`MARCH 1981
`
`
`
`__ ,......-11L P. -0.3
`1-4|s~.g..=..»c-’;s‘ LIBRARY
`
`arthrifis
`and
`eumansm
`
`
`
`Official Jo—urnal of the Amgerictan Rheumafism Associatioti
`Se%c:ti-on of the Arthritis Foundation
`
`- N‘
`
`Page 1 of 12
`
`Artihritis Foundation
`Atlanta, Gefor.gjia
`
`HOLOGIC EXHIBIT 1006
`Hologic V. Enzo
`
`
`
`__
`'. "
`
`Page 1 of 12
`
`HOLOGIC EXHIBIT 1006
`Hologic v. Enzo
`
`
`
`ARTHRITIS AND RHEUMATISM
`
`Editor
`
`Nathan J. Zvaifler. MD.
`
`University of California Medical 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. M.D.
`James W. Hollingsworth. M.D.
`Donald L. Resnick. M.D.
`Michael H. Weisman, M.D.
`
`Editorial Board
`
`K. Frank Austen. M.D.. Boston
`
`‘Rodney Bluestone. M.D.. Los Angeies
`Giles G. Bole. .lr.. M.D.. Ann Arbor
`Alan S. Cohen. M.D.. Boston
`
`John 1.. Decker. M.D.. Bethesda
`Virgil Hanson. M.D.. Los Angeles
`Edward D. Harris. Jr., M.D., Hanover
`
`Evelyn V. Hess. M.D., Cincinnati
`David S. Howell. M.D.. Miami
`Gene G. Hunder. M.D.. Rochester
`
`David Koffler. M.D., Philadelphia
`Stephen M. Krane. M.D., Boston
`Henry G. Kunkel. 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. McDufl°te. M.D., Atlanta
`
`Stephen E. Malawista. M.D., New Haven
`Man Mannik. M.D., Seattle
`William Martel. M.D.. Ann Arbor
`Jane H. Morse. M.D., New York
`
`Carl M. Pearson. M.D.. Los Angeles
`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.. Danville
`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.. M.D., Albuquerque
`Robert J. Winchester. M.D., 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
`1. Claude Bennett, M.D.
`University of Alabama
`Birmingham. Alabama
`
`Second Vice-President
`
`Mary Betty Stevens, M.D.
`Johns Hopkins University
`Baltimore, Maryland
`
`Secretary-Treasurer
`James Klinenberg. MD.
`Cedars-Sinai Medical Center
`
`Los Angeies, California
`
`Executive Secretary
`Lynn Bonfiglio
`
`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., Birmingham
`Ronald P. Messner, M.D., Minneapolis
`Paul H. Plotz. M.D., Bethesda
`
`Production Stall’
`
`Daphna Gregg, Managing Editor
`Jerelyn Jordan. Consulting Editor
`Drema McCord, Circulation
`Avis Bradshaw, Editorial Assistant
`Elizabeth Thurlow, Editorial Assistant
`
`Published monthly by the Arthritis Foundation. 3400 Peaehtree Rd. NE.. Atlanta, Georgia 30326. Printed in the United States.
`"Second Class Postage paid at Atlanta. Georgia. and additional oifices. ISSN 0004-359l.
`I SUBSCRIPTION RATES: $20.00 per year for members, as part of the yearly dues, $35.00 for nonmembers within the U.S.A.. and
`$50.00 for nonmembers elsewhere. Students. fellows. intern
`s, and residents in North America: $20.00 per year. (A letter giving
`qualifying data must accompany such orders.) Single copies: $5 .00, except for special issues. Copyright I980 the Arthritis Founda-
`‘ lion, Atlanta. Georgia. All rights reserved.
`
`Page 2 of 12
`
`Page 2 of 12
`
`
`
`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 aflinity 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 bufl'er 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 38 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 "5235.
`Supported by USPHS research grant No. AM [8505 and an
`Arthritis Foundation Clinical Study Center Grant.
`Fall: 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
`Han~y Hines Boulevard, Dallas. Texas 75235.
`Submitted for publication September 10. I980; accepted Oc-
`tober 23. I980.
`
`Arthritis and Rheumatism, Vol. 24. No. 3 (March 1981}
`
`Page 3 of 12
`
`SLE sera, I of 25 treated SLE, 4 of 35 rheumatoid ar-
`thritis. 3 of 35 scleroderma, and I of 13 polymyositis
`sera demonstrated positive anti-dsDNA activity. The
`anti-dsDNA values obtained in the radioimmunoassay
`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 (15) have their inherent deficiencies: they may de-
`tect proteins other than immunoglobulins interacting
`with DNA 06,17); the radioactively labeled DNA may
`be subjected to radiation damage (12); and the methods"
`may not be sensitive enough to measure low concentra-
`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-
`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.-5
`it is possible to determine the lg class of the anti-DNA:
`antibody by this technique.
`_
`In spite of these theoretical advantages of solid.
`
`Page 3 of 12
`
`
`
`MICRORADIOIMMUNOASSAY FOR ANTI-dsDNA
`
`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 difficulty 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 scleroclerma, 13 with
`polymyositis or dermatomyositis, and 38 normal individuals
`were also examined. 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-
`iayed for anti-DNA antibodies by Chubick et al (25)
`:_e,inploying the Crithidia 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
`:e_ollected from laboratory stall‘.
`_
`Antisera. Goat anti-human IgG directed against
`heavy and light chains was produced by Miles Yeda, Rehovot,
`ilsrael. As determined by immunoelectrophoresis.
`this anti-
`
`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 (27).
`lmmunoabsorbents. Sepharose 4B (Pharmacia, Upp-
`sala, Sweden) was activated with cyanogen bromide by the
`method of March et al (28). 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-
`C1118.
`
`Affinity 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 mp was less than 0.01. The bound antibody
`was eluted with 0.5N 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 l0 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 chloraniine T method (30) using one atom of
`carrier iodide per molecule of protein {3|). 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 til of solution containing 1 mCi ""1-sodium
`iodide (13-17 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 terminated 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 0.7 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 bisulfite. 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-x10 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 _ttl aliquots.
`Nucleic acids. The synthetic double stranded copoly«
`mer of deoxyadenosine and deoxythyniidine (poly dA-dT)
`and the synthetic homopolymers 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 of 5 Am units] ml (ap-
`proximately 250 ,ug/ ml) and stored at —20°C. Denatured calf
`thymus DNA was prepared by heating a 0.] mg/ml solution
`
`Page 4 of 12
`
`
`
`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 "SI-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/ml. 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 ‘H-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 butler 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 microtitration‘
`
`trays containing alternate rows of DNA-coated u-.2
`DNA-uncoated wells. After 3 hours’ incubation at r a n.
`
`temperature.‘ the incubation mixtures were removed an
`the trays washed. Twenty-five microliters of '”I-ant
`diluted 1:100 in Tris buffer containing either 2% B
`or 2% BGG plus I% BSA were then introduced into -
`the wells of the trays, which were then incubated ov .'
`night at room temperature. Thereafter, the trays w
`washed and counted. The results, presented in Table
`show that under the conditions employed in this ex -«
`
`'
`
`Table 1. Binding of 3H-poly dA-dT to polyvinyl microtitration trays
`% bound to
`
`3H poly dA-d'I‘ added
`(I-I8/rnlr
`2.5
`5
`10
`
`Untreated wells
`0.2
`0.1
`0.04
`
`PLL coated wells
`l9.'l'
`l'i'.5
`l[}.3
`
`- so ,ul/well in Tris-HCI (pH 7.3).
`
`of calfthyrnus DNA (Sigma) in water for l5 minutes at l00°C
`followed by rapid cooling in an ice bath. 3H-poly dA-dT (0.05
`p.Ci/mg) was obtained from Miles Laboratories.
`DNA coated microtitration trays. A 50 pg/ml solution
`of poly-I.-lysine (PLL type VII-B, Sigma) in 0.lM 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 _u.l 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 bufl"er 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 mp 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 buffer, 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 of the ml-anti lg, di-
`lu ted 1 : 100 in 2% BGG, 1% BSA were then added to all wells.
`The tray was covered tightly with Paraftlnt (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 Autogamma 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
`
`Table 2. DNA binding versus surface binding of immunoglobulin from normal and SLE sera
`
`Anti-lg
`diluted in
`
`2% BSA
`
`1% BSA plus
`2% BGG
`
`—
`
`-1-
`—
`
`cpm of '2‘l-anti-lg bound per well‘ at
`104 dilution of
`10" dilution of
`IO”5 dilution of
`
`SLE
`serum
`
`28,159
`72,834
`
`22.172
`44,322
`
`Normal
`serum
`
`3303
`51.l79
`
`I53?
`22,846
`
`SLE
`serum
`
`4-022
`28,176
`
`2799
`l4,6l3
`
`Nonnal
`serum
`
`SLE
`serum
`
`Normal
`serum
`
`I559
`l4,9l0
`
`SI 1
`5633
`
`136?
`7572
`
`632
`1806
`
`I084
`50ll
`
`401
`l 106
`
`“‘ Total added: 23.66”.-'9 cpm/well (20 ng of anti-lg).
`
`tnent, 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 immunoglobulin, namely
`"BGG, to the labeled anti-lg resulted in an overall re-
`duction in the binding of this reagent. Since the addi-
`_l:_i0n of BGG to the '”I-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 ‘Z51-anti-lg was diluted in
`f_-2% BGG, 1% BSA in all further experiments.
`The efleet of unrelated Ig on the DNA specificity
`pr SLE and normal sera. In the preceding experiments,
`surface binding of lg of both SLE and normal
`',s'_erum was observed both in the presence and absence of
`is DNA coat. On the possibility that this resulted from
`Laonspecific interaction of‘ the Ig in these sera with the
`-. ‘d phase, an effort was made to competitively inhibit
`binding by the addition of nonrelated lg. In this ex-
`gteriment the addition of BGG to the BSA “blocking
`I;.'coat” on the surface of the well had no conspicuous ef-
`
`fect 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 lg 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 BGG on the radioirnmunoassay of anti-DNA antibodies in SLE and normal sera
`
`cpm of ml-anti-lg bound per well‘ at
`I0" dilution of
`I0” dilution of
`
`l()‘5 dilution of
`
`SLE
`serum
`
`24,085
`43,13 I
`
`23,75 2( I )1‘
`44, 194(0)
`
`20,696(l4)
`2l65(95)
`
`Normal
`serum
`
`2196
`29,0ll
`
`SLE
`serum
`
`3 149
`15,209
`
`Normal
`serum
`
`SLE
`serum
`
`Nonna!
