Serum antibodies to DNA by counterimmunoelectrophoresis (CIE)

CLINICAL

IMMUNOLOGY

AND

2,510-518

IMMUNOPATHOLOGY

(1974)

Serum Antibodies to DNA by Counterimmunoelectrophoresis

(CIE)l

JOHN S. DAVE? AND JOHN B. WINFIELD~ University of Virginia Charlottesville, Received

School Virginia July

of Medicine, 22901

9,1973

Counterimmunoelectrophoresis (CIE) is rapid, convenient, and a relatively sensitive method for detection of anti-DNA antibodies. Specificity of anti-DNA antibodies for SLE as measured by CIE is only fair with unfractionated DNA as antigen. The relative lack of specificity is related to contaminating denatured DNA in our preparation. Specificity regarding detection of anti-DNA is good, although there was a disturbingly high incidence of sera positive by CIE and negative by Farr assay (11% of sera tested). Again, these false positives occurred when commerical unfractionated DNA was used, and this problem was largely overcome by use of fractionated or purified DNA. Data obtained using methylated albumin-kieselguhr native DNA fractions suggest that anti-nDNA is quite specific for SLE. There remain, however, occasional patients who give positive reactions with native DNA by CIE, but who apparently have illnesses other than SLE.

Antibodies to nuclear constituents may be found in a variety of conditions; they are usually analyzed by immunofluorescence techniques that are now routine in many institutions. Because antinuclear factors may be found in so many different pathological conditions, and indeed may be found in low titer in some apparently normal individuals, a more definitive screening test for certain connective tissue diseases, especially systemic lupus erythematosus (SLE), seemed desirable. Antibodies to native DNA (nDNA) have been reported to occur mainly in patients with active SLE (l-3). Current methods to determine the presence of anti-DNA include Ouchterlony double diffusion, hemagglutination, complement fixation, and variations of the Farr binding technique (4-7); each of these methods has certain disadvantages as a routine procedure. For the past 2 yr we have been screening sera for anti-DNA antibodies by the technique of counterimmunoelectrophoresis (CIE), a method that has been widely used for the assay of Australia antigen (8). We have further compared the technique ’ This work was supported in part by a grant from the United States Public Health Service (5ROl-AM11766) and in part by a grant horn the John S. Hartford Foundation, Inc. Presented in part at the Southern Society for Clinical Investigation Meeting, New Orleans, LA in January, 1973. 2 Division of Rheumatology, Department of Medicine, University of Virginia School of Medicine, Charlottesville, Virginia 22901. 3 At the time this work was done, Dr. Winfield was a Fellow in Rheumatology at the University of Virginia. Present address: The Rockefeller University, New York, New York 10021. 510 Copyright All rights

@ 1974 by Academic Press, Inc. of reproduction in any form reserved.

ANTIBODIES

with the Farr method sitivity.

DNA

in order to more precisely

MATERIALS A. Patients

TO

AND

BY

511

CIE define

its specificity

and sen-

METHODS

and Sera

Patient sera were obtained primarily from the Lupus and Arthritis Clinics as well as the Medical Service of the University of Virginia Hospital. Normal control sera were obtained from the University of Virginia blood bank. Patients diagnosed as having SLE fulfilled at least four of the preliminary criteria of the American Rheumatism Association Committee (9). In general patients with disorders other than SLE were seriously ill and/or were suspected of having some form of vasculitis or connective tissue disease. Sera were heatinactivated at 56°C for 30 min before use. B. CZE Kodak Lantern Slides (8.3 x 10.2 cm) were coated with Seakem agarose (manufactured by Marine Colloids, Inc., Rockland, ME) made up to 1% in barbitone acetate buffer (Colab Laboratories, Chicago Heights, IL), pH 8.6, 0.05 ionic strength. Parallel rows of wells, 4 mm in diameter, were punched with a template. Worthington (Freehold, NJ) calf thymus DNA diluted to 4 pg per ml in saline-citrate buffer (0.015 M NaCl and 0.15 M trisodium citrate, pH 7.0) was placed in the cathodal wells while test sera were placed in the anodal wells. One half (15) of the cathodal wells were filled with saline as a control for nonspecific precipitation in the agarose. A positive control serum was run with each assay. Electrophoresis was carried out at room temperature with cold-water cooling for 30 min at 40 mA per slide. Slides were read immediately. If nonspecific precipitation occurred, sera were rerun; rare sera continued to show a hazy precipitate that made interpretation by CIE difficult. Specificity was determined on several occasions by preliminary incubation of DNA with DNAse. Sera were run undiluted and results expressed as negative or I+ to 3+; serial dilutions of sera were also assayed in many cases with titer expressed as the highest dilution of sera still showing a precipitin line. C. Farr Binding

Technique

Farr assay for quantitation of anti-DNA antibodies. 14C-Labeled KB cell DNA was a gift from Dr. Tom Chused of the National Institutes of Health. Preparation and characterization of this radiolabeled DNA has been previously described (5). The 260 :280-nm ratio was 1.9. Specific activity was 8000 counts per minute per microgram of DNA with a counting efficiency of 75%. The assay procedure was as described by Pincus tit al. (6,7) with the following modifications. An aliquot (100 ~1) of the supematant after centrifugation was dissolved in 1 ml of NCS solubilizer (Amersham/Searle). Scintillation cocktail, butyl-PBD/toluene (Beckman) was added and the radioactivity was determined with a Beckman model LS250 liquid scintillation counter.

512

DAVIS

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WINFIELD

Percentage of radioactivity bound was determined by the formula T - 2S/T x 100 = percent DNA bound, where T = total counts added to each tube and S = counts per minute in 100 h of supematant. Methylated albumin-kieselguhr chromatography. Calf thymus DNA was fractionated by methylated albumin-kieselguhr (MAK) chromatography by a modification of the method of Mandell and Hershey (10,ll). Methylated albumin (Sigma) was mixed with kieselguhr (Hyflo Super-Cel, Johns Manville) suspended in 0.1 M NaCl, 0.05 M NaH,P04, pH 6.7, and packed in small columns (capacity = 100 pug DNA per ml of packed MAK). Elution of native DNA was carried out by increasing the molarity from 0.3 M NaCl to 0.5 M NaCl. Denatured DNA which was not eluted as the molarity was increased further (to 0.9 M (12,13) or even to 5 M NaCl with calf thymus DNA) was then eluted by raising the pH of the column to 9-10 with 1.0 M saline-phosphate buffer, pH 11.6. Molar@ of the fractions was measured with a Bausch and Lomb refractometer. The fractions were dialyzed against saline-citrate buffer and concentration of DNA was adjusted to 4 pg per ml. The amount of denatured DNA present in our commercial calf thymus preparation was substantial (- lo-15% as estimated by OD at 260 nm and equating OD of 0.02 equal to 1 pg native DNA). Some assays were performed with calf thymus DNA further purified by the method of Marmur (14). In this procedure the DNA solution is treated with an equal volume of chloroform-isoamyl alcohol in which the upper aqueous phase contains the nucleic acids; these are precipitated by 2 vol of ethyl alcohol, the DNA solution is treated with ribonuclease, again deproteinized, and reprecipitated with isopropyl alcohol. RESULTS Sensitivity

and Specificity

of Method

With DNA at a concentration of 4 pg/ml, a strong lupus anti-DNA serum continued to show a clear precipitin line at a 1: 8 serum dilution (Fig. 1). The lowest concentration of DNA giving a visible precipitin line against undiluted sera with strong anti-DNA activity was 0.2 pug/ml. Treatment of the DNA preparation with DNAse abolished the precipitin line. Clinical

Survey

Sera numbering 1766 were assayed including those from 99 patients with SLE (Table 1). All 60 normal sera were negative. Of 766 sera from 99 lupus patients, 37% were positive; 65% of the SLE patients exhibited anti-DNA on at least one occasion. Seventy-five of 940 other disease sera (8%) were also positive. Connective tissue disease was either diagnosed or suspected in most of these patients. Table 2 shows a summary by disease category of the patients who were positive by CIE for anti-DNA activity with three different DNA preparations. With unfractionated DNA, 330 sera from 101 patients diagnosed as having a connective tissue disease were positive; 36 of these patients had diseases

ANTIBODIES

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DNA

BY

CIE

513

i:2

1:16

1:32

FIG. @g/ml).

1. Serial dilutions of lupus serum electrophoresed with a constant DNA is in cathodal wells and lupus serum is in anodal wells.

amount

of DNA

(4

other than SLE. Diagnosis in 11 of these patients was drug induced LE; in 22, rheumatoid arthritis-vasculitis. Twenty-four patients were included in a miscellaneous category including cancer, hepatitis, nephritis, amyloidosis, and other nonrheumatic diseases. Only one of these nonlupus sera gave a 3+ reaction with unfractionated DNA in CIE, and this was from a patient with rheumatoid vasculitis. Sera from 22 of the 65 anti-DNA-positive lupus patients were reexamined for anti-DNA using the native DNA (nDNA) and the denatured DNA (dDNA) MAK fractions. All 22 sera were positive with dDNA and 17 of 22 were positive with nDNA. Of the 38 positive nonlupus sera, 33 failed to give a precipitin line against the nDNA fraction; 30 of these nonlupus sera continued to give a precipitin line against dDNA. TABLE ANTI-DNA No.

