Proc. Natl. Acad. Sci. USAVol. 84, pp. 4979-4983, July 1987Immunology

Analysis of anticentromere autoantibodies using clonedautoantigen CENP-BWILLIAM C. EARNSHAW*, PAULA S. MACHLINt, BONNIE J. BORDWELLt, NAOMI F. ROTHFIELD*,AND DON W. CLEVELANDtDepartments of *Cell Biology and Anatomy, and tBiological Chemistry, The Johns Hopkins University School of Medicine, 725 N. Wolfe Street, Baltimore,MD 21205; and tDivision of Rhetzmatic Diseases, Department of Medicine, University of Connecticut Health Center, Farmington, CT 60305

Communicated by John W. Littlefield, March 16, 1987 (receivedfor review January 16, 1987)

ABSTRACT A cDNA clone encoding CENP-B, the 80-kDahuman centromere autoantigen, was used to construct a panelof hybrid proteins containing four different regions of CENP-B. These have allowed us to identify three independent epitopeson CENP-B that are targets of autoantibodies. Two of these arerecognized concurrently in -90% of patient sera containinganticentromere autoantibodies (ACA), conclusively demon-strating that this autoimmune response is polyclonal. Whenpresent and previous data are combined, ACA are shown torecognize at least five independent epitopes on CENP-B. Aradioimmunoassay based on cloned CENP-B has demonstratedthat sera from 296% of patients with ACA recognize thecloned antigen, thus defining a region of the protein that isrecognized by virtually all patients with ACA. These rmdingshave significant implications for models that seek to explain theorigin of ACA and for the future detection of this group ofautoantibodies in the clinical setting.

The rheumatic diseases are characterized by the productionof autoantibodies directed against nuclear and cytoplasmicautoantigens (reviewed in refs. 1-8). The reasons forautoantibody expression are generally unknown, and manytheories seeking to explain the phenomenon are currentlyunder consideration. In particular, it is not known if theautoimmune response results from a classical antigen-drivenimmunization or is a result of aberrations of the mechanismsthat normally control the immune system.We have chosen the anticentromere autoantibody (ACA)

response for study since this involves the production ofhigh-titer, high-affinity autoantibodies that recognize proteinantigens. ACA were discovered in 1980, when it was foundthat certain patients with the calcinosis/Raynauds phenome-non/esophogeal dysmotility/sclerodactyly/telangiectasiae(CREST) variant of scleroderma produce autoantibodies thatrecognize the centromere region of chromosomes (9-11).Though ACA are closely associated with the CREST syn-drome, the only clinical finding common to all ACA+ indi-viduals is Raynauds phenomenon (12).Our prior immunoblotting analysis revealed that >96% of

a test group of 39 ACA+ sera recognized three chromosomalpolypeptides of 17 kDa (CENP-A), 80 kDa (CENP-B), and140 kDa (CENP-C) (refs. 12 and 13; see also refs. 14-19).Antibodies affinity purified from CENP-B cross-reacted withCENPs A and C, indicating that these antigens are structurally related (13). Antibodies to CENP-B are present at hightiter in all ACA+ sera examined, whereas the titer ofantibodies to CENPs A and C is occasionally lower (12).We describe below a detailed examination of the binding of

ACA to subdomains of CENP-B that have been cloned andexpressed in bacteria. Our experiments reveal that theautoimmune response against this protein is multifocal: as

many as five distinct determinants are recognized. Contraryto our prior expectations, the data suggest that ACA arisefrom a specific polyclonal immune response directed againstcentromeres.

MATERIALS AND METHODSGeneral Methods. All cloning methods and procedures

have been described in detail elsewhere (20). NaDodSO4/PAGE was performed using the method of Lewis andLaemmli (21). Electrophoretic transfer of proteins to nitro-cellulose (22) was performed at 340 mA for 6 hr at 4°C. Theimmunoblotting protocol has been described (12), as has themethod for affinity purification of antibodies from nitrocel-lulose strips (13). The antibody-blocking experiments weredescribed in ref. 20.RIA. Fusion protein granules were isolated by differential

centrifugation from induced (23, 24) cultures of lysogenX-CENP-B1 (20, 36). Granules were then solubilized in hoturea and dialyzed into 10 mM Tris-HCl, pH 7.7/50 mMNaCl/2 mM EDTA. The yield was 1 mg of Cte,,nCENP-B[,8-gal] per liter of bacterial culture. CtlmCENP-B[p-gal] in 10mM imidazole buffer was adsorbed to microtiter plates (0.1,ug per well; Removawell strips, Immulon) that were thenprobed with a 1:500 dilution of patient serum followed by1251I-labeled protein A; radioactivity was determined in a ycounter as described (25). All assays were performed intriplicate and sera were used in random order.

RESULTSMolecular Cloning of CENP-B. We previously obtained a

series ofoverlapping cDNA clones corresponding to =95% ofthe mRNA encoding CENP-B (20). The availability of theclones permitted us to produce the following series ofchimeric proteins as fusions with the bacterial TrpE protein(using the pATH plasmid series of expression vectors, gift ofT. J. Koerner, Duke University). With the exception ofCtermCENP-B[,8-ga1] (described below), all chimeric proteinsused in the studies presented here were TrpE fusions. (Therelative sizes and locations of these proteins are presented inFig. 5.)

CtermCENP-B[13-gal] consists of the amino-terminal 113-kDa portion of P-galactosidase linked to the carboxyl-terminal 147 amino acids of CENP-B. This hybrid gene wascarried by bacteria lysogenic for the recombinant X bacteri-ophage originally detected by autoantibody (20). CtemCENP-B[,B-gal] was used to elicit production of two monoclonalACA, m-ACA1 and m-ACA2, which recognize two nonover-lapping determinants on CENP-B (20).

Abbreviations: ACA, anticentromere autoantibody(ies); CREST,calcinosis/Raynauds phenomenon/esophogeal dysmotility/sclero-dactyly/telangiectasiae.

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CtermCENP-B consists of the 147 carboxyl-terminal aminoacid residues of cloned CENP-B fused to the bacterial TrpEprotein. CtermCENP-BL and CternCENP-BR were producedby further subdividing the human portion of CtermCENP-Binto segments of 104 amino-terminal and 43 carboxyl-termi-nal amino acid residues, respectively, and expressing these asTrpE fusion proteins.NproxCENP-B is comprised of TrpE linked to 347 amino

acid residues from the amino-terminal region of CENP-B(20). (The cDNA encoding the -50 amino-terminal residueshas not yet been isolated.) The human portion of the proteinencoded by this clone is separated in the CENP-B sequenceby 98 amino acid residues from that encoded by CtermCENP-B (Fig. 5).

Localization of Centromere Epitope 1 (CE1), a MajorAutoepitope in the Carboxyl-Terminal Region of CENP-B. Toidentify the epitope(s) in CENP-B that are targets for auto-antibodies, we have examined the interactions of a panel of37 ACA+ patient sera and 3 ACA- control sera with thecloned CENP-B polypeptide. [All patient sera were previ-ously characterized by indirect immunofluorescence andimmunoblotting against the proteins of isolated mitotic chro-mosomes (12).] Remarkably, all 37 ACA' sera bound signif-icantly to CtermCENP-B (Fig. 1A). Strong binding was evi-dent for 86% (32 sera) and weaker binding characterized theremaining 14% (5 sera). Control sera (lanes 12, 18, and 40) didnot bind the cloned antigen.

In our previous studies of cloned CENP-B, we isolated amurine monoclonal antibody, m-ACA1, that recognizes a site(CE1) present in CtermCENP-B (20). We now show that thesite recognized by m-ACA1 overlaps a major autoepitope onCENP-B. Nitrocellulose blots containing CtermCENP-B werecut into 40 strips, each ofwhich was incubated overnight witha 1:50 dilution of human serum. Each strip was then probedwith m-ACA1, the binding of which was detected with125I-labeled goat anti-mouse IgG (Fig. 1D). Pretreatment ofthe blot strips with any of the 37 ACA+ sera blocked thebinding of m-ACA1 to some extent, with 19 sera showing.80% blocking (Fig. 1D). The ACA+ sera least effective atblocking the binding ofm-ACA1 (sera 8, 20, and 33) were also3 of the poorest at binding to CtermCENP-B (Fig. LA),suggesting that the variability of blocking of m-ACA1 may belargely due to variations in the titer of the anti-CtermCENP-Bautoantibodies.CE2, a Second Autoepitope in the Carboxyl-Terminal Region

of CENP-B. A minority of the panel of patient sera recognizes

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at least one other epitope (CE2) in CterrCENP-B (identifiedby immunoblotting of bacterial lysates expressing CtermCENP-BL, Fig. 1C). Sera 6 and 9 recognized this hybridprotein (and a proteolytic fragment) strongly, whereas weak-er binding was exhibited by sera 3, 13, 19, 24, 32, and 38(making 22% in all).The location of CE2 was defined by a second murine

monoclonal antibody, m-ACA2, previously shown to bind toCtermCENP-BL, (20). Three patient sera showed significantblocking of the binding of m-ACA2 (Fig. 1E, lanes 6, 9, and19). The other five sera that bound to CtermCENP-BL inimmunoblots failed to block the binding of m-ACA2 to asignificant degree, perhaps due to low titers of anti-CE2antibodies.Even though all ACA' sera bound to CtermCENP-B, only

a handful (22%) recognized CtemCENP-BL and none recog-nized CterCENP-BR (not shown). Thus, most ACA' seraapparently recognize only one epitope in CtermCENP-B.CE3, an Epitope Present on N-Proximal CENP-B. To de-

termine whether additional autoantibody binding sites occuroutside the carboxyl-terminal region of CENP-B, we exam-ined the binding of the panel of 40 sera to NprOXCENP-B inimmunoblots (Fig. 1B). Ninety percent (33) of the ACA' serashowed significant binding, although the intensity was muchmore variable than that observed with Cte~rmCENP-B (com-pare Fig. 1 A and B). Thus, a third autoepitope(s), CE3, islocalized within the amino-terminal 60% of CENP-B. Theprecise location and number of epitopes recognized in thisregion are not known.

Titer of ACA. We have measured the titers of antibodiesagainst chromosomal CENPs A, B, and C and against tworegions of cloned CENP-B for one serum (KG). The titer ofantibodies specific for each antigen was determined byprobing parallel blot strips of chromosomal proteins withserial dilutions of the patient serum (Fig. 2). Positive signalswere obtained in overnight exposures for the following serumdilutions: CENP-A, 1:1,638,400; CENP-B, >1:3,276,800;and CENP-C, 1:12,800 (lanes 1-9). Thus, though the titers ofantibodies against CENPs A and B are comparable, antibod-ies to CENP-C are less abundant (roughly by a factor of 200).(Note, however, that the three chromosomal antigens may bepresent in differing amounts.) When the serum was titeredagainst cloned CtrmCENP-B, a positive signal was obtainedat an antibody dilution of 1:4,096,000 (Fig. 2, lane 14). Thetiter of antibodies recognizing NproxCENP-B was also high,with a positive signal being observed at an antibody dilution

FIG. 1. Binding of a panel of 37CermCENP-B ACA' sera and 3 control sera (lanes 12,

18, and 40, indicated by a *) to variouse

regions of cloned CENP-B. (A-C) Bind--*, * ^ ^ @ | a *@ " "* * 2 * ing of the sera to blots containing

NproxCENP-B CtermCENP-B (A), NPrOXCENP-B (B),and Ctr,,CENP-BL (C) (see Fig. 5 for a

s diagram polypeptides).

XllA|XS|| § case a nitrocellulose strip cut from a blot"" of the appropriate bacterial lysate was

C emCENP-BL incubated with the indicated patient se-rum. Blots were processed for antibody

5 10 15 20 25 30 35 40 detection as described (12). Only the>** region of antibody binding is shown. (D_ * _ _ * and E) Binding of the autoantibodies to

sites on CtermCENP-B defined by mono-m-ACA1 clonal antibodies m-ACA1 (D) and m-

ACA2 (E). In these blocking experi-me. ***||* eee ._ 00._ ments, a strong signal is seen when the

* ***humanserum does not recognize the site

(i.e., does not block the binding of them-ACA2 monoclonal antibody).

20 25 30 35 40

x easeo so*a a0a es a

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CENP-C .

CENP-B'. _

CENP-B * 0e -CENP-B. -

I 10 11121314

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CENP-A@ ee

1 2 34 5678 9 15 161718

FIG. 2. Titer ofautoantiserum KG against chromosomal antigensand cloned CENP-B. Lanes 1-8, mitotic chromosomes isolated fromHeLa cells probed with the following dilutions of antibody: 1:800,1:3200, 1:6400, 1:12,800, 1:102,400, 1:204,800, 1:409,600, and1:819,200. Lane 9, Coomassie blue-stained gel. Lane 10, Coomassieblue-stained gel of Ct,,mCENP-B. Lanes 11-14, parallel nitrocellu-lose strips probed with antibody dilutions 1:64,000, 1:512,000,1:2,048,000, and 1:4,096,000. Lane 15, Coomassie blue-stained gel ofNproxCENP-B. Lanes 16-18, parallel nitrocellulose strips probedwith the following antibody dilutions: 1:128,000, 1:256,000, and1:512,000.

of 1:512,000 (Fig. 2, lane 18). The extraordinary titersmeasured in these experiments are substantially greater thanthe 10- to 30-fold increase observed subsequent to a nonspe-cific polyclonal lymphocyte activation (26, 27), suggestingthat ACA do not arise as a result of generalized derepressionof the immune system.Development of a Solid-Phase Binding Assay to Detect ACA.

We wished to determine whether a solid-phase binding assayusing cloned CENP-B might be suitable for detection ofACAin the clinic. We therefore purified CternCENP-B[f-gal] to>40% homogeneity (Fig. 3 Inset). [Details of the procedureare described elsewhere (36).] Immunoblotting experimentsconfirmed that the soluble CtmCENP-B[P-gal] retains reac-

tivity with the autoantibodies (Fig. 3 Inset, lane 7).We then used a solid-phase RIA to screen 48 ACA+ patient

>6

6

5

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sera for binding to Cte,,CENP-B[,8-gal]. We also analyzed 20sera from normal individuals and 92 ACA- patient sera. Thelatter included all those available to us from patients withRaynauds phenomenon and scleroderma (with or without fullor partial CREST) as well as randomly chosen sera fromindividuals with systemic lupus erythematosus and Sj0grensyndrome. Virtually every ACA' patient serum binds to thisportion of CENP-B [Fig. 3, except for two sera, bothcharacterized by the presence of substantial levels of anti-CtermCENP-B of the IgG3 subtype, which is not recognizedby protein A (R. A. Eisenberg, B.J.B., W.C.E., and N.F.R.,unpublished data)]. The average values for the three serumclasses shown were normal control sera, 195 ± 100 cpm;ACA- patient sera, 284 ± 187 cpm; and ACA' patient sera,6013 ± 4809 cpm (a 22-fold stimulation for ACA' sera overACA- control sera).

In a control experiment, the binding of a number ofrandomly chosen ACA' and ACA- sera to CtemCENP-B[,P-gal] and to f-galactosidase was examined (Fig. 4A). (Thelatter comprises 87% of the mass of CtermCENP-B[,8-gal]). Arabbit antiserum (elicited by injection with CtemCENP-B[PB-gal], ref. 20) was included as a positive control. This serumbound to both substrates in this assay (Fig. 4A, lane 4).However, none of the patient or control sera exhibitedsignificant binding to 8-galactosidase.A quantitative immunoblotting assay also confirmed that

the binding observed in the RIA was specific for the humanportion of Cte,,CENP-B[,3-gal]. Bacterial lysates containingCte1,CENP-B (fused to TrpE and therefore containing nobacterial sequences in common with CtemCENP-B[,8-gal])were subjected to NaDodSO4/PAGE and immunoblotting(Fig. 1A). The region of the nitrocellulose containing thefusion protein with its bound autoantibody and 1251-labeledprotein A was excised and counted in a y counter. The resultsfor the panel of 40 sera are shown in Fig. 4B along with thevalues obtained for these same sera by RIA. The striking

10 A S B13- GALACTOSIDASE_8 EDC CENP-BItermx 6

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FIG. 3. Use of C,,,.,,CENP-B[3-gal] in a RIA to detect ACA. TheRIA shows binding ofvarious patient and control sera to CtermCENP-B[3-gal]. Each dot is the average of three measurements for theserum from a different individual. (Inset) Isolation of partly purifiedCtrn,CENP-B[P-gal] from induced lysogens. Lane 1, marker proteins(molecular masses indicated in kDa to the left of the gel). Lanes 2-4,Coomassie blue staining of proteins of whole cell lysate (lane 2),soluble protein fraction (lane 3), and final fraction (lane 4). Lanes5-7, immunoblotting analysis of the samples of lanes 2-4 usingpatient serum GS (1:1000).

5 30 35 4010 15 20 25SERUM NUMBER

FIG. 4. Demonstration that binding in the RIA is specific forcloned human CENP-B. (A) Test for the binding of a random panelof control and patient sera to 3-galactosidase and CtemnCENP-B[f-gal] by RIA. The sera used were normal controls (1-3), serum froma rabbit immunized with CtermCENP-B[P-gal] (contains both anti-CENP-B and anti-/3-galactosidase) (4), ACA- patient control sera

(5-9), and ACA+ patient sera (10-21). (B) Comparison of the RIA ofFig. 3 and the immunoblotting results of Fig. 1A. The relevantportions of the immunoblots shown in Fig. 1A were excised andcounted in a y counter. The two sets of results were normalized togive an equivalent average value.

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correlation exhibited by the two data sets confirms that theRIA detects the binding of autoimmune sera to the humanportion of CENP-B.

DISCUSSION

Classes of ACA. Our analysis of the binding of ACA tocloned portions of CENP-B expressed in bacteria identifiesthree epitopes, CE1, CE2, and CE3, that are targets for theACA autoimmune response. These results, in conjunctionwith our previous work, define at least six independentclasses of ACA (summarized in Fig. 5).CE1 is located in the carboxyl-terminal region of CENP-B

and overlaps the site of binding of a murine monoclonalantibody, m-ACA1. Because disruption of CtermCENP-B at asite 43 amino acids from the carboxyl terminus destroys theepitope recognized by m-ACA1 (20), we postulate (Fig. 5)that CE1 may span this region of the protein (although loss ofantigenicity could also result from a protein folding defect).Because of the close correspondence between the levels ofbinding observed with CtermCENP-B[,8-gal] by RIA and withCtermCENP-B[TrpE] by immunoblotting (Fig. 4B), it is likelythat CE1 is the principal determinant being recognized in theRIA. CE1 is recognized by virtually all ACA' patient serapreviously identified by indirect immunofluorescence.CE2 is located within a 104 amino acid stretch starting 43

amino acids upstream from the carboxyl terminus of CENP-B. This epitope is recognized by only =20% of the patientsera tested, and, even in the sera that interact most signifi-cantly with it (Fig. 1C, lanes 6 and 9), anti-CE2 comprisesonly a small fraction of the antibodies that recognizeCtermCENP-B.CE3 is located somewhere within the amino-terminal 60%

of CENP-B. It is strongly recognized by a minority of ACA+sera, but lower titers of anti-CE3 ACA are present in most (orall) of the remaining ACA+ sera. Overall, -90% of the ACA+sera exhibit detectable binding to CE3. CE1 and CE3 aredistinct structural determinants, since affinity-purified anti-CE1 and anti-CE3 do not cross-react (20).

CLONED ANTIGENS: |

CENP-aB (547 aa)

";`3 CR CENP-B

t term

N CENP-B i (147 aa)prox

(347 aa)

C CENP-B c CENP-Bterm L term R

(104 aa) (43 aa)

ICHROMOSOMAL ANTIGENS:I6 5

CENP-C (140 kDa)

CENP-B (80 kDa)

4CENP-A (17 kDa)

FIG. 5. Distribution of epitopes recognized by ACA. Clonedantigens: All are present only on CENP-B and are localized to thehybrid proteins as shown. The precise location of the epitope withina given shaded segment is not known. aa, Amino acids. Chromo-somal antigens: These determinants are defined by our earlier studies(13). None of them is present on cloned CENP-B, although epitopes4 and 5 are present on chromosomal CENP-B (13). None of thesedeterminants has been precisely mapped.

Three additional autoepitopes recognized by ACA werepreviously identified by analysis of the binding of variousaffinity-purified patient sera to chromosomal antigens inimmunoblots (13). The first of these, CE4, is present onCENPs A and B and is absent from CENP-C (defined by seraGS and SN, ref. 13). We demonstrated that -97% (38/39) ofa panel of 39 ACA' patient sera had anti-CENP-A detectableby immunoblotting (12). We assume that all sera binding toCENP-A bind to CE4, although the structure of this antigenmay be more complex. Epitope CE5, shared by CENPs B andC, was defined by antibodies from serum JR, affinity-purifiedfrom CENP-C, that were subsequently found to cross-reactstrongly with CENP-B (13). Finally, CE6, found solely onCENP-C, was defined when antibodies were affinity purifiedfrom CENP-C (using serum GS) that showed no rebinding toCENP-B (13). The pattern of binding observed using anti-bodies affinity purified from CE1, CE3, CE4, CE5, and CE6indicates that all of these determinants are structurallyindependent (13, 20).

Solid-Phase Binding Assay for ACA. Antibodies to CENP-Bappear to be diagnostic for ACA, since all ACA' patient serawe have tested (105 sera, from Farmington, Baltimore,Montreal, Houston, La Jolla, Nijmegen, and Heidelberg),recognize chromosomal CENP-B in immunoblots. We haveyet to observe anti-CENP-B in any ACA- patient (365 tested)or normal control (32 tested) serum (W.C.E., B.J.B., andN.F.R., unpublished). Moreover, a RIA based on clonedCtermCENP-B[,8-gal] provides a sensitive, reliable method forthe detection of ACA. This assay may eventually provide analternative method for screening large numbers of patientsera for ACA in the clinic.

Origin of ACA. Although much progress is being made inidentifying and characterizing autoantigens, the origin ofantinuclear autoantