VDLUHE 24. HO. 3
`MARCH ‘I981
`
`rthrifis T
`
`and
`eumatism
`
`of the A.m~:eri::an .Rheumatism Association
`SE-tiifln of the Arthriiis Foundation
`
`Aflhrijlis Foufidatian
`Atlanta, Georgia
`
`Page 1 of 12
`I r‘. Page1of12
`
`BD EXHIBIT 1006
`BD EXHIBITIOO6
`
`
`
`

`
`ARTHRITIS AND RHEUMATISM
`
`Editor
`
`Nathan J. Zvaifler, M.D.
`
`University of California Medicai Center
`225 West Dickinson Street
`
`San Diego, California 92103
`
`Associate Editors
`
`Wayne H. Akeson, MD.
`Michael A. Becker, M.D.
`Han'y G. Bluestein, M.D.
`F. Richard Convery, MD.
`James W. Hollingsworth, M.D.
`Donald L. Resnick. MD.
`Michael H. Weisman, M.D.
`
`Editorial Board
`
`K. Frank Austen, M.D.. Boston
`
`Rodney Bluestone. M.D.. Los Angeies
`Giles G. Bole. Jr.. MD.. Ann Arbor
`Alan S. Cohen, M.D.. Boston
`
`John L. Decker. M.D.. Bethesda
`Virgil Hanson. M.D.. LosAnget'es
`Edward D. Harris. Jr., M.D.. Hanover
`
`Evelyn V. Hess. M.D., Cincinnati
`David S. Howell. MI}. Miami
`Gene (3. Hunder. M.D.. Rochester
`
`David Koffler. M.D., Philadelphia
`Stephen M. Kranc. M.D., Boston
`Henry G. Kunltel, M.D.. New York
`E. Carwile LeR0y. M.D., Charleston
`Michael D. Lockshin. M.D.. New York
`Donald E. McCollum. M.D., Durham
`Frederic C. McDuffie. M.D.. Atianta
`
`Stephen E. Maiawista, M.D., New Haven
`Mart Mannik. M.D.. Seattie
`William Martel, M.D.. Ann Arbor
`Jane H. Morse. M.D., New York
`
`Carl M. Pearson. M.D.. Los Angetes
`Gerald P. Rodnan. M.D.. Pittsburgh
`Shaun Ruddy, M.D.. Richmond
`Jane G. Schaller. M.D.. Seattle
`Peter H. Schur, M.D., Boston
`
`John T. Sharp. M.D.. Danviite
`Clement B. Sledge. M.D., Boston
`Ralph Snyderman. M.D.. Durham
`Leon Sokoloff. M.D.. Stony Brook
`Mary Betty Stevens, M.D., Baltimore
`Norman Talal. M.D.. San Francisco
`
`Eng M. Tan. M.D.. Denver
`Ralph C. Williams. Jr.. MD.. Albuquerque
`Robert J. Winchester. MD.. New York
`
`AMERICAN RHEUMATISM
`ASSOCIATION
`
`A Section of the Arthritis Foundation
`
`3400 Peachtree Rd. N.E.. Atlanta. Georgia 30326
`
`President
`
`Giles G. Bole. .lr., MD.
`University of Michigan
`Ann Arbor, Michigan
`
`Vice-President and President-Elect
`J. Claude Bennett, MD.
`University ofAiaban1a
`Birmingham. Aiabama
`
`Second Vic-e—Presldent
`
`Mary Betty Stevens, M.D.
`Johns Hopkins University
`Baltimore, Maryland
`
`Secretary-Treasurer
`James Klinenberg. M. D.
`Cedars-Sinai Medical Center
`
`Los Angeies, Caii ornia
`
`Executive Secretary
`Lynn Bon figlio
`
`Assistant Executive Secretary
`Angel Fortenberry
`
`Committee for the Publication of
`Arthritis and Rheumatism
`
`E. Carwile LeRoy, M.D., Chairman, Charleston
`Edgar S. Cathcart, M.D., Boston
`Andrew H. Kang, M.D., Memphis
`William J. Koopman, M.D., Birrninghant
`Ronald P. Messner, M.D., Minneapolis
`Paul H. Plotz, M.D., Bethesda
`
`Production Stafl‘
`
`Daphna Gregg, Managing Editor
`Jereiyn Jordan, Consuiting Editor
`Drema McCord, Circulation
`Avis Bradshaw, Editoriai Assistant
`Elizabeth Thurlow, Editorial Assistant
`
`Published monthly by the Arthritis Foundation. 3400 Peachtree Rd. NE.. Atlanta. Georgia 30326. Printed in the United States.
`"Second Class Postage paid at Atlanta. Georgia. and additional otfices. ISSN 0004-359l.
