Journal oflmmunological Methods, 28 (1979)149—162
`© Elsevier/North-Holland Biomedical Press
`
`149
`
`MEASUREMENT OF ANTI-DOUBLE-STRANDED DNA ANTIBODIES IN
`MAJOR IMMUNOGLOBULIN CLASSES
`
`SHINICHI AOTSUKA, MASAKO OKAWA, KENJI IKEBE and RYUICHI YOKOHARI
`
`Division of Clinical Immunology, Clinical Research Institute, National Medical Center
`Hospital, Shinjuku-ku, Tokyo 162, Japan
`
`(Received 2 October 1978, accepted 23 January 1979)
`
`A solid-phase radioimmunoassay for quantitating anti-double-stranded deoxyribonu-
`cleic acid antibodies (anti-dsDNA) in IgG, IgM and IgA classes has been devised. A dis-
`tinct feature of the method is an application of polystyrene tubes coated with poly-L-
`lysine, through which dsDNA could be bound firmly to a solid phase. Studies on patients’
`sera as well as normal sera revealed that anti-dsDNA was not qualitatively but quantita-
`tively characteristic of systemic lupus erythematosus (SLE) and that IgG anti-dsDNA
`levels correlated well with the disease activity.
`
`INTRODUCTION
`
`Antibodies against double-stranded DNA (dsDNA) are thought to be char-
`acteristic of systemic lupus erythematosus (SLE) and are generally associated
`with the disease activity. Measurement of anti-dsDNA gives accurate diag-
`nosis and close monitoring of the course of SLE. In recent years, using radio-
`actively labeled DNA, sensitive techniques such as the ammonium sulfate
`precipitation method (Wold et al., 1968) and the Millipore filter assay
`(Ginsberg and Keiser, 1973) have been available for detection of dsDNA
`binding activity by serum. However, these methods have some disadvantages:
`they may detect non-specific DNA binding proteins (Aarden et al., 1975)
`and do not provide any information about immunoglobulin (Ig) classes of
`the antibodies. There have been accumulated evidences that, as compared
`with IgM anti-dsDNA, IgG anti-dsDNA is more characteristic of SLE, corre-
`lates better with the disease activity (Clough, 1977; Pennebaker et al., 1977)
`and plays a more important role in the pathogenesis of lupus nephritis
`(Koffler et al., 1967). Therefore, it is of value to quantitate anti-dsDNA of
`each Ig class. On the other hand, naturally derived native DNA used in assay-
`ing these antibodies are virtually always contaminated with small but
`immunologically significant amounts of single-stranded DNA (ssDNA) deter-
`minant. This may result
`in frequent occurrence of anti-native DNA anti-
`bodies in the diseases other than SLE as well as normals (Hasselbacher and
`LeR0y, 1974; Rochmis et al., 1974). Purification of native DNA on
`methylated albumin kieselguhr (MAK) column has been used for improving
`
`Page 1 of 14
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`
`BD EXHIBIT 1018
`BD EXHIBIT 1018
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`

`
`150
`
`the specificity of anti-dsDNA binding assay (Winfield and Davis, 1974;
`Samaha and Irvin, 1975). Recently, a synthetic polynucleotide, self-comple-
`mentary alternating copolymer of deoxyadenylate and deoxythymidylate
`(dA-dT) was proposed to be antigenically equivalent to dsDNA while lacking
`significant contamination with single-stranded determinant (Steinman et al.,
`1977). Native DNA treated with endonuclease specific for ssDNA also
`appears to be ideal dsDNA, although care should be taken to avoid its dena-
`turation during handling and storage.
`In the present study, we have devised a solid-phase radioimmunoassay for
`quantitating anti-dsDNA in the major Ig classes. A distinct feature of this
`method is an application of polystyrene tubes coated with poly-L-lysine
`(PLL), through which dsDNA could be bound to a solid phase. After exam-
`ining technical conditions, sera from patients as well as normals were assayed
`by this method.
`
`MATERIALS AND METHODS
`
`Patients
`
`Nineteen patients with SLE, 1 patient with rheumatoid arthritis (RA), 5
`patients with progressive systemic sclerosis (PSS), 3 patients with Sj6gren’s
`syndrome (SS), 1 patient with mixed connective tissue disease (MCTD) and
`1 patient with lupoid hepatitis were subjected to this study. Patients with
`SLE satisfied the preliminary criteria for the classification of SLE (Cohen et
`al., 1971), a patient with RA was classical on ARA criteria (Ropes et al.,
`1958), patients with PSS met Medsger’s criteria for PSS (Medsger and Masi,
`1971). Sj6gren’s syndrome was ascertained by a sialography and/or a labial
`biopsy. MCTD was diagnosed according to Sharp’s description (Sharp et al.,
`1972). Eleven patients with diseases other than SLE were selected on the
`basis of their having high dsDNA binding activities on the Millipore filter
`assay.
`
`Disease activity score
`
`At the time sera were drawn, patients with SLE were assigned a disease
`activity score according to Pennebaker et al. (1977). It represented the sum
`of the following disease features then present:
`fever, alopecia, arthritis,
`serositis, leukopenia, abnormal urinary sediment, erythrocyte sedimentation
`rate greater than 50 mm/h and CH50 less than 20 units (normal range: 30-
`45 units). Disease activity score more than one was arbitrarily judged as
`active .
`
`DNA preparations
`[”C]DNA was extracted, by the method of Marmur (1961), from nuclei
`of E. coli cultured in the presence of [”C]thymine, treated with ssDNA
`specific endonuclease (nuclease S1, Seikagaku Kogyo Co., Tokyo) and frac-
`tionated on MAK column chromatography by the method of Sueoka and
`
`Page 2 of 14
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`
`

`
`151
`
`Cheng (1962). Fractions eluted with 0.05 M phosphate buffer, pH 6.7, con-
`taining 0.6 M NaCl were used as [1"C]dsDNA, whose molecular weight was
`about 100 X 10“ as judged on CsCl gradient ultracentrifugation. Its specific
`activity was 1600 cpm/pg. [”C]dsDNA did not react with rabbit anti-ssDNA
`antibodies on the Millipore filter assay.
`Calf thymus DNA (CT-DNA) was purchased from Boehringer Mannheim
`Co., G.F.R. Treatment with nuclease S1 followed by fractionation on MAK
`column, was carried out to obtain purified CT-dsDNA.
`DNA labeled internally with 1251 was obtained from Radiochemical Centre
`Co., U.K. and treated with nuclease S1 followed by fractionation on MAK.
`
`Millipore filter assay
`DsDNA binding activity by serum was determined with a modification of
`the Millipore filter method of Ginsberg and Keiser (1973). Five pl of serum
`inactivated at 56°C for 120 min were incubated at 45°C for 15 min with 195
`
`pl of 0.15 M ’I‘ris-HCl buffer, pH 7.6, containing 0.4 ,ug of [”'C]dsDNA. The
`reaction was stopped by addition of 3 ml of Tris-HCl buffer, and the mixture
`was passed through a prewetted Millipore filter (HAWP 02500) under gentle
`suction. After washing two times with Tris-HCl buffer and once with dis-
`tilled water, the filter was dried, transferred to a glass scintillation vial and
`measured for radioactivity in a liquid scintillation counter. The result was
`expressed as per cent [”C]dsDNA retained on the filter.
`
`Immunoglobulins
`IgG was purified from human fraction II of a commercial source
`(Midorijuji Co., Japan) by DEAE-cellulose chromatography. IgM was pre-
`pared from euglobulins of serum from a patient with macroglobulinemia by
`Sephadex G-200 gel filtration. IgA was prepared from serum of a patient
`with IgA myeloma by the method of Vaerman et al. (1963). The prepara-
`tions were tested for purity by Ouchterlony gel precipitation. The concentra-
`
`tion of Ig in each preparation was measured by a single radial immunodif-
`fusion method.
`
`Antibodies specific for IgG, IgM and IgA
`Antisera specific for IgG, IgM and IgA (anti—IgG, anti-IgM and anti-IgA,
`respectively) were purchased from Behringwerke AG, G.F.R. Globulins of
`antisera, obtained by rivanol fractionation and Sephadex G-25 gel filtration,
`were applied to Sepharose CL-2B coupled with purified Ig of the corre-
`sponding class. Specific antibodies were eluted with 0.5 M glycine-HCI
`buffer, pH 3.0, immediately mixed with 2.0 M Tris-HCl buffer, pH 8.0 and
`dialyzed against saline. Purified specific antibodies were then labeled with
`1251 by the method of Klinman and Taylor (1969).
