)" of Cali]ornia. lr~'hre. California and Orange Count), Medical Center. Orange. California. and.

Divisioll of Allergy and Imnntttolog),. Departtnent of l:~'perirnental PatholoKv, Scripps CTinic attd Research Fot~ndation, La Jolla, Cali]brnia. U.S.A.

Received 12 March 1973

Accepted 7 June 1973

A practical micro-titration hemagglutination test which can be helpful in the detection of either anti-I)NA antibodies or circulating free DNA is described. ]'he procedure utilizes formalinized human erythrocytes coated with sonically disrupted DNA which are capable of reacting with antibodies to both native and single-strand DNA and show minimal non-specific agglutination. By an inhibition assay, the test could be adapted to detect circulating DNA antigen. Eleven of 51 SLE sera revealed anti-DNA antibody titer of 1 : 4 - 1 : 512 and 9 of the 40 sera showing no anti-DNA antibody had circulating DNA levels ranging from 0.8--25 tag/ml. The test provides a reliable method to determine circulating DNA antibody or antigen and may be used to l\~llow the course of the disease and response to therapy in patients with systemic lupus erythematosus.

1. INTRODUCTION In the various collagen diseases, a n t i b o d i e s to m a n y d i f f e r e n t nuclear antigens are seen. Several i m p o r t a n t nuclear antigens are d o u b l e - s t r a n d native d e o x y r i b o n u cleic acid ( N - D N A ) , d e n a t u r e d or single-strand d e o x y r i b o n u c l e i c acid (SS-DNA), d e o x y r i b o n u c l e o p r o t e i n i d e n t i f i e d as D N A - h i s t o n e c o m p l e x , saline e x t r a c t a b l e antigens (Sin and RNP), and nucleolar ribonucleic acid antigen ( R N A ) (Stollar and

* Supported hy NIH grants AMI2198 and A100214 and by Research and Education Foundation of Orange County Medical Center Attending Staff, Inc., grant 71-5. ** Senior Investigator, "[he Arthritis Foundation. This is publication No. 686 from tile DepartThis is publicatit~n No. 686 from the Department of Experimental Pathology. 287

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Levine, 1961: Beck, 1963: tlohnan. 1965:Tan and Kunkel. lt)b6:Tan et al., 1966: Arana and Seligmann, 1967: Tan. 1967a: ('ohert et al.. 1971 : Kol'fler et al., 1971, 1073:Sharp et al., 1¢)71). A sensitive and practical test for all types of antint,clear antibodies such as the indirect immunoflt, orescent (Roitt and Doniach. 196t~: Beck. 1~)7(): Bundell. 1970) or peroxidase-labeled antibody tests (Nakane and Pierce. 1967: Avramez, s and Bouteille. 1908: Benson and Cohen. 1970: Pelts and Roitt. 197t) would be preferred as tile initial screening tests for the collagen diseases. However. tests for antibodies to specific nuclear antigens st, oh as DNA arc helpful in: (a) establishing specil+ic diagnosis: {b) monitoritlg the cot, rse of therapy. ,rod (c) predicting prognosis and response to therapy. In systemic lupus erythetnatosus (SI_E). the impt~rlance of antibodies to DNA m pathogenesis of tissue injury has been established (Tan el al.. 1966: Koffler el al.. 1967. 1973: Krishnan and Kaplan. 1067: Andres et al.. 1970). The presence of I)NA and antibodies to DNA in circulatitm ,,','as the initial evidence which suggested that this immune complex might be important in kidney disease (Tan et al.. 1966). Iiistotogic and inmlunologic findings on renal biopsies reported by Andres el :1t. (1970) and Koffler et al. (1967) clearly demollstrafed dtat DNA was present on glomerular basement nlembranes and distribuled in a tllatitaer sinlitar Io tllat for inanlttnoglobulins. [:urthcrmore. ehlates t'rom kidneys of S[_E patients were comprised of immunogh~lmlins with high levels of specific antibody activity Io nuclear antigen (Koffler et al.. 1967: Krishnan and Kaplan. 1967). The radiolabeled-l)NA antigen I+inding test is a sensitive assay for detection t~t+ ,mti-DNA antibody {\Void ct al.. 1968. C a r r e t al.. 1c~69: Pinct, s e t al.. 1909) ht, t is not readily available in many clinical laboratories. Previously reported DNA agglutination tests utilized either native I)NA or heat-denatured-(single-strand) DNA coated cells {Jokinen and Julkunen. 1965: Burns and Rheins. 1966: Koflle, et ,tl.. 1971 1973. Sharp el al. 1971). A specific agglutination test with smgle-strandDNA coaled cells may demonstrate auto-agglutination unless co[llplexed with actinomycin 1) (Kofl'ler et al.. 1971 ). The present reporl describes a sensilive micro-hemagglutmatiotl test utilizing sonicated-DNA coated red cells which is able to detect antibodies to both native and single-sltand I)NA and shows minimal tier'l-specific agghttitlatitm. The variables such as the salt ,rod protein concentration, specificity and sensitivity of the test have been explored.