`. SUBSCRIPTION RATES: $20.00 per year for members, as part of the yearly clues, $35.00 for nonmembers within the U.S.A.. and
`for nonmembers elsewhere. Students. fellows. interns, and residents in North America: $20.00 per year. (A letter giving
`qnahfying data must accompany such orders.) Single copies: $5 .00, except for special issues. Copyright I980 the Arthritis Founda-
`tion, Atlanta, Georgia. All rights reserved.
`Page 2 of 12
`Page2of12
`
`
`
`_
`
`"
`
`

`
`534
`
`A SENSITIVE SOLID PHASE
`MICRORADIOIMMUNOASSAY FOR
`ANTI-DOUBLE STRANDED DNA ANTIBODIES
`
`FALK FISH and MORRIS ZIFF
`
`A sensitive solid phase microradioimmu-
`noassay has been developed for measurement of anti-
`double stranded DNA (dsDNA) antibodies. In this pro-
`cedure, advantage has been taken of the capacity of
`poly-L-lysine (PLL) to facilitate the binding of pure
`dsDNA to plastic surfaces. In the absence of PLL, bind-
`ing did not occur. Diluted sera were incubated in PLL-
`treated dsDNA-coated microtitration trays and anti-
`dsDNA lg was measured using allinity purified “’I-anti-
`lg of high specific activity. The synthetic DNA, poly dA-
`dT, was used as a model for dsDNA. in initial experi-
`ments, specific anti-DNA binding could not be demon-
`strated because of high background binding of patient lg
`to PLL—treated surfaces. This was reduced by diluting
`test sera and anti-lg in buffer containing 2% BGG and
`1% BSA. Specificity of the assay for DNA was demon-
`strated by absorbing the anti-DNA activity on DNA-
`coated plastic. The binding of systemic lupus erythema-
`tosus (SLE) patient serum lg to poly dA-dT coated
`trays did not diminish after digestion with nuclease S..
`suggesting that the synthetic polymer is an appropriate
`model for dsDNA. Patient and normal sera were
`screened for anti-dsDNA activity using poly dA-dT as
`antigen. None of the 33 normal sera, 23 of 35 active
`
`From the Department of Internal Medicine. Rheumatic Dis-
`eases Unit. University of Texas Health Science Center at Dallas. Dal-
`las, Texas 75235.
`Supported by USPHS research grant No. AM [8505 and an
`Arthritis Foundation Clinical Study Center Grant.
`Fallt Fish. PhD: Faculty Associate; Morris Zifl‘, MD. PhD:
`Professor and Chief. Rheumatic Diseases Unit. Department of inter-
`nal Medicine, University of Texas Health Science Center at Dallas.
`Address reprint requests to Morris Ziff, MD. Department of
`Internal Medicine. University of Texas Health Science Center, 5323
`Hany Hines Boulevard, Dallas. Texas 15235.
`Submitted for publication September 10. I930; accepted Oc-
`tober 23. I980.
`
`Arthritis and Rheumatism, Vol. 24, No. 3 (March 1981)
`
`Page 3 of 12
`Page 3 of 12
`
`SLE sera, I of 25 treated SLE, 4 of 35 rheumatoid ar-
`thritis. 3 of 35 scleroderma, and l of 13 polymyositis
`sera demonstrated positive anti-dsI)NA activity. The
`anti—dsDNA values obtained in the radioimrnunoassay
`correlated significantly with those obtained in the Cri-
`rhidia luciliae assay.
`
`Antibodies to double-stranded DNA (dsDNA)
`are generally considered to be specific for systemic
`lupus erythematosus (SLE) (1-3) and the SLE-like syn-
`drome of some autoimmune mouse strains (4). These
`antibodies have been considered the principal factor in
`the pathogenesis of lupus nephritis (5,6) and are of im-
`portance in assessing the state of the disease (7-1 1). The .
`occurrence of anti-dsDNA in other disease states re-
`ported by some workers has been attributed to the mo-
`lecular form of the DNA preparations used in the dif-
`ferent assays (12,13).
`The current techniques commonly used for mea-
`suring anti-dsDNA, namely, the membrane filter bind-
`ing assay (14) and the ammonium sulfate precipitation
`assay (l5) have their inherent deficiencies: they may de-
`tect proteins other than immunoglobulins interacting
`with DNA (16.17); the radioactively labeled DNA may '
`be subjected to radiation damage (12); and the methods"
`may not be sensitive enough to measure low concentra-5
`tions of antibody in dilute fluids, such as tissue culture;-.
`supernatants, without concentration before assay (18).-.
`The solid phase type of immunoassay offers high sensi--I
`tivity because of the amplification of the measurements.
`by the isotope or enzyme-labeled secondary anti-lg‘
`antibody utilized in this type of procedure. In addition.-.,.
`it is possible to determine the lg class of the anti-DNA:
`antibody by this technique.
`In spite of these theoretical advantages of solid
`
`

`
`MICRORADIOIMMUNOASSAY FOR ANTI-dsDNA
`
`535
`
`phase immune assays, there are only a few reports of at-
`tempts to adapt this type of method, using DNA coated
`plastic tubes as the solid absorbent, to the measurement
`of anti-dsDNA antibodies (19-22). Pesce et al (20) have
`used the ELISA version of the solid phase immunoassay
`and reported poor reproducibility. Lange et al (21) have
`used radioimmunoassay to measure anti-DNA and de-
`tected increased binding of nonspecific lg from autoim-
`mune sera to the gelatin coated plastic. These reports
`have led Pesce et al (23) to conclude that technical and
`biologic problems interfere with the application of solid
`phase assays to the measurement of anti-DNA anti-
`bodies.
`
`A reason for technical difliculty has been re-
`cently identified. Pure double-stranded DNA does not
`bind to plastic unless the surface is first treated with
`poly-L-lysine (PLL), a positively charged polymer (22).
`At the same time there is binding of high affinity lg in
`autoimmune sera to the solid phase (21). Therefore
`solid phase immunoassays attempting to demonstrate
`specific anti-dsDNA must include a basic specificity
`control for each sample tested, namely, the binding of
`lg to a surface not coated with DNA, but otherwise
`treated identically with the DNA-coated surface.
`The present communication describes a method
`for the measurement of specific anti-native DNA anti-
`body in which the high surface binding of immunoglob-
`ulin from autoimmune sera has been reduced. Under
`
`'
`
`this assay is specific and
`the conditions employed,
`highly sensitive. Sensitivity has been increased by the
`use of affinity purified anti-lg iodinated to high specific
`activity by a simple and reproducible radioiodination
`method.
`
`MATERIALS AND METHODS
`
`Sera. Sera was obtained from 35 patients with active
`‘systemic lupus erythematosus (SLE) and 25 patients with
`treated SLE. All patients met the American Rheumatism As-
`. sociation (ARA) criteria for systemic lupus erythematosus
`_ (24). The criteria for the activity of the SLE patient have been
`previously reported (25). Sera from 35 patients with definite
`‘or classic rheumatoid arthritis, 35 with scleroderma, 13 with
`polymyositis or dermatomyositis, and 38 normal individuals
`were also examitted. Most of the sera of patients with con-
`nective tissue disease were obtained from the inpatient serv-
`ices of Parkland Memorial Hospital and were previously as-
`itayed for anti-DNA antibodies by Chubiclt et al (25)
`i_'e;_Inploy'tng the Crirhidia Iucilfae method (26). Some sera were
`provided by Dr. S. Cohen from patients of the St. Paul Hospi-
`inpatient and outpatient services. The nonnal sera were
`:collected from laboratory stall‘.
`_
`Antisera. Goat anti-human IgG directed against
`heavy and light chains was produced by Miles Yeda, Rehovot,
`%lsrael. As determined by imrnunoelectrophoresis.
`this anti-
`
`Page 4 of 12
`Page 4 of 12
`
`serum demonstrated activity against IgG and also some activ-
`ity against IgM.
`IgG. Crude human IgG was precipitated from human
`Cohn fraction II (Sigma. St. Louis, Missouri) by ammonium
`sulfate (23-').
`lmmunoabsorbents. Sepharose 4B (Pharmacia, Upp-
`sala. Sweden) was activated with cyanogen bromide by the
`method of March et al (23). The freshly activated resin was
`then reacted with a 10 mg/ml solution of human IgG. After
`incubation for 16 hours at 5°C, the resins were washed and
`the remaining active groups were neutralized with 1.0M gly-
`cme.
`
`Aflinity purification of anti-IgG antisera. One milliliter
`of heat inactivated anti-human IgG (S6°C, 30 minutes) was
`incubated for 2 hours at room temperature with 2-4 ml of ap-
`propriate immunoabsorbent. The resin was then washed with
`phosphate-buffered saline (PBS) until the optical density of
`the wash at 280 Inn was less than 0.01. The bound antibody
`was eluted with 0.51)! acetic acid (29) at room temperature.
`The eluate was neutralized and dialyzed overnight at 5°C
`against PBS. After centrifugation at 10,000 g for 10 minutes,
`the purified antibody was concentrated by ultrafiltration to 1
`mg protein/ml. Aliquots, 0.1 ml, of this preparation were
`stored at —20°C.
`
`Radioiodination of purified anti-IgG. This was per-
`formed by the chloramine T method (30) using one atom of
`carrier iodide per molecule of protein {.3 I). To 100 pl of the
`purified antibody solution (1 mg/ml PBS) reagents were
`added in the following order: 1) [0 pl potassium iodide (6.25
`x l0‘5M). 2)l0 pl of solution containing 1 mCi ""1-sodium
`iodide (13-1? mCi/pg l. Amersham Co., Arlington Heights.