`
`Buffers
`
`0.15M Tris-HCl buffer, pH 7.6, containing 1% bovine serum albumin
`(BSA) and 0.1% Triton X-100, was used for incubation in the solid-phase
`
`Page 3 of 14
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`

`
`152
`
`radioimmunoassay (incubation buffer). 0.15 M Tris—HCl buffer, pH 7.6, con-
`taining 0.1% BSA and 0.1% Triton X-100, was used for washing in the solid-
`phase radioimmunoassay (washing buffer).
`
`Poly-L-lysine (PLL)
`Poly-L-lysine hydrobromide, type IB, was purchased from Sigma Chemical
`Co., U.S.A.
`
`Coatingpolystyrene tubes with calf thymus dsDNA (dsDNA tubes)
`Polystyrene tubes (11 mm X 80 mm) were purchased from Eiken Kizai
`Co., Japan. The tubes were filled with 1 ml of 40 ,ug/ml PLL in Tris-HCI buff-
`er. After 30 min at room temperature, the PLL solution was aspirated and the
`tubes were washed 3 times with Tris-HC1 buffer (PLL tubes). One ml of 10
`pg/ml CT-dsDNA in Tris—HCl buffer was next added to the tubes, while to
`the control tubes was added 1 ml of 10 U/ml heparin in Tris-HC1 buffer.
`After 60 min’s rotation at 37°C, the tubes were washed 3 times with Tris-
`HCl buffer and filled with 2 ml of Tris-HCl buffer containing BSA, i.e., incu-
`bation buffer, and rotated at 37°C for 60 min to coat any active sites of the
`tubes with BSA.
`
`Radioimmunoassay for anti-dsDNA in major Ig classes (standard assay)
`Two pl of test serum diluted in 1 ml of incubation buffer were added to
`each of 6 dsDNA-tubes and 6 control tubes. After incubation at 37°C for
`
`120 min, the serum was aspirated and the tubes were washed 3 times with
`washing buffer. 1251-labeled anti-Ig (about 10,000 cpm) in 1 ml of incubation
`buffer was next added so that 2 dsDNA tubes and 2 control tubes received
`
`each of specific anti-IgG, anti-IgM and anti-IgA. The tubes were incubated
`overnight at room temperature and washed 3 times with washing buffer. The
`radioactivities retained on the tubes were then measured in an auto gamma
`spectrometer. The results were corrected for non-specific binding of radio-
`activity by subtracting appropriate control counts and for dilution by mul-
`tiplying the count by appropriate dilution factor. The amount of anti-
`dsDNA was expressed as pg/ml, using a conversion factor described in the
`following standardization of the assay.
`
`Standardization of the assay
`Purified IgG,
`IgM and IgA were labeled with 1251 by the method of
`Klinman and Taylor (1969). After sufficient dialysis, each of them was
`diluted and specific activity was determined. Three concentrations of each
`labeled Ig were coupled to polystyrene tubes, 4 of which were for each con-
`centration. The tubes were washed 3 times with washing buffer and treated
`with incubation buffer to coat any active sites with BSA. To two of the
`tubes for each concentration was added ”5I-labeled anti-Ig in 1 ml of incuba-
`tion buffer, while the other tubes (control tubes) were not exposed to ‘Z51-
`labeled anti-Ig. After rotating overnight at room temperature, the tubes were
`
`Page 4 of 14
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`
`

`
`153
`
`washed 3 times with washing buffer. The amount of Ig coupled to the tubes
`was determined by radioactive counting of the control tubes. Calculation of
`specific anti-Ig binding by subtracting radioactivity of the control tubes
`revealed a conversion factor for each batch of labeled anti~Ig to estimate
`micrograms of Ig.