1 BY

CIE” No. positive

Percent

positive

-6

Total determinations SLE sera SLE patients Other disease sera Normal sera ” Summary from patients

1766 766

358 283

(65)

(99) 940 60

of results of anti-DNA determinations with diseases other than SLE were

20.3 36.9 (65.7)

75 0 by CIE positive.

8.0 0.0 on 1766

sera. Eight

percent

of sera

514

DAVIS

AND

WINFIELD

TABLE ANTI-DNA Unfractionated DNA

Diagnosis Connective tissue (a) SLE (b) Drug-induced (c) Rheumatoid vasculitis” (d) Polyarteritis (e) Scleroderma Miscellaneous Normal

2 BY CIE”

diseases

Native DNA

Denatured DNA

101 65 11 22

LE arthritisnodosa

2 1 24 0

17122” z/11

22122” ll/ll

318

318

N.D.’ N.D. o/19

N.D. N.D. 16/19

” Diagnoses of patients with sera positive for anti-DNA by CIE using three different DNA preparations. There were 1766 total sera. Drug-induced LE and rheumatoid arthritis-vasculitis comprise the largest individual groups positive with each preparation other than SLE. JJNumber positive/total examined. (’ Not done. ” These patients with rheumatoid arthritis had severe disease; there was often evidence of vasculitis.

Five hundred twenty-four lupus sera were assayed for anti-DNA activity by double diffusion in Ouchterlony plates; 36 (6.9%) were positive. This compares with 283/766 (36.9%) lupus sera positive by CIE (see Table 1). The same Worthington calf thymus DNA was used in the two systems, although the concentration of DNA in the Ouchterlony wells was 1 mg/ml. All sera positive by Ouchterlony were also positive by CIE and usually the reactions were 2-3+. The lower limit of sensitivity by the Ouchterlony method with our strongest positive SLE serum was 3-5 pg/ml giving this method about 1/20th the sensitivity of CIE in detecting DNA. Comparison

with

the Farr

Technique

In an attempt to assess the significance of rating CIE determinations as O-3+ in terms of relative amount of antibody present, a number of sera were examined by both CIE and the Far-r assay. Data are shown in Fig. 2. The mean binding for 60 healthy, nonhospitalized subjects was 8%. The upper limit of normal for ‘“C-DNA binding was arbitrarily set at 2 standard deviations above the mean (26%). One hundred sixty-five serum samples from 78 total patients were compared (66 SLE, 12 drug-induced lupus). Sixty-six sera negative for anti-DNA by CIE (negative group) gave a mean ‘C binding of 21% in the Farr assay. However, 19/66 sera (29%) exhibited significant (> 26%) binding. Sera giving l+ or 2+ precipitin lines by CIE did not differ significantly in binding and were, therefore, combined as a single positive group. Mean binding of 82 sera in this group was 56%. For 17 sera giving 3+ (very positive) precipitin lines by CIE, mean binding was 60%.

ANTIBODIES

TO

DNA

BY

CIE

515

100 -

90 -

. .

80 -

70 -

. ci z

60 -

2 ; P 5 6 Ly L

50-

;

40-

30-

20” . 10 -

t .

. .

POSITIVE (1+-z+)

STRONG POSITIVE

ONEGATIVE

t3+1 C. I. E. FIG. 2. Comparison of results of anti-DNA determinations Sera are from patients with SLE or drug-induced lupus. tionated.

by CIE and the Fan binding assay. DNA used in these assays is unfrac-

Eleven of 99 SLE sera exhibiting precipitin lines by CIE in the positive or very positive groups failed to bind 14C KB DNA significantly in the Farr assay. When these sera were reexamined by CIE using the nDNA MAK fraction (nDNA) or the Marmur purified DNA, none gave precipitin lines. Binding of 14C-DNA by both positive groups was highly significantly different from the negative group (P < 0.00001). The positive group did not differ significantly from the very positive group (P = 0.6). However, if the 11 anomalously low binding values for sera positive for anti-DNA by CIE are omitted, positive and very positive groups do differ withP = 0.06. DNA binding by sera giving l+ precipitin lines with CIE was very significantly different from binding of CIE 3+ sera (P = 0.005). Thus, CIE failed to detect anti-DNA in 29% of sera having significant activ-

516

DAVIS CIE:

AND ttt

WINFIELD ++t

ttt

+t

+

100 a

90

2

80

d u

70

f

60 ‘ii P 5 .E m

50 40 30

Z 8

20

b LL

10

I

L undild

I

I

I

I

J

1:2

I:4

1:s

Id6

1:32

Serum FIG. diluted.