`Illinois), and 3) 10 pl of freshly prepared chloramine T solu-
`tion (10 mg/‘ ml) (Eastman, Rochester, New York). The mix-
`ture was agitated for 30 seconds at room temperature. The io-
`dination reaction was tenninated by the successive addition of
`0.1 ml of a freshly prepared solution of sodium meta bisulfite
`(2 mg/ml). 0.1 ml fetal calf serum, and 0.".-' ml PBS.
`The iodination process was monitored with potassium
`iodide-starch indicator paper after the addition of the chlora-
`mine T for the existence of excess of chloramine T and ab-
`sence of unreduced chloramine T after the addition of sodium
`meta bisultite. In addition, a 2 pl sample was removed after
`completion of the reaction and added to I ml of 2% bovine
`serum albumin (BSA) and precipitated with 1 ml
`l0% TCA.
`The suspension was spun at 4,000g and the incorporation of
`the radioactive isotope into protein then determined. The in-
`corporation values were consistently about 90%. Unreacted io-
`dide was then removed by incubating the mixture with 0.2 ml
`of washed Dowex 1-xlfl beads for 10 minutes at room temper-
`ature. After this treatment
`the amount of TCA non-
`
`precipitable radioactivity was reduced to less than 2% of the
`total. The radiolabeled anti-IgG preparation was stored at
`—20°C in 50 pl aliquots.
`Nucleic acids. The synthetic double stranded copoly-
`mer of deoxyadenosine and deoxythymidine (poly dA-dT)
`and the synthetic hotnopolymers of deoxyadenosine (poly dA)
`and deoxycytidine (poly dC) were purchased from Miles Lab-
`oratories (Elkhart, Indiana). They were dissolved in PBS con-
`taining 25% ethanol to a concentration of5 Am units/ml (ap-
`proximately 250 pg/ml) and stored at —20°C. Denatured calf
`thymus DNA was prepared by heating a 0.} mg/rnl solution
`
`

`
`FISH AND ZIFF
`
`activity bound to DNA— wells from that bound to DNA+
`wells. Negative values were considered equal to zero. The re-
`sults were expressed either as counts per minute or as the
`amount of '2-‘I-anti lg specifically fixed to DNA.
`Nuclease S, treatment of DNA coated trays. Nuclease
`S. {from Aspergillus oryzae, Type III, Sigma) was diluted in
`0.1M NaCl, 0.05M acetate pH 4.6 to a final concentration of
`2600 units/rnl. Fifty microliters of the enzyme solution were
`introduced into DNA+ and DNA‘ wells immediately after
`the DNA coating phase of the microtitration tray preparation.
`After 20 minutes incubation at 37°C, the tray was washed in
`saline 3 times and covered with 2% BSA as usual. The enzyme
`nontreated wells were incubated in the same buffer solution
`{NaCl. acetate pH 4.6) containing 5% glycerol {to compensate
`for the glycerol present in the nuclease preparation).
`
`
`
`RESULTS
`
`The effect of PLL treatment on DNA surface
`
`binding. To examine the effect of PLL on DNA binding,
`the wells of microtitration trays were incubated with 50
`pl aliquots of a solution of PLL (50 pg/ml) in Tris buf-
`fer. After 45 minutes at room temperature the PLL solu-
`tion was removed and the trays were washed three times
`in normal saline. Fifty microliter aliquots of solutions of
`-‘H-labeled poly dA-dT at various concentrations in Tris
`buffer were pipetted into both PLL-treated and PLL-
`untreated wells in the microtitration tray. After 60 min-
`utes at room temperature, the solution was removed and
`the tray was again washed and the wells counted. The
`percent of added 3H-poly dA-dT bound to the wells was
`calculated. The results are summarized in Table 1. It is
`
`seen that the synthetic DNA, poly dA-dT, did not bind;
`to the polyvinyl surface unless this surface was first
`treated with PLL. This confirms the observations of.‘
`
`Aotsuka et al (22) and extends them to the binding of’
`poly dA-dT to polyvinyl surfaces.
`Inability to demonstrate specificity for DNA hr;
`SLE serum diluted in bufler alone. As an initial ap-
`proach to the assay of anti-dsDNA antibodies in human"
`serum, tested sera were diluted in Tris bulfer. Twenty"-“
`five microliter aliquots of the serum dilutions were in-
`troduced into the wells of DNA-coated microtitratioii
`
`trays containing alternate rows of DNA-coated u-.-
`DNA-uncoated wells. After 3 hours’ incubation at r a n.
`
`"
`
`temperature,‘ the incubation mixtures were removed '
`the trays washed. Twenty-five microliters of '”I-ant
`diluted 1: 100 in Tris buffer containing either 2% BS
`='_-
`or 2% EGG plus I% BSA were then introduced into =
`the wells of the trays, which were then incubated ov ._
`night at room temperature. Thereafter, the trays we :
`washed and counted. The results, presented in Table
`show that under the conditions employed in this ex - -« "
`
`536
`
`Table 1. Binding of 3H-poly dA-dT to polyvinyl microtitration trays
`% hou nd to
`
`3H poly dA-dT added
`(its/ml)‘
`2.5
`5
`10
`
`Untreated wells
`0.2
`0.1
`0.04
`
`PLL coated wells
`l9.'J'
`l'l‘.5
`l0.3
`
`* so ,ul/well in Tris-HCl (pH 7.3).
`
`of calfthymus DNA (Sigma) in water for 15 minutes at l00°C
`followed by rapid cooling in an ice bath. ‘H-poly dA-dT (0.05
`p.Ci/mg) was obtained from Miles Laboratories.
`DNA coated microtitration trays. A 50 pg/ml solution
`of poly-L-lysine (PLL type VII-B, Sigma) in [HM Tris-HCl
`buffer, pH 13 (32), was prepared immediately before use.
`Twenty-five microliter aliquots of the PLL solution were in-
`troduced into each well of a V-shaped polyvinyl micro-
`titration tray (Dynatech, Alexandria, Virginia). Special care
`was given to spread the solution over the bottom of the well
`by lightly tapping the plate. After 45 minut_es of incubation at
`room temperature the trays were washed three times in nor-
`mal saline and 25 pl of poly dA-dT solution, 10 pg/ml, in Tris
`buffer (optical density at 260 mp. = 0.2} were introduced into
`each well of alternate rows in the tray. These wells were desig-
`nated DNA+ wells. The wells in the other rows were in-
`
`cubated with Tris bufler only (DNA- wells). After 60 min-
`utes‘ incubation at room temperature, the trays were washed
`three times in normal saline and coated with 2% BSA (Frac-
`tion V, Sigma) in Tris buffer for 30 minutes at room temper-
`ature as a means of blocking protein binding sites on the plas-
`tic surface ("blocking coat”).
`Single stranded DNA coated trays. A mixture of poly-
`dA (5 pg/ml) and poly-dC (5 pg/ml) in Tris bullet was in-
`troduced into PLL-coated microtitration trays as described
`previously. Heat denatured calf thymus DNA was suspended
`in Tris buffer to an optical density at 260 mo of 0.2 and simi-
`larly introduced into the microtitration trays.
`Assay for anti-DNA antibodies in human serum. The
`serum to be tested was heat inactivated at 56°C for 30 minutes
`
`and serially diluted in the reaction buffer solution; the starting
`dilution for screening human sera was 1:100. The buffer solu-
`tion consisted of 0.1M Tris bufier, pH 7.2, containing 2% w/v
`bovine gamma globulin (BGG. Cohn’s Fraction II, Sigma}
`and 1% w/v BSA (Cohn‘s fraction V, Sigma). The BSA solu-
`tion was then removed from the DNA coated tray previously
`prepared and the tray was washed once with normal saline.
`Twenty-five microliter aliquots of the diluted serum were de-
`livered into the DNA+ and DNA- wells. After 3 hours’ in-
`
`cubation at room temperature. the tray was washed five times
`in normal saline. Twenty-five microliters ofthe ml-anti lg, di-
`[med 1 : [00 in 2% BGG, l% BSA were then added to all wells.
`The tray was covered tightly with Parafilrn {American Can
`Co, Greenwich, Connecticut) or with wet paper towel and in-
`cubated overnight at room temperature. The assay was tenni-
`nated by washing the trays five times in normal saline and
`drying over a warm hotplate.
`The bottoms of the wells were cut from the dried trays
`with scissors and the bound radioactivity was measured in a
`Packard Autoganima Spectrometer model 5230. The specific
`anti-DNA activity was calculated by subtracting the radio-
`
`
`
`Page 5 of 12
`Page 5 of 12
`
`

`
`MICRORADIOIMMUNOASSAY FOR ANTI—dsDNA
`
`537
`
`Table 2. DNA binding versus surface binding of immunoglobulin from normal and SLE sera
`
`cpm of"2‘I-anti-Ig bound per well’ at
`
`l0‘3 dilution of
`
`10-‘ dilution of
`
`HTS dilution of
`
`Anti-lg
`DNA on
`SLE
`Normal
`SLE
`Normal
`SLE
`Normal
`diluted in
`surface
`serum
`serum
`serum
`serum
`serum
`serum
`
`
`2% BSA
`
`+
`—
`
`28,159
`72,834
`
`3303
`51. I79
`
`4022
`28,1136
`
`I559
`l4,9I0
`
`136?
`7572
`
`I084
`50H
`
`401
`I532
`5! 1
`2799
`I537
`22,172
`+
`1% BSA plus
`
`
`
`
`
`
`
`— 44,322 21846 14,613 5638 18062% B-GG I106
`
`“‘ Total added: 23,6679 cpm/well (20 ng of anti-lg).