`
`RESULTS
`
`PLL concentration used for coating the tubes
`Polystyrene tubes were filled with 1 ml of PLL solution of different con-
`centrations in Tris-HCl buffer. After 30 min at room temperature, the PLL
`solution was aspirated and the tubes were washed 3 times with Tris-HCl
`buffer. One ml of [”5I]dsDNA (10 ug/ml, 1500 cpm/pg) in Tris-HC1 buffer
`was next added to the tubes. After 60 min rotation at 37°C, the ["51]-
`dsDNA solution was aspirated and the tubes were washed 3 times with Tris-
`HC1 buffer. The radioactivities retained on the tubes were then measured in
`
`an auto gamma spectrometer. As shown in Fig. 1, more than 5 pg/ml of PLL
`were sufficient for dsDNA coating. For the assay, a concentration of 40 ,ug/
`ml of PLL was chosen.
`
`Antigen concentration used for coating PLL tubes
`One ml of [”5I]dsDNA solution of different concentrations in Tris-HCl
`buffer was added to PLL tubes. After 60 min rotation at 37° C, the [1251]-
`
`°
`
`0
`
`2.0
`
`f'\
`2.
`no
`“’
`
`2
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`52‘
`fl
`‘.3
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`S
`
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`
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`
`2.0
`
`3
`3
`ca
`
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`a
`ca
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`Q
`
`0
`
`U
`
`10
`
`20
`
`30
`
`H0
`
`0
`
`0
`
`10
`
`20
`
`30
`
`H0
`
`50
`
`CONCENTRATION OF DSDNA (pa/ML)
`CONCENTRATION OF PLL (pa/ML)
`Fig. 1. Concentration of PLL required for coating tubes with [”5I]dsDNA was exam-
`ined. More than 5 ,ul/ml PLL were sufficient for coating tubes with [”5I]dsDNA. With-
`out PLL [‘25I]dsDNA was not bound to the tubes.
`
`Fig. 2. Concentration of [‘25I]dsDNA suitable for coating PLL tubes was examined. Effi-
`cient coatingreached a maximal value at approximately 10 pg/ml of [”5I]dsDNA.
`
`Page 5 of 14
`Page 5 of 14
`
`

`
`154
`
`dsDNA solution was aspirated and the tubes were washed 3 times with Tris-
`HCl buffer. The radioactivities retained on the tubes were then measured in
`an auto gamma spectrometer. As shown in Fig. 2, efficient coating reached a
`maximal value at approximately 10 pg/ml of dsDNA.
`
`Stabflity of dsDNA on a solid phase
`Tubes coated with [”5I]dsDNA were rotated with 1 ml of incubation
`buffer containing different amounts of serum or heparinized plasma at 37°C
`for 120 min and Washed 3 times with washing buffer. The tubes were further
`treated in the same manner as in the standard assay, except that unlabeled
`anti-Ig was used and the radioactivities retained on the tubes were measured.
`As shown in Fig. 3, less than 50 ,ul of serum eliminated none of the [1251]-
`dsDNA from the tubes during the treatment of the standard assay, while
`heparinized plasma took off dsDNA from the tubes to a certain degree. This
`effect was found to be due to heparin by another experiment (data not
`shown).
`
`Inhibition studies
`
`Thirty pl of SLE serum were incubated with 1.5 ml of different concen-
`
`
`
`
`
`RADIOACTIVITYRETAINED(Z)
`
`CPMBoumu
`
`100
`
`U
`
`5
`
`2
`5
`SD
`AMOUNT OF DSDNA Us D FOR lNHII3ITI0N
`OF ANTI—DsDNA lips/pr SERUM)
`AMOUNT OF SERUM/PLASMA (pr)
`Fig. 3. The stability of dsDNA coupled to a solid phase was examined. Different amounts
`of serum or heparinized plasma were treated in the same manner as In the standard #:1355337.
`substituting dsDNA for ['25I]dsDNA and [”5I]anti-Ig for unlabeled anti-Ig.
`[
`I l-
`dsDNA retained on a solid phase was expressed as per cent radioactivities counted after
`the treatment.
`
`
`
`0
`
`Fig. 4. Anti-dsDNA levels in the sera absorbed with dsDNA were measured with the solid-
`phase radioimmunoassay, Anti-dsDNA levels were expressed in cpm.