`serum
`
`822
`6877
`
`694
`2529
`
`5 1 I
`I03".-'
`
`l640(2S)
`27,7s7(4)
`
`2398(8)
`I0.786(29)
`
`5'?-4(30)
`4 l45(40)
`
`6l2(‘i-'2}
`5ss(9s}
`
`21550 1)
`46292)
`
`4s9(44)
`24106}
`
`644(7)
`l5 199:0)
`
`57307}
`21392)
`
`421(3)
`977(6)
`
`4oa(2o)
`22503)
`
`DNA
`on
`sur-
`face
`
`+
`—
`
`+
`-
`
`+
`-
`
`Treatment
`
`None
`(control)
`
`2% BGG in
`last coat
`
`2% BGG in
`diluting bulfer
`
`‘ Total added: 230.980 cprn/well (20 ng of‘ anti-lg).
`f Numbers in parentheses represent the percent reduction in binding compared to the untreated corre-
`sponding control.
`
`Page 6 of 12
`
`Page 6 of 12
`
`
`
`FISH AND ZIFF
`
`Specificity for dsDNA demonstrated by nuclease-
`S. treatment of DNA. Nuclease S,
`is a hydrolytic en---
`zyme specific for terminal and internal phosphodiester
`bonds of single-stranded DNA and RNA (33). As such,_
`this enzyme can be used as a probe for assessing the pu--
`rity of the double-stranded synthetic DNA used in
`assay. Accordingly, PLL treated wells were coated with-
`one of the following nucleic acids: poly dA-dT, a mix-.
`ture of poly dA + poly dC and heat-denatured calf 5
`thymus DNA. Half of the nucleic acid coated wells were :5
`subjected to nuclease S. digestion. Sera from 2 SLE pa»:
`tients were diluted 1:100 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 be:
`seen that nuclease S, treatment had no effect on the
`binding of SLE lg 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 hydrolytic 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 (M 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: 100 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 the:
`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 di-:-
`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 simi-'
`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
`
`cpm ml-anti-lg bound per well‘
`10*’ dilution
`10" dilution
`
`SLE
`serum
`
`[1010
`3442
`
`4806
`3033
`
`l6,42T
`I959
`
`Normal
`semm
`
`SLE
`serum
`
`Nonnal
`serum
`
`til I
`853
`
`48 l
`793
`
`6E4
`482
`
`2511
`465
`
`74'?
`488
`
`248’?
`419
`
`250
`260
`
`321
`235
`
`243
`22’?
`
`Treatment
`
`DNA
`
`Controlf
`
`DNA
`absorbed
`
`PLL
`absorbed
`
`+
`—
`
`+
`—
`
`+
`—
`
`‘ Total added: 153,038 cprn/well (20 ng anti-lg).
`T Sera uncubated overnight at 5°C in untreated microtitration trays.
`
`immunoglobulin to the solid phase, the following proce-
`dures were screened:
`
`1. 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 1M
`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
`
`Specific onti-DNA Binding
`125
`
`I-onti-lg bound/ml 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 wells was digested with nuclease 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 '2’!-anti-lg bound per milliliter of 1: 100
`diluted serum.
`
`cifie binding to dsDNA in low frequency (4 of 35 for
`:'RA; 3 of 35 for scleroderma; 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-
`isue 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
`ithigh frequency of positive reactions in the normal sera
`"[21/30) as well as in sera of patients with other diseases.
`Sinding to this type of single-stranded nucleic acid
`Liherefore appears to have no diagnostic value.
`1
`Correlation between the radioimmunoassay and
`{the Crithicfia Iueilae assay. For this study, 15 Crirhidia
`assay positive sera were selected. All were diluted 1:100
`n 2% BGG, 1% BSA and tested against poly dA-dT in
`his solid phase radioimmunoassay. The relationship be-
`iween the results of the radioimmunoassay and the Cri-
`:.~ ' test is plotted in Figure 5. A positive (r = 0.27)
`'_
`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 (2I,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.
`
`Page 8 of 12
`
`Page 8 of 12
`
`
`
`
`
`
`
`
`
`
`
`ng125i-oniilgboundtopolydA—-dT/ml1:100serum
`
`Poi»;-
`Sclerodermcl mwsm-5
`
`on0II¢00t
`
`FISH AND ZIFF=-
`
`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-
`munoglobulin in the test serum probably by com-
`petition for nonspecific binding sites. The binding of
`specific anti-DNA to DNA was not alfected, 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 noneflective 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 by many authors (l2,l3,35,36). We
`have used poly dA-dT, the synthetic copolymer of '
`deoxyadenosine and deoxythymidine, as a sourc