2. MATERIALS AND METHOI)S

2.1. Buffer designation I'BS(I): Phosphate (0.075 M) b u f f c r - N a C l (0.45%). pH 7.3: PBS(II): Phosphate (0.01 M) bt, ffcr NaCI (0.84%),pH 7.3:PBS(IlI): Phosphate(0.01 M) buffer-NaCl

(0.6%).pH 7.3:Mcllvaine: Critic acid (0.05 M) Na211PO 4(0.1 M).plt4.9.

D,VA and antibodies to DNA by micro-hemagglutination

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2.2. Source o f sere Positive sera were obtained from patients showing active clinical symptoms of SLE. Some of these were referred samples. For non-SI,E samples, apparent normal specimens were taken at random from the clinical laboratory, specimens in which the sera exhibited no abnormal chemistries. In these so called normal sera the disease states were unknown. Both the positive SLE and normal sera were collected and processed in the manner usually employed in the clinical laboratory without tmdue precautions. Serum samples were inactivated by heating at 56°C for 30 rain, preserved with 0.15~ sodium azide and stored at 4°C or frozen at 20°C. 2.3. hnmunological techniques The Ouchterlony immunodiffusion method was employed to establish anti-DNA antibodies in several SLE scra. Diffusion plates were prepared with 0.6% agarosc (Seakem distributed by Bausch and Lomb) in PBS(ll) containing 0.1% sodium azide. To improve clarity, the hot agarose solution was passed through coarse filter paper. Petri dishes (100 X 15 ram) containing 25 ml of agarose were used. Usually, the wells were 7 mm in diameter and separated 4 mm apart. Immunological reactions were allowed to proceed overnight at room temperature and observations for precipitin lines were made the next morning and for two additional days. At the end of the third day, the plates were discarded if no lines appeared. 2.4. DNA sokttion Stock solution of native DNA was prepared by dissolving call" thymus DNA (Worthington Biochemical Corporation) in PBS(ll) or in Mcllvaine buffer (pH 4.9) at a concentration of 1 mg/ml. Single-strand DNA was prepared daily by heating the 1.0 mg/ml native DNA solution for 10 rain in a boiling water bath and cooling immediately in an ice bath. Sonicated DNA (So-DNA) was obtained by sonically disrupting native DNA, 0.5 mg/ml, for 2 rain using a power sonifier ( Branson Sonic Power Sonifier, Model 91 I0). The beaker containing the solution was placed in an ice bath and sonication was ca,ried out for 1 rain: the heat developed was permitted to dissipate and sonication was continued for another minute. 2.5. Formalinization o f eo,thro~3,tes Fresh hnman type O Rh negative blood from blood bank was centrifuged at 800 g for 4 rain and the plasma carefully removed with an aspirator. The packed red cells were washed 4 times, each time with 5 8 vol of PBS(I). Care was taken to remove any buffy coat that was observed on the surface of the packed cells. After the fourth washing, the cells were suspended to z, concentration o t 10~,~:in PBS(I). Stock 37% fornraldehyde solution (J.T. Baker) was diluted to 3.75~ with PBS(1) and