`
`Page 6 of 14
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`
`

`
`155
`
`trations of dsDNA and filtered through a Millipore filter to eliminate
`dsDNA—~anti-dsDNA complexes formed. Aliquots of the filtrates were tested
`for anti-dsDNA by the standard assay. As shown in Fig. 4, essentially none
`of anti—dsDNA of the 3 lg classes was measured in the serum treated with
`dsDNA of 5 ug/pl serum.
`
`Kinetics of binding of anti—dsDNA to dsDNA tubes
`Two ,ul of SLE serum diluted in 1 ml of incubation buffer were added to
`dsDNA tubes and were then treated in the same manner as in the standard
`
`assay. As shown in Fig. 5, binding of antibodies to the antigen on a solid
`phase was almost completed within 120 min.
`
`Kinetics of binding of anti-Ig to anti—dsDNA on dsDNA tubes
`dsDNA tubes, which had been treated with SLE serum, were rotated with
`“SI-labeled anti-Ig in 1 ml of incubation buffer at room temperature for 2, 4,
`8 and 18 h. The tubes were washed 3 times with washing buffer and the
`radioactivities were measured. As shown in Fig. 6, a relatively long period
`was required for sufficient binding of anti-Ig to antibodies on the dsDNA
`
`CPMBoumn
`
`0
`
`30
`
`60
`
`120
`
`180
`
`TIME (Mm)
`
`TIME (HR)
`
`Fig. 5. Kinetics of binding of anti-dsDNA to dsDNA tubes. Binding of anti-dsDNA to
`dsDNA on a solid phase was almost completed within 120 min.
`
`Fig. 6. Kinetics of binding of anti-Ig to anti-dsDNA on dsDNA tubes. Binding of anti-Ig to
`anti-dsDNA was not completed in a period examined.
`
`Page 7 of 14
`Page 7 of 14
`
`

`
`156
`
`TABLE 1
`
`EFFECT OF RA SERA ON MEASUREMENT OF ANTI-dsDNA LEVELS IN SLE
`SERUM
`
`RA sera
`
`1
`
`40 X
`
`0.65 C
`0.58
`
`2
`
`40 X
`
`0.50
`0.30
`
`3
`
`80 X
`
`0.87
`0.90
`
`4
`
`n1 3
`
`320 X
`
`--
`
`0.93
`0.55
`
`0.96
`0.89
`
`Latex fixation test 13
`
`Effect on:
`
`IgG anti-dsDNA
`IgM anti-dsDNA
`
`3 Normal serum control.
`
`17 Highland’s ‘RA test’ used.
`0
`anti-dsDNA level in the mixture of SLE and RA sera
`
`anti-dsDNA level in SLE serum + anti-dsDNA level in RA serum‘
`
`tubes. However, an overnight incubation (18 h) was done for practical pur-
`poses.
`
`Effect of rheumatoid factor
`Two ,ul of SLE serum were mixed with equal amounts of each of 4 RA
`sera containing rheumatoid factor with various latex fixation titers, and the
`anti-dsDNA levels in the mixtures were compared with anti-dsDNA levels in
`SLE serum plus those in RA serum. As shown in Table 1, addition of RA
`serum decreased both IgG and IgM anti-dsDNA levels.
`
`Studies on patients’ sera
`Thirty sera obtained from 19 patients with SLE, 11 sera from 11 patients
`with diseases other than SLE (others), which had shown significant dsDNA
`binding activities on the Millipore filter assay, and 8 sera from healthy con-
`trols were studied. Twenty-two sera from patients with SLE were obtained
`during active phase as judged by a disease activity score and 8 sera during
`inactive phase. The results were shown in Fig. 7 and the statistics in Table 2.
`Most of the normal sera showed low levels of anti-dsDNA, the majority of
`which was IgM although IgG and IgA were also detectable. Sera from
`patients with active SLE contained large amounts of anti-dsDNA distributed
`among the 3 lg classes in different proportions, though no significant differ-
`ence was revealed between IgG and IgM anti-dsDNA levels. Sera from
`patients with inactive SLE also contained some amounts of anti-dsDNA in
`the 3 Ig classes. The IgG anti-dsDNA levels were significantly lower than
`those in active SLE, while IgM anti-dsDNA levels were not significantly dif-
`ferent from those in active SLE. Anti-dsDNA levels in sera from diseases
`
`other than SLE were elevated predominantly in IgM class, although anti-
`dsDNA levels in each class were significantly higher than those of normal
`controls .
`
`Page 8 of 14
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`
`

`
`157
`
`.
`
`Active SLE
`
`Inactive
`
`Normals
`
`0
`
`.
`
`'
`
`0
`
`o
`
`C C
`
`,
`
`1
`'C
`
`o
`
`80
`
`70
`
`60
`
`50
`
`40
`
`30
`
`20
`
`10
`
`\U
`
`E
`
`‘
`3-‘
`CD
`v—l
`93G,
`94
`
`E
`D
`U)
`
`"'3
`~H
`4-’
`C
`
`C‘
`
`'!'..i....+
`o L
`IgG IgM IgA TotalIgG IgM
`I9
`
`T t 1
`IgM IgA ° a
`19
`
`-r__«~_.o.4_m
`T t 1
`IgG IgM IgA ° '3
`19
`
`Fig. 7. Results of measurement of anti-dsDNA levels in IgG, IgM and IgA on sera from
`patients with SLE, those with diseases other than SLE (others), whose dsDNA binding
`activities were high on the Millipore filter assay, and normal controls. Total anti-dsDNA
`levels (total Ig), i.e., IgG + IgM + IgA, were also shown.
`
`Comparison with the Millipore filter assay
`Fifty-one sera were examined with both the Millipore filter assay and the
`method we have developed. The results were shown in Fig. 8. Significant cor-
`relations were revealed for IgG and total Ig anti-dsDNA levels, as compared
`with dsDNA binding activities on the Millipore filter assay. It is noted that 8
`sera from patients with high dsDNA binding activities on the Millipore filter
`assay showed total Ig anti-dsDNA levels below the upper limit of normal,
`namely, 4.99 ng/ml (the mean plus 2 S.D. for normal sera). On the other
`hand, none of sera with normal dsDNA binding activities on the Millipore
`
`Page 9 of 14
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`
`

`
`158
`
`TABLE 2
`
`ANTI-dsDNA LEVELS IN PATIENTS WITH SLE, PATIENTS WITH OTHER DISEASES
`AND NORMALS
`
`Active SLE, n = 22; inactive SLE, n = 8; others, n = 11; normals, n = 8.
`
`Mean + S.D. 3
`
`Inactive SLE
`
`Others
`
`Normals
`
`(Hg/ml)
`
`IgG
`
`Active SLE
`Inactive SLE
`
`15.8
`3.31
`
`i 16.2
`i 4.02
`
`P < 0.05 b
`-
`
`P < 0.025
`NS ‘?
`
`IgM
`
`IgA
`
`Others
`Normals
`
`2.34 i .115
`0.575 i 0.277
`
`Active SLE
`Inactive SLE
`Others
`Normals
`
`Active SLE
`Inactive SLE
`Others
`Normals
`
`8.77 i 10.4
`5.55 i 11.4
`10.4
`i 9.39
`0925 i 1.02
`
`i 1.55
`1.91
`0.750 i 0.634
`1.47 i 1.16
`0.425 i 0.441
`
`NS
`—
`
`NS
`—
`
`Active SLE
`IgG +
`M + A Inactive SLE
`Others
`Normals
`
`i 20.6
`26.5
`9.24 i 12.4
`142
`i 9.80
`1.95 i 1.53
`
`P < 0.05
`-—
`
`a S.D. = standard deviation.
`b P values for significant differences, based on Student t-test.
`c
`_
`.
`.
`.
`NS — not significant.
`
`—
`
`NS
`NS
`—
`
`NS
`NS
`—
`
`NS
`NS
`~
`
`P < 0.005
`NS
`
`P < 0.05
`—
`
`P < 0.05
`NS
`P < 0.025
`—
`
`P < 0.025
`NS
`P < 0.025
`—
`
`P < 0.005
`NS
`P < 0.005
`—
`
`filter assay (less than 10%) showed total Ig anti-dsDNA levels higher than the
`upper limit of normal.