`
`
`
`ment, specificity for DNA could not be demonstrated in
`-the SLE serum. Both the normal and SLE sera exhib-
`ited greater binding to DNA— than to DNA+ surfaces.
`The addition of an unrelated inimunoglobulin, namely
`EGG, to the labeled anti—ig resulted in an overall re-
`duction in the binding of this reagent. Since the addi-
`tion of BGG to the '25]-anti-lg had a greater effect at the
`-"higher dilutions of the tested sera than at the lower dilu-
`tions, it is likely that the BGG competitively blocked
`_the direct binding of the labeled reagent to the solid sur-
`face when the concentration of human serum was low.
`in view of these results, the "51-anti-lg was diluted in
`2% BGG, 1% BSA in all further experiments.
`The effect of unrelated lg on the DNA specificity
`SLE and normal sera. In the preceding experiments,
`"-high surface binding of 1g of both SLE and normal
`Jiserum was observed both in the presence and absence of
`.—;'a DNA coat. On the possibility that this resulted from
`gnonspecific interaction of the Ig in these sera with the
`lid phase, an effort was made to competitively inhibit
`' binding by the addition of nonrelated lg. In this ex-
`‘
`,-- ‘en! the addition of BGG to the BSA “blocking
`gcoat” on the surface of the well had no conspicuous ef-
`
`feet on the specificity of binding of Ig either of the SLE
`or the normal serum (Table 3). However, diluting the
`tested sera in a buffer containing 2% BGG and 1% BSA
`resulted in a marked improvement in the specificity of
`binding (Table 3). Under these conditions the binding
`of the SLE lg to the DNA-coated wells was more than
`9.6 times greater than the binding to DNA-- wells at
`10*’ dilution and 4.6 times greater at 10“ dilution
`whereas the differences in binding between DNA+ and
`DNA— wells for the normal serum were only 1.1 at 10"
`dilution and 1.9 at 10“ dilution.
`The differences in binding between the SLE and
`normal serum almost disappeared at the 10" dilution.
`This dilution probably represents the endpoint for this
`particular SLE serum which had a 1:600 titer in the
`Crithidia assay. Thus it appears that the presence of an
`unrelated Ig in the buffer diluting the test sera interferes
`effectively with the nonspecific binding of human lg to
`the solid phase and this effect is more marked on the
`nonspecific binding of SLE sera than on that of normal
`sera.
`
`Other attempts at reducing background binding.
`In attempts to further reduce the nonspecific binding of
`
`Table 3. The effect of B66 on the radioimrnunoassay of anti-DNA antibodies in SLE and normal sera
`
`cpm of '25]-anti-lg bound per well‘ at
`
`ID" dilution of
`IO" dilution of
`I0” dilution of
`DNA
`on rm“? T T
`sur-
`SLE
`Normal
`SLE
`Normal
`SLE
`Normal
`face
`serum
`serum
`serum
`serum
`serum
`serum
`
`+
`—
`
`+
`—
`
`24,085
`43,13 I
`
`2196
`29,01 1
`
`3149
`15,209
`
`822
`6877
`
`694
`2529
`
`51 I
`I03”.-'
`
`23,?S2( I )i'
`44,19-1(0)
`
`16-10(25)
`2?,?S?(4)
`
`2898(8)
`10.78609)
`
`5'?4(30)
`4l45(40}
`
`644(7)
`I5 I 9010]
`
`421(8)
`977(6)
`
`Treatment
`
`None
`(control)
`
`2% BGG in
`last coat
`
`408{2fl)
`57 3(l‘?]
`459044)
`21650 I}
`6l2{‘i'2}
`20,696(l4)
`+
`2% BGG in
`
`
`-diluting buffer 22608) 2 l65(95) 56S(98) 462(92) 241(96) 2I3(92)
`
`
`
`
`
`‘ Total added: 230,980 cpm/well (20 ng of anti-lg).
`‘t Numbers in parentheses represent the percent reduction in binding compared to the untreated corre
`sponding control.
`
`Page 6 of 12
`Page 6 of 12
`
`

`
`538
`
`FISH AND ZIFFH
`
`
`
`Specificity for dsDNA demonstrated by nuclease?
`S. treatment of DNA. Nuclease S,
`is a hydrolytic en-v
`zyrne specific for terminal and internal phosphodiestcrv
`bonds of single-stranded DNA and RNA (33). As such,
`this enzyme can be used as a probe for assessing the pu-i -
`rity of the double—stranded synthetic DNA used in
`assay. Accordingly, PLL treated wells were coated
`one of the following nucleic acids: poly dA-dT, a
`ture of poly dA + poly dC and heat-denatured calf‘_
`thymus DNA. Half of the nucleic acid coated wells were”-
`subjected to nuclease S. digestion. Sera from 2 SLE pa.--=_
`tients were diluted l:l00 in 2% BGG, 1% BSA buffer-
`and incubated in enzyme treated and untreated wells.-_.
`The net amount of Ig bound to each nucleic acid coated"
`well was assayed by the standard method described
`above. The results are presented as Figure 1. It can beef
`seen that nuclease S, treatment had no effect on the‘
`binding of SLE Ig to poly dA-dT coated wells, thus in»-
`dicating that this DNA preparation was indeed wholly
`double-stranded. On the other hand, the binding of lg-
`to heat-denatured DNA was almost completely abol-s
`ished by the enzymatic digestion. This positive control
`for the nuclease S, activity suggests that single-stranded
`nucleic acid, bound to PLL treated plastic, remains sus--
`ceptible to the ltydrolytic activity of the enzyme.
`Of some interest was the binding of SLE lg to-
`synthetic single-stranded deoxynucleic acids. The bind-
`ing to a mixture of untreated poly dA and poly dC was .
`considerably lower than the binding to denatured calf
`thymus, probably because the repertoire of single or-
`ganic bases was incomplete and base-sequence determi-
`nants were lacking (1). The synthetic nucleic acids were.-_
`not completely sensitive to the degradative activity of:
`the nuclease S., probably because of secondary struc-
`ture formation.
`
`Measurement of anti-DNA activity in sera. All
`sera were heat inactivated (56°C, 30 minutes) diluted-.'
`1: 10-0 in 2% BGG, 1% BSA and assayed for anti-DNA:
`activity. employing poly dA-dT as the antigen. The net:-'
`specific binding to DNA was calculated by subtracting",
`the counts per minute bound to DNA— wells from me:
`binding to DNA-+ wells. Negative values were consid--_
`ered zero. The results are presented in Figure 2. There‘
`was complete absence of net anti-DNA activity in
`]_
`sera of the 38 normal individuals tested at the 1: 100 die‘-
`lution. Twenty-three of 35 patients with active treat
`SLE gave evidence of the presence of anti-DNA in the,
`serum. Only I of 25 treated SLE patients showed simia.
`lar activity. Sera derived from patients with other rheu-'
`matic diseases (rheumatoid arthritis [RA], sclerode
`and polymyositis-dermatomyositis) demonstrated spe-
`
`Table 4. Solid phase absorption of normal and SLE sera
`
`cprn ‘Z51-anti-lg bound per well‘
`10*’ dilution
`10“ dilution
`
`SL E
`scrum
`
`I7,-310
`3442
`
`4806
`3033
`
`l6,42'.-'
`I959
`
`Normal
`serum
`
`SLE
`serum
`
`Norm al
`serum
`
`61 I
`853
`
`48 I
`‘I93
`
`6E4
`482
`
`25! 1
`465
`
`74‘?
`488
`
`248?
`419
`
`250
`260
`
`321
`235
`
`243
`22‘!
`
`Treatrne nt
`
`DNA
`
`Control1'
`
`DNA
`absorbed
`
`PLL
`absorbed
`
`+
`—
`
`+
`—
`
`+
`—
`
`‘ Total added: 153,038 cpm/well (20 ng anti-lg).
`1‘ Sets; uncubated overnight at 5°C in untreated microtitration trays.
`
`irnmunoglobulin to the solid phase, the following proce-
`dures were screened:
`
`l. The DNA coated microtitration trays were
`coated with polyanions (poly-L-glu and hepa-
`rin) at concentrations that were pre-
`determined to neutralize all the charges on
`PLL heated wells.
`
`2. Trays were washed after the incubation with
`test sera in high salt concentrations (up to IM
`NaCl) and in normal saline containing 0.05%
`Tween-20 or 10% glycerol.
`
`Although the manipulations in 1) reduced the
`nonspecific binding of immunoglobulin to PLL coated
`wells, they were inferior in their effectiveness to diluting
`the test sera in 2% BGG and 1% BSA. The manipula-
`tions in 2) demonstrated almost no beneficial effect.
`DNA specificity of the assay demonstrated by
`absorption. To further demonstrate the specificity of the
`anti-DNA assay, preabsorption studies were under-
`taken. SLE and normal sera were diluted 1: 100 in Tris
`
`buffer and incubated overnight at 5°C in DNA-coated
`or DNA uncoated PLL treated wells and in untreated
`
`wells. Alter absorption, the sera were further diluted in
`2% BGG and 1% BSA and tested for anti-DNA activity
`
`by the standard method. The results are presented in
`Table 4. It is seen that a single absorption of the SLE
`serum in DNA-coated wells resulted in 72% reduction
`
`in the binding of DNA-coated wells. Absorption of the
`same serum in wells coated with PLL alone resulted in
`
`only 3% reduction in binding to DNA coated wells. Ab-
`sorption of the normal serum with DNA or PLL re-
`sulted in minor reductions in binding.
`
`
`
`Page 7 of 12
`Page 7 of 12
`
`

`
`MICRORADIOIMMUNOASSAY FOR ANTI-dsDNA
`
`539
`
`Specific onli-DNA Binding
`125
`
`int:
`
`I-onii-lg bound/mi 1-.100 serum)
`
`poly dA + poly dC
`
`denatured DNA
`
`0
`
`100
`
`200
`
`Figure l. The effect of nuclease S1 treatment of DNA preparations on the binding of immuno-
`globulin of 2 SLE sera. PLL treated microtitration wells were coated with various preparations of
`double~stranded and single-stranded DNA. A portion of the welis was digested with nuciease S,
`before the addition of the patient sera. The binding to intact DNA (empty bars) and to nuclease
`treated DNA (black bars) is expressed as nanograms of '1’!-anti-lg bound per milliliter of l: 100
`diluted serum.
`
`Page 8 of 12
`Page 8 of 12
`
`
`
`cific binding to dsDNA in low frequency (4 of 35 for
`ERA; 3 of 35 for scleroderrna; 1 of 13 for polymyositis).
`The reactivity of the various sera with denatured
`-calf thymus DNA is depicted in Figure 3. In this case,
`'_'.3l of 35 SLE sera and 11 of 35 normal sera demon-
`=-strated positive reactions. There was also an increase in
`‘the incidence of positive sera in the other connective tis-
`.=_sue diseases. Of interest also was the binding pattern to
`-the mixture of synthetic single-stranded nucleic acids
`{poly cm and poly dC) depicted in Figure 4. There was
`it high frequency of positive reactions in the normal sera
`"[21/30) as well as in sera of patients with other diseases.
`{Binding to this type of single-stranded nucleic acid
`jhcrefore appears to have no diagnostic value.
`i
`Correlation between the radioimmunoassay and
`Crirlddia Iucilae assay. For this study, 15 Crirhidia
`assay positive sera were selected. All were diluted 1:100
`2% BGG, 1% BSA and tested against poly dA-dT in
`ihe solid phase radioimmunoassay. The relationship be-
`" een the results of the radioimmunoassay and the Cri-
`'
`'
`test is plotted in Figure 5. A positive (r = 03?)
`highly significant (P < 0.001) correlation between
`two assays was calculated.
`
`DISCUSSION
`
`Solid phase immune assays for antibody activ-
`ity offer many advantages over other methods. Aside
`from simplicity of performance and ease in separating
`the bound from the free antibody (2l,22), this type of
`assay is independent of secondary phenomena such as
`complement fixation or precipitation. Moreover,
`the
`sensitivity can be tailored to the needs of the investiga-
`tor by manipulating the specific activity of the isotope
`or enzyme labeled secondary antibody. However, since
`all antigen-antibody reactions take place at the solid
`phase, two basic requirements must be met: 1) the anti-
`gen must adhere to the solid phase for the duration of
`the assay; and 2) immunoglobulin, both specific anti-
`body and nonrelated immunoglobuljn, must not bind
`nonspecifically to the solid phase.
`The first condition was not met in early attempts
`(19-21) to establish a solid phase assay for antibodies to
`dsDNA. As has been reported by Aotsuka et al (22) and
`confirmed by us, pure double-stranded DNA does not
`bind to plastic surfaces. Therefore, the antibody that
`was measured by these groups was directed against ei-
`ther denatured DNA or an associated protein.
`
`

`
`540
`
`FISH AND ZIFF=-
`
`
`
`ng125!-onlilgboundtopolydrt-dT/ml1:100serum
`
`
`
`
`
`
`
`Active
`SLE
`
`Tr
`
`otd
`L
`
`Nor mols
`
`Poly-
`Scleroderrno mwsi ,1-5
`
`100
`
`control for background binding to PLL coated tubes.
`By conducting the assay in a bovine gamma.-
`globulin (BGG) rich medium we succeeded in reducing
`the background binding of immunoglobulin in SLE
`sera,
`thus enabling us to demonstrate DNA specific
`antibody. The high concentration of the BGG in the re- '
`action mixture reduced the nonspecific binding of im-
`munogiobulin in the test serum probably by com-
`petition for nonspecific binding sites. The binding of
`specific anti-DNA to DNA was not aifected, presum-
`ably because of its higher aflinity for the related anti-
`gen. Other agents employed to reduce background
`binding, namely, poly-L-glu, heparin, concentrated salt
`solutions, or detergents were either nonefifective or infe-
`rior in reducing background binding.
`The choice of antigen is of major importance in
`developing an assay for anti-DNA. Different DNA-
`preparations may lead to contradictory results (12,13).
`The importance of using pure double-stranded DNA
`has been stressed