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the pit adjusled lo 7.3. ILqual vt~hunes of the 10
2.7. Orating o.f cell.~ Fomlalinized tanned cells were coated with either native, single-s/rand or sentcared DNA. DNA solution in Mcllvaine huller (p|l 4.9 at a cotlcentralioH of 50 /ag/ml) was added to the packed cells to give a 4<~ cell suspension. This mixture was incubated at .~7 ( for 1 hr with frequent shaking. The cells were packed and washed 4 limes with PBS(II) and resuspended in this buffer cent,lining 0.075; bovine serum albunlin (BSA) (llyland Laboratories) to a final concentr;ition of 1.3';. These coated cells were stored at 40( ` and used for lhree wceks wilhoul loss t~l reactivity. 2.8. Micro-titralirm hemagghttinations The sera to be tested were serially diluted with a 25 ~1 inicro-dih, ter (('ookc Engineering ('onlpany) in micro-titration V-plates (Linbro of tile Pacific. Los Angeles). The diluent was PBS(III) containing 0.2":~ BSA. DNA-coated cells were then dispensed into each well using a 25/.d pipet dropper (Coole Engineering ('o.). The plates were tapped several times to achieve complete mixing of the solutions and covered with transparent sealers. Hemagglutination liters were read 1 - 4 h r after titration. A smooth mat at the bottoln of the wells was recorded as positive (+) and a clearly defined button of cells centered at the bottom of the well was designated negative (--); intermediate reactions were assigned (-+). The last well (highest dilutiorl) showing positive re~,ctiori was taken as the endpoint.

DNA and atttibodies to DNA by micro-hemagglutination

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2.9. Specilh'i O, -lhe specificity of the hemagglutination reaction was determined by inhibition sit, dies. In a parallel row of wells. 25/11 of antigen at a concentration of 100/ag/ml in PBS(III) containing 0.2~7~ BSA was placed m all but the first well. To the first well were added 25/al of antigen at a concentration of 200/ag/ml in PBS(III) and 25 ~1 of lest sert, m, the solutions were mixed and serially diluted in subsequent wells conlainmg constant levels of antigen. Thus all wells contained a final amount of 2.5 ,ug DNA as added inhibitor. The plates were shaken and allowed to stand at room temperatt, re for approximately 20 min. Then. DNA-coated cells were added. the plate tapped and covered. Decrease in the hemagglutination liter compared to the lest without added DNA confirmed the presence of DNA antibodies in the test serum. 2.10. Determinatkm o f circulating DNA A quantitative inhibition technique was used to determine the circulating DNA levels in SLE sera. The reference assay employed known quantities of DNA to inhibit hemagglutination and was performed as follows : Twenty five microliters of DNA solution at 200 og/ml were serially diluted in PBS(III) containing 0.2% BSA. Reference antiserum (Ab) known to contain antibodies to both native and singlestrand DNA was diluted with PBS(II) to a concentration approximately two times that indicated by the hemagglutination endpoint (in this instance 1 : 32 dilution of serum Ab). Twenty five microliters of 1 : 32 dilution of Ab serum was added to each well containing the serially diluted DNA. The plate was allowed to stand at room temperature for 20 min before the addition of 25/al of DNA-coated cells. Hemagglutmation was read 1--4 hr later. Concentration of DNA was calculated from the last well which inhibited hemagglutination. The detection of circulating DNA in SLE serum involved the same steps as those described in the previous paragraph with one exception, namely, that 25 ,ul of test serum was serially diluted in place of the DNA solution. After tile addition of the diluted reference antiserum, the plate was allowed to stand at room temperature for 20 min. Then, DNA-coated red cells were added and the hemagglutination inhibition titer was read 1 - 4 hr later. From this information, in conjunction with the sensitivity of the DNA assay, the amount of circulating DNA in the SLE serum was calculated by the followmg relationship: Circ. DNA (ug/ml) = (DN-A~)I lT S where: Circ. DNA = Circulating DNA in test serum: (DNA) = DNA concentration (200,ug/ml) that was used in sensitivity determination: 1s = Inhibition tiler obtained from reference assay with DNA: IT = Inhibition titer of test serum.

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3. RESUI.FS As an initial step in this work. unti-DNA antibodies in several scru (AF. BW. EL) taken frolll Ski" palients were characterized by the use of the O u c h l e r l o n y double immunodiffusicm technique in agarose gel. A photograph of an agar plate is given ill fig. I to show tile prccipilm reactions of BW and AF c (AI- concentrated 3 I by v;.lctlut'~l dialysis) with I mg/ml mttive DNA :,nd 1 mg/ml heat-denatured DNA. In a separate experiment it was learned tha! u n c o n c e n t r a t e d AF reacts with native DNA It) give a single precipitin l i n e however, no lines were observed to heal-denatured I)NA. Serum BW. known !o contain anti-DNA antibodies specific to single-strand DNA. logically gave a strong precipi~m line to heat-denatured DNA. A faint reaction line is seen between BW and N-I)NA. In other such i m m u n o d i f f u s i o n studies. more frequently than not. this weak line has not been observed. Significant ;.llllOUtllS of single-strand DNA in native DNA solution would have resulted in a precipitin line with lhis serum. A single reaction line of AI:. with native DNA (N-DNA) and two trees with heat-demltt, red DNA (IID-DNA) are seen. The stronger line against HD-DNA fused with lhe N-DNA line suggesling lhat serum AFc contained antibodies to DNA which ~eact to recognize identical antigenic determinants on native and singlestrand DNA. This is the more frequent type of a n t i b o d y that has been described in the literature (Tan and Kunket. 1966: Arana and Seligmann, 1967: ('ohen et al.. I ~771 : Kotfler el al.. t q71 ). althot, gh there have been anli-DNA antibodies reacting OIIIV wilh native DNA. As to the wec,k reaction line of AFc with HD-DNA. its identification has not been elucidaled. Fig. 1 further reveals that the precipilin lines belween BW and tID-DNA and between AFc and I-]D-DNA are not lines of identity. This has been repealedly c o n l n m e d m many such immunodit'fusion stt, dies. Thus. AF c