Dilutions

3. Correlation of CIE and the Farr binding A good quantitative relationship is noted.

(l-Fold) assay

when

a single

SLE

serum

is serially

ity by Farr assay. Eleven percent of sera assayed by the Farr method and thought to be positive by CIE against standard commercial calf thymus DNA were in fact negative for anti-DNA by the Farr assay and by CIE when either fractionated nDNA, dDNA, or purified (Marmur) DNA was used. These sera were also negative with denatured DNA in the Farr assay. Grading the precipitin lines l-3+ allows only the least and most positive to be reliably distinguished with regard to the actual amount of anti-DNA binding activity present. In Fig. 3 a dilution curve for anti-DNA activity of one representative SLE serum is shown by both CIE and Fan binding; good correlation between the two techniques is demonstrated. DISCUSSION A simple method to detect antibodies to DNA is desirable in view of reports that antibodies to native DNA are relatively specific for SLE (l-3). The finding of antibodies to native DNA would tend to establish a diagnosis of SLE in patients with positive ANF activity and would be an adjunct to treatment. Since ANF assays are now generally available as a screening test for SLE, a method for detection of anti-DNA which is simple, specific, and sensitive should have widespread usefulness. The CIE technique itself has been well established as a screening method for Australia antigen (8). In addition, recent studies suggest a usefulness 6f CIE for detection of bacterial antigens in biological fluids (X5-19). Radioactive reagents and expensive equipment are not required. The entire assay takes less than an hour. Koffler and colleagues, using a hemagglutination technique with sheep red cells sensitized with various DNA preparations, found that antibodies to denatured DNA were present in sera from patients with a wide variety of

ANTIBODIES

TO

DNA

BY

CIE

517

rheumatic and nonrheumatic diseases, as well as in 4% of healthy individuals (20); these investigators and others (3,7,21,22) have described occasional patients with diseases other than SLE who also exhibited antibodies to “native” DNA. It is clear that a number or our positive sera from non-SLE patients (8%) contained antibody reacting with denatured DNA and were giving precipitin lines with a “contaminating” denatured DNA subfraction in our commercial “native” DNA. There remain, however, a few nonlupus patients who appear to have antinative DNA by this technique. Our data, however, support previous work (2-3) indicating high, although perhaps incomplete, specificity of antinative DNA for SLE. Recent data of Samaha and Irwin in which human KB DNA was fractionated by MAK chromatography indicates that a putative “native” DNA may be significantly contaminated with denatured material (23); Tan had previously shown this to be the case for Worthington DNA as well (24). Using MAK chromatography, we also found our ‘4c-KB DNA to be significantly contaminated with denatured material (25). Use of unfractionated 14C-DNA in the Farr assay in the present study in effect detected antibodies to both native DNA and denatured DNA. We suggest that future investigations using CIE or the Farr assay for measurement of anti-nDNA antibodies in human disease be carried out with DNA which has been critically examined for partial denaturation. A consequence of increasing the specificity of CIE for SLE by using either fractionated native DNA or Marmur-purified DNA is a loss in sensitivity for detection of anti-DNA. Only three-fourths of SLE sera giving a 2+ or 3+ precipitin line against unfractionated calf thymus DNA remain positive against the more native preparations. Weakly positive sera usually no longer give a line. This observation undoubtedly obtains, in part at least, because some of the antibody with specificity only for dDNA is no longer measured. As a practical consideration, specificity of a positive CIE assay for SLE can also be increased without further fractionation or purification of the DNA by simply diluting the sera or rating as positives with undiluted sera only 2+ or 3+ reactions. When data used for Table 1 were reanalyzed with I+ reactions considered “negative,” only 3.6% of nonlupus sera were still “positive.” But again, with either manipulation sensitivity is decreased. The reason for positive precipitin lines against unfractionated DNA in sera apparently lacking antibody to both nDNA and dDNA is unclear. These “false positives” may be related to nonspecific protein-protein or protein-DNA interactions. A limitation of CIE for measuring anti-DNA is that the technique is only semiquantitative. The data shown in Fig. 2 comparing a truly quantitative technique (Far-r assay) with CIE indicate, however, that grading of precipitin lines as slightly positive (l+) or strongly positive (3+) is of value. We have found that for severely ill SLE patients (where quantitation of anti-DNA is of most significance), titers of anti-DNA may be easily determined. Correlation of the dilution curve for CIE with a curve derived from the Far-r assay is good (Fig. 3). It should be kept in mind, however, that CIE measures precipitating

518

DAVIS

AND

WINFIELD

antibodies while the Farr method detects binding cies in correlation may reflect true differences

antibodies; thus, discrepanin the antibodies detected.

ACKNOWLEDGMENTS The authors thank Mrs. Emma Pryor, Mrs. Stephania McGehee for their excellent technical

Linda Kaufman, assistance.

and Miss

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