`
`Relationship of anti-dsDNA levels to the disease activity of patients with
`SLE
`
`Anti-dsDNA levels in each of 3 lg classes and total Ig were examined for
`relations to disease activity scores, which patients had at the time sera were
`drawn (Fig. 9). Statistically significant correlations with disease activity
`scores were revealed for anti-dsDNA levels in IgG and total Ig.
`
`DISCUSSION
`
`A solid-phase radioimmunoassay or a radioallergosorbent test (RAST) ori-
`ginally described by Wide et al. (1967) provided a highly sensitive method
`to quantitate antibodies in any of Ig classes,
`if an antigen is effectively
`coupled to a solid phase. As to antibodies against DNA, Shimizu et al.
`(1975) reported the measurement of IgG antibodies to ssDNA by modifica-
`tion of the RAST procedure. Clough (1977) described a radioimmunoadsor-
`
`Page 10 of 14
`Page 10 of 14
`
`

`
`0
`
`o
`
`0
`
`o
`oo
`o
`O
`
`159
`
`IQM
`NS
`
`so
`
`.
`
`O
`
`93
`
`0 .
`o
`
`0
`O
`
`O
`
`O .
`
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`0'
`<9
`0
`.
`0
`o
`O
`‘a_<*_o§aa_e=2es_:>
`O0
`50
`100
`
`IgG
`r=O.538
`p<0.005
`
`O
`
`O
`
`O Cb
`o
`3
`o o
`O
`0
`.&m_9
`so
`100
`
`50
`
`00
`
`>
`
`2Hm0
`
`U1 0
`
`
`
`Anti—dsDNALevels(pg/ml)onSolidPhaseAssay
`
`
`
`
`
`% dsDNA Binding on Millipore Filter Assay
`
`Fig. 8. Anti-dsDNA levels in IgG, IgM and IgA as well as total Ig measured with the solid-
`phase radioimmunoassay were compared to dsDNA binding activities measured with the
`Millipore filter assay. 0, SLE; 0, others; A, normals. Significant correlations were revealed
`for IgG and total Ig anti-dsDNA levels as compared to dsDNA binding activities on the
`same assay.
`
`bent assay for quantitating specific DNA binding Ig in IgG, IgM and IgA
`class. One of the problems in their techniques is repeated troublesome centrifu-
`gations for washing, since antigens are coupled to particles such as microcrys-
`talline cellulose (Shimizu et al., 1975) or formalinized human erythrocytes
`(Clough, 1977). We tried, therefore, to coat polystyrene tubes with dsDNA,
`which do not require centrifugations. Although Tan and Epstein (1973)
`
`Page 11 of 14
`Page 11 of 14
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`

`
`160
`
`50
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`
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`Anti-dsDNALevels(Jug/ml)
`
`190
`r=0.582
`
`p<0.01
`
`IgM
`r=-0.025
`
`.
`
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`:=o_275
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`Disease Activity Score
`
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`Fig. 9. Anti-dsDNA levels in each of 3 lg classes and total Ig were examined for relations
`to disease activity scores (n = 30). Statistically significant correlations were revealed for
`anti-dsDNA levels in IgG and total Ig.
`
`succeeded in coating plastic cups with hydroxyapatite-purified dsDNA, we
`failed until PLL was found to be a useful reagent for coupling dsDNA to
`polystyrene tubes. The reason why we could not couple dsDNA to poly-
`styrene tubes was not clear. A material of the plastic used might be different.
`At any rate, we think it is reasonable to apply PLL, basic amino acid poly-
`mer, for coupling strongly charged antigens such as dsDNA to a solid phase.
`Since the bonding of PLL and dsDNA is ionic, there is a possibility that high
`ionic strength of buffers and more strongly charged substances interfere with
`the bonding. However, examination using ["5I]dsDNA revealed that dsDNA«
`on a solid phase of polystyrene tubes was quite stable under the condi-
`tions used, except for heparin, an anticoagulant, which took dsDNA off
`from a solid phase. Therefore, heparinized plasma cannot be tested with this
`method. Another possibility is also conceivable that binding of dsDNA to
`PLL creates a nucleoprotein linkage which might pick up antibodies other
`than anti-dsDNA. It was ruled out by performing inhibition studies using
`dsDNA in solution to eliminate all activities picked up by this method.
`Clough (1977) claimed that rheumatoid factors interfere with his test pro-
`ducing either suppression or occasionally enhancement. We also examined
`RA sera for the effect on our assay. The results were almost the same as
`Clough’s, although none of our RA sera produced enhancement of anti-
`dsDNA levels in either IgG or IgM. It was not determined whether the sub-
`stances in RA sera, which interfered with the assay, were really rheumatoid
`factor. There was lack of correlation between the suppressive effect and titer
`of rheumatoid factor. Furthermore, among 11 patients with diseases other
`
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`

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`161
`
`than SLE and high dsDNA binding activities on the Millipore filter assay, 7
`with rheumatoid factor had rather higher IgM anti-dsDNA levels than 4 with-
`out rheumatoid factor on this assay (data not shown). Those findings sug-
`gested that rheumatoid factor may not be responsible for the suppression.
`In the experiment on the comparison with the Millipore filter assay, it was
`found that some sera from patients with high dsDNA binding activities on
`the Millipore filter assay showed total Ig anti-dsDNA levels below the upper
`limit of normal measured by the method we have developed. The Millipore
`filter assay might depend less on the Ig nature of the dsDNA binding mate-
`rial detected, although results by both methods correlated significantly.
`However,
`the possibility that our method is
`less sensitive cannot be
`excluded.
`
`Studies on patients’ sera revealed that anti-dsDNA levels of all of the 3 lg
`classes were elevated in patients with active SLE. Anti-dsDNA levels of any
`of 3 lg classes in patients with inactive SLE were not significantly higher
`than those in normals, although IgG anti-dsDNA levels in 5 out of 8 patients
`were over the upper limit of normal. Anti-dsDNA levels in patients with dis-
`eases other than SLE, whose dsDNA binding activities were high on the Milli-
`pore filter assay, were elevated predominantly in IgM class. Most of the nor-
`mal sera showed low levels of anti-dsDNA, the majority of which was IgM
`although IgG and IgA were also detectable. These results seem to indicate
`that anti-dsDNA, even in IgG class,
`is not qualitatively but quantitatively
`characteristic of SLE. The findings are in agreement with the observation by
`Bankhurst and Williams (1975) that DNA binding B lymphocytes exist in the
`peripheral blood of both normals and SLE patients. They postulated that
`anti-dsDNA antibody response may be initiated by the stimulation of pre-
`existing DNA specific B lymphocytes by several mechanisms. Our data may
`suggest that the mechanisms of stimulation in active SLE are different from
`those involved in diseases other than SLE, since the elevation of anti-dsDNA
`levels in the former was observed in both IgG and IgM, whereas that in the
`latter was predominantly in IgM.
`Studies on the relation of anti-dsDNA levels to the clinical status of
`
`patients with SLE revealed that IgG anti-dsDNA levels correlated well with
`disease activities. The results were consistent with the observation by Penne-
`baker et al. (1977). They further showed that the occurrence of chiefly IgM-
`native DNA binding activity in the serum was associated with milder clinical
`disease and more benign renal histologic findings than observed in the
`patients with a majority of their native DNA binding in IgG class. Since we
`had only a few patients with predominantly (86-98%, as they defined) IgM
`anti-dsDNA in the serum, the same investigation as theirs could not be car-
`ried out. However, their concept may be supported by our findings that anti-
`dsDNA of patients with diseases other than SLE and no renal involvement
`was predominantly IgM.
`Although significantly higher than in normals, IgA anti-dsDNA levels in
`active SLE were neither remarkably elevated nor associated with the clinical
`
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`162
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`status of the patients. The measurement of IgA anti-dsDN A seems to be clin-
`ically less significant than that of IgG anti-dsDN A levels.
`In summary, our results were almost consistent with previous observations
`that IgG anti-dsDNA is characteristic of SLE and correlates well with the
`disease activity. However, much remains to be elucidated especially as to the
`clinical or pathogenetic significance of anti-dsDNA in Ig classes other than
`IgG. Our method will offer a useful tool for such investigations.
`
`ACKNOWLEDGEMENT
`
`This study was supported by the research grants for Autoimmune Diseases
`from The Ministry of Health and Welfare and for Etiology of Systemic
`
`Lupus Erythematosus of The Ministry of Education, Japan.
`
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