N-DNA

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HD-DNA

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N-DNA

Fig. I. lmmunodiffusion in agarose showing reaction of t~vo SLI~ sera with difterent preparations of DNA. nalivc (N-DNA) and heat-denatured (HD-DNA). Serum AFc cont:,ms antibodies to DNA which react to recognize identical d¢lerminants on N-DNA and IlD-DNA. as shown by identical precipitin lines. "l'hc precipitin lines between BW and IID-DNA and between AFc and IID-DNA arc not linc~, of identit.',, indicating that BW recognize antigenic determinants only in SS-DNA.

DNA and antibodies to DNA b)' micro-hemagglutination

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HD-DNA So-DNA N-DNA Fig. 2. lmmunodiffusion in agarose showing reaction of sonically disrupted (So-DNA), native tN-DNA) and heat denatured (IID-DNA) DNA with sera BW and AF,,. AFt reacts with both native and sonicatcd DNA with precipitin lines which suggests identity. Lack of identity of precipitin lines of A I c and BW with sonicated I)NA is seen, indicating reaction with different antigenic determinants. Furthermore. So-DNA and ltD-DNA have common antigenic determinants as shown by the line of identity to serum BW.

sera such as BW recognize antigenic determinants present only in SS-DNA; whereas, AFc recognize antigenic determinants present on both native (double-strand) and single-strand DNA. Another serum EL behaved almost exactly as BW, revealing a strong line to single-strand DNA and none to native DNA. AFc reacts with both native and sonicated DNA with precipitin lines which suggested identity although in repeated experiments it was difficult to obtain complete fusion of the two lines. However, crossing or spur formation of the two lines was not observed. Serum BW however, reacted with both heat-denatured and sonicated DNA to give lines of complete fusion. A further feature illustrated in fig. 2 is the lack of identity of precipitin lines of AF c and BW with sonicated DNA, suggesting reaction with different antigenic determinants. Since studies of the type illustrated in fig. 1 demonstrated that BW reacted only with single-strand DNA. and not with native DNA, sonicated DNA must contain antigenic determinants of singlestrand DNA as well as native DNA. A F and EL (a second serum with antibodies to SS-DNA) by virtue of their high antibody content and their relative abundance, were used as reference materials to characterize the conditions optimum in the adaptation of micro-hemagglutination procedure to the deteclion of DNA antibodies. Since sonicated DNA was demonstrated to contain both single and double-strand DNA determinants, this antigen was used to sensitize formalinized, tanned, type O Rh negative human erythrocytes. Using the coated cells, the micro-hemagglutination method was evaluated with A F and EL. Typical henlagglutination patterns for these sera are shown in figs. 3 and 4. Specificity of tim agglutination was confirmed by inhibition experiments. The first row in fig. 3 shows the hemagglutination pattern of 8 wells (DNA antibody liter of 1 : 256) for EL. The behaviour exemplified in the second and third rows illustrates that in the presence of added single-slrand

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Fig. 3.1lcmagglutination patterns for serum I'll. against red L.'cllscoated with sonicatcd I)NA. Ist rm~ (11. + PBS) shows positi',c hclnag.,.2hltmation to dilution o i ] : 256 (v, ell 8). hi second alld third rows, the test Was performed m the presence c)t"added single-strand L)NA (SS-I)NA) and ,~onicatcd I)NA (,So-DNA) and sho,as inhibition or" agghltinalion. Addition of nmtive I)NA (N-DNA) shows no inhibition, demonstrating that specificity of I'L serum is ag:.tinM SS-I)NA.

DNA and sonicated DNA, inhibition of agglutmatiot~ was achieved. The pattern given in the fourth row ittdicates that m) inhibititm was obtained with the addition of native DNA. These results confirmed tire information derived from double intmtnnodiffusion experiments, namely, that EL contains DNA antibodies only it) single-st rand DNA. The hemaggtutint, tion results for A F are given in fig. 4. Agglutination is seen to 6 wells (dilution ()t" 1 : 64). In the next three rows. test p e r f o r m e d in the presence of added single-strand DNA (SS-DNA). sonicated DNA (So-DNA) and native DNA (N-DNA) shows that agglutination was inhibited COlnpletely. These results are consistent with the data frot'q O u c h t e r h m y phttes which showed AF to coiHailt antibodies with specificity to single-stratld and native DNA. Red cells coaled with native DNA and single-strand DNA were also evaluated. Although. at times, native DNA-coated red cells served equally well in the detection of DNA antibodies as sonicated D N A - c o a t e d red cells, preparations of native DNA-

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Fig. 4. Ilemagglutination patterns for serum AI- against red cells coated with sonicated DNA. lsl row (AF + PBS) shows positive hemagglutination to dilution of 1 : 64. In the next three rows. the test was performed in the presence of added single-strand DNA (SS-DNAL sonicated DNA (So-DNA) and native DNA (N-DNA) and shows inhihition of agglutination, demonstrating that specificity of AF serum is against single-strand, sonicatcd, and native DNA.

D,VA and antihodies to DNA h)' micro-hemagghttiuation

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sensitized cells from different batches were found to give varying a n t i b o d y titers with the same serum. Moreover. native DNA-coated cells often st, ffered from tendency toward non-specific agglutination and low stability, whereas, rest, Its from sonicated I)NA-coated cells were consistent and highly reproducible. Red cells coated with single-strand DNA were found to demonstrate non-specific agglutination. These problems with single-strand DNA-coated cells were ~,lso reported by Koftqer el al. (1971). They observed that non-specific agglutination could be removed if single-slrand DNA is first coupled to a c t i n o m y c i n - D and then attached to red blood cells. The micro-titration technique using sonicated DNA-coated red cells was applied to 103 non-Sl.E or negative sera and 51 sert,m specimens from clinically active lupus patients. The results are depicted in fig. 5. in which the reciprocal of tile hemagglutination titers for the non-SLE (NITS) and SLE sera are plotted. Ilemaggh, tination was not observed in most of the negative sera: those which showed sli~lt agglutination, the reaction was restricted to tile first well (dilution of 1 ' 2). This indicates that serum which demonstrates a titer of 1 : 4 or greater is significant. Eleven of the 51 SLE sera exhibited DNA a n t i b o d y titer ranging from 1 : 4 to 1 : 512. Seven showed specificity to single-strand DNA only (:::): the remaining four had specificity to both double and single-strand DNA (./",). A t t e n t i o n was then directed to the determination of circulating DNA. Sample AF was used as :, source for the reference antiserum t A b ) because large quantities of serum were available and it contained antibodies to both single- and double-

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Fig. 5. 1temagglutination test on 103 normal human sera (NTIS) against So-DNA-coated red cells shows anti-DNA antibody titers of less than 1 : 4. Eleven of 51 SLE sera iSLE) revealed ttA titers ranging from 1 : 4-1 : 512, seven showing specificity for single-strand DNA (,:)and four showing specificity for both native and single-strand DNA (:.).

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Y.II. INAM! et al.

s t r a n d DNA. S t a n d a r d s o l u t i o n s o f D N A were p r e p a r e d in PBS(III). each c o n t a i n i n g 200 p g j t n l o f e i t h e r heat d e n a t t , r e d , native or s o n i c a t e d DNA. Similar s o l u t i o n s were m a d e in n o r m a l h t , m a n serum. Q u a n t i t a t i v e itlhibition e x p e r i m e n t s were carried out using these solutions. T h e degree o f i n h i b i t i o n was essentially the same tol the three kinds o f DNA mid i n d e p e n d e n t o f the media in w h i c h the DNA s o l u t i o n s were prepared. T h e negative sela a n d the 51 Ski- sera used in the search for a n t i - D N A were t i t r a l e d for c i r c u l a t i n g DNA. All sera s h o w i n g significant titer for a n t i - D N A anlibodies were negative for circulating DNA. h l h i b i t i o n titers for the n o n - S L E sera were f o u n d t o be m~ greater than I " 2. T h u s . it/hibititm tilers o f 1 • 4 a n d greater are significant :is positive tests. Nine o f the SLE sera had i n h i b i t i o n liters ranging from 1 : 4 to 1 : 128. By c o m p a r i s o n with a test run s i n t u l t a n e o u s l y in w h i c h k n o w n a m o u n t s o f D N A were added, it c o u l d I~e calculated that the range o f circulating DNA was 0.8 25,ug/ml. By following carefully lhc p r o c e d u r e as ot, tlined, it was possible to achieve clearly defined h e m a g g l u t i n a t i o n p~,tterns w h i c h indicate the presence or absence o f e i t h e r DNA a t t t i b o d i e s or circt, lating DNA. As a,t illustrative e x a m p l e , a p h o t o g r a p h o f a t i t r a t i o n plate is given in fig. 6. First row ( A b + PBS) s h o w s positive hemagglutinatioll to dih, tion o f 1 : 6 4 . Negative h e m a g g l u t i n a t i o n in the s e c o n d row indicates a b s e n c e o f D N A a n t i b o d i e s in n o n - S L E serum JP. In the n e x t six rows.

N)4.PB8 JP÷PBS So-DNA4.PBS÷Ab IHtPBS~.Ab JC ~.PB84.Ab RS+PBS.14ib C,8.~PBS,,Ab MB,,.PBS÷Ab I'ig. 6. Photograph of a bemagglutination titration plate incorporating red cells coated with sonicated DNA. 1st row {Ab + PBSI shows positive hemagglutination to dilution of 1 : 6 4 . Negalive hemagglutination m 2nd row indicates absence of DNA antibodies in non-Sl.l serum JP. In the nexl six rows. quantitative inhibition test was performed using reference antiserum diluled I : 32. 3rd ro~ shows with 200 tag/ml So-DNA serially diluted. IlA inhibition of IO wells was achieved. Rows 4 and 6 illustrate respectively the presence of 25 and 1.6,ug/m} of circulating DNA. In the 5th row. no inhibition is observed because the serum contains antibodies to DNA. Row 8 exemplifies that nonspecific inhibition of one well may be observed m Solne normal sera: ill others, as demonstraled by ttle pattern in row 7. no inhibition is observed.

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a quantitative inhibition lest was performed by adding reference antiserum Ab dih, ted 1 : 32 to each well containing serially diluted DNA or serum solutions. At 1 : 32 dilution the DNA antibody level corresponds to slightly more than that needed to agghttinate the cells. Row 3 shows that in the test in which 200 #g/'ml So-DNA were serially diluted, a hemagglutmation inhibition of 10 wells was achieved. The sensitivity of the assay using antiserum Ab is designated as that amot, nt of DNA which is just sufficient to inhibit hemagglutination. Inhibition to 10 wells gives a sensitivity of 0.2/ag/ml. Rows 4 and 6 illustrate respectively the presence of 25/ag/ml and 1.6 #,g/ml of circt, lating DNA. In the 5th row, no inhibition is observed because serum JC had been demonstrated to contain antibodies to DNA. Row 8 exemplifies that non-specific inhibition of one well may be observed in some normal sera:m others, as demonstrated by the pattern shown in Row 7. no inhibition is observed.

4. DISCUSSION The present ]mnlaggh, tination technique which incorporates the use of sonicated DNA-coated type O, Rh negative formalinized human erythrocytes offers several advantageous features in DNA immunoassay: a) Tire method may be performed with a micro-titration apparatus using small quantities of serum. Also the titration steps are greatly simplified. b) Preadsorption of the test serum samples is eliminated with the use of human O. Rh negative cells. c) With the use of sonicated DNA antigen, the test will detect antibodies to both native and single-strand DNA. The specificity of the antibodies can be determined by running simultaneously, inhibition with native and single-strand DNA: d) An inhibition procedure can be used to test for DNA in circulation capable of detecting 0.2/ag DNA per ml. e) The incidence of non-specific agglutination resulting with test serum samples is practically eliminated with the use of sonicated DNA. Methods which utilize native or single-strand DNA-coated cells tend to show an increased incidence of non-specific agglutination. In the adaptation of hemagglutination technique to the detection of DNA antibodies, several factors important for the reproducibility of the method were revealed and are discussed in the following paragraphs. 4.1. Optimal conditions fi)r hemagglutination titration

4.1.1. ltuman erythrocytes The blood should be washed and formalinized on tile same day that it is drawn from the donor. These formalin-treated cells when examined under subdued light at magnification 450× should appear as smooth bi-concave discs. Such cells worked

298

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,,veil in hemagghltination experiments, ltowever, t'ormalmized red cells prepared froln an outdated Hood hank unit gave extensive non-specific agglutination. 4.1.2. Salt concentration ot diluent In the early phase of this study+ barhital Imft'er (0.05 M. pit 7.3) with 0.217; BSA was used as the diluent. Extensive non-specific agglutination was observed. B\.' increasing the ionic strength of the Ixtrbital buffer solution with the ~,ddition of sodiuln chloride (0.075 M g.ltld higher) the non-specific reaction was etimim, ted. A similal phenomenon was encountered m the use of O.01 M phosphate buft~'r as diluent. Sa,line concentration ofO. I M was optimum: higher concentration resuhed in lower tilers and lower concentrations produced non-specific agglutination. Phosphate buffered saline was preferred over barbital buffered saline because the latter was fottnd to form precipitates at refrigeration telllperatures. 4.].3. Protein concentration Usually, 0.25~ BSA was added to phosphate buffered sz,line. sometimes 0.075.; was ft)und tt)he adequate. When nt) protein was used, some non-specific agglutination resulted. 4.1.4. Antigen concentration Fifty micrograms of DNA were used to sensitize the cells contained in I ml of a 47f suspension. In a single experiment 25 ~g level was tried and found to give rest,Its (tiler and sensitivity) comparable to those derived from higher antigen level: however. the cells coated at the lower antigen concentration soon losl their immunological activity. 4.1.5. ('ell concentration One 1.3 and 2~)~ cell suspensions were evaluated. Endpoint titer was lower by one well when the 291 suspension was used. Although, higher titers were obtained with the 15:{-suspension, the buttons formed on the hottoms of the micro-titration plates were rather small. One (/, and 1.3% suspensions produced the same titer but the latter cell concentration gave easily readable and well defined buttons. 4.1.6. Collection of serum Blood is drawn and collected in a clean tube. As soon as the clot is t'ormed, the cellular elements are removed by centrifugation. Sodium azide at a concentration of O.l'.~ is added to the serum, which is stored at 4°C or frozen at 20o( ". The usefuhless of sonicated DNA-coated cells m the detection of antibodies to DNA m SLE sera has been demonstrated, tlemagglutination tests have shown that such cells are able to react with both single and double-strand DNA, eliminating the need for two separate groups of sensitized cells, one coaled with single-strand DNA and the other coated with double-strand DNA. The sensitivity of sonicated DNAcoated cells to both types of DNA antibodies was verified by immunodiffusion

D:VA atul antibodies" to D:VA IU" micro-hemagghttittation

299

e×perinlents. SLE sera having high henaagghitination titers invariably gave positive gel diffusion rest, Its: some showed precipitin lines to both SS-DNA and N-DNA and others only to SS-DNA. Specificities as shown by these assays were in agreement with those found with hemagglutination inhibition tests. In tile daily performance of micro-titration laemagglutination test it is strongly reconunended that tile coated cells be tested with the use o l ' a negative and a positive control. Specificity of tile agghitinations should be checked by rutming an inhil)ition experiment. If only a few samples are involved, the hamagglutination and inhibition determinations can be performed m the same run. However, where large numbers of samples tire involved it is perhaps more e×peditious to screen initially for positive hemagglutination and conduct inhibition experiments only o n those specimens which exhibit significant anti-DNA antibody titers. Specimens iioI demonstrating antibodies to DNA might be tested for circulating DNA. Ill our laboratory sera from patients with clinical symptoms suspect of SI,E arc initially screened for anti-nuclear antibodies by indirect immunofluorescence. Positive sera tire then tested bv micro-hemagglutination for anti-DNA antibodies. This is important because in the pathogenesis of SLE the important pathogenic antibodies appear to be anti-DNA antibodies (Tall et al.. 1%6: Koflqer ctal.. 1 % 7 Krishnan and Kaplan, 1967:Andres et al., 19701. lfanti-DNA antibodies tire not obserced. these sera and those SLE sera which demonstrated negative anti-nuclear antibody test by indirect immunofluorescent method tire examined for circulaling DNA using the micro-hemagghltination inhibition procedure becat.se tictive cases of SLE may have circulating DNA and/or DNA antigen antibody complexes which will result in a negative in vitro test for anti-DNA antibody. Clinical trial by micro-hemagglutination on 51 SLE scra showed 22'); had antiDNA antibody w i t h titers ranging from 1 : 4 1 : 512, and 18",',, had circulating DNA of 0.8 25/ag/ml. Because circulatingDNA was detected by inhibition of an antibody reactive with both native ancl SS-DNA, the incidence (18~,/~) represents the sum total of the occurence of both native and SS-DNA m SLE sofa. The separate incidence of native and SS-DNA antigens could be determined by using appropriate sera containing antibodies reactive only with native or SS-DNA respectively. In this connection, Koft'ler et al. (1973) have reported tile frecluent occurrence of SS-DNA in sera of SLE and other diseases. Further studies with this micro-hemagglutilaation systein are in progress to provide niore information concerning the character of circulating DNA in disease states and tile possible relationship of this finding with pathogenesis.

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Avramcas, A. and M. L:louteille. 1968. l'ixptl. Cell Rcs. 53. 166. Beck. J.S.. 1963. Scot. Mcd. J. 8. 373. Beck. J.S., 1970, Mayo Clin. Proc. 4 4 , 6 0 0 . Bcnson. M.I).and A . S . ( ' o h c n , 1970 Ann. lnternalMed. 7 3 , 9 4 3 . Bundell. G.P., 1970. m: Progress in clinical pathology ed. M. Slefanini, vol. 3 q(;runc and Sttatton. New York)p. 211. Burns, R.M. and M.S. Rhcins. 1966, Proc. Soc. l-xptl. Biol. Med. 1 2 2 , 7 1 4 . Carr. R.I., D. Koffler, V. Agnello, and I1.(.;. Kunkel, 1969. Clin. Exptl. h n m u n o l . 4. 527. ( o h e n . S.A., G.R.V. Ilughes, G . L Noel and C.I,. Christians. 1971. ('lin. Exptl. h n m u n o l . 8, 551. llolman. II.R., 1965. Ann. N.Y. Acad. Sci. 124.8(1(I. Jokmcn. E.J. and It. Julkuncn, 1965, Ann. Rheumatic Discascs 24. 477. Kofllcr. D..P.lt. S h u r a n d l t . G . Kunkcl, 1967. J. Exptl. Mcd. 126 607. Kofflcr. D.. R.I. Carr, V. A~ncllo and II.G. Kunkcl, 1969. l,cdcration Proc. 28. 486. Koftler, D.. V. Agnello, R. T h o b u r n , and t|.G. Kunkcl, 1971, J. Exptl. Med. 134, 169s. Kofflcr, I)., R. Cart. V. Agncllo. R. T h o b u r n and II.G. Kunkcl, 1971. J. Exptl. Meal 134. 294. Kc~ftlcr. D . V. Agncllo, R. Winchcstcr and II.G. Kunk¢l, 1973. J. Clin. Invest. 52. 198. Krishnan. ('. and M.It. Kaplan, 1967..I. ('lin. Invest. 4 6 , 5 6 9 . Nakanc. P.K. and G.B. Picrcc. 1967. J. ('ell Biol. 33. 307. Pctts. V. and I.M. l~,oitt. 1971, ('lin. I,~xptl. h n m u n o l . 9. 407. Pincus. T.. P.It. Schur. J.A. Rosc, J.L. Decker ;anti N. Talal, 1969, New l:'ng. J. ~.led. 2 8 1 , 7 0 1 . Roitt. 1.31. and D. Doniach. 1969, World Ilealth Organization tlandbook. Sharp. G.C., W.S. Irvin, R.I,. l.aRoquc. C. Voice, V. l)aly. A D . Kaiser and If.R. llolman. 1971, J. Clin. lnvcst. 50, 350. Stollar. D. and L. l,cvinc. 1961. J. h n m u n o l . 87. 477. Tan. L.M.. 1967a. J. ('lin. Invest. 46. 735. Tan, E.M.. 1967b. J. l,ab. ('lin. Mcd. 70. 800. Tan. I,L.M. and II.G. Kunkcl. 1966. J. lmmunol. 96,464. Tan. E.M. and P.G. Natali. 1970..I. lmmt.nol. 104. 9 0 2 Tan. I-.M.. P.tt. Shur. R.I. ('arr and II.G. Kunkcl. 1966..I. Clin. Invest. 45, 1732. Wold. R.T.. I'.E. Young, I;.M. Tan and R.S. l:arr. 1968, Science 161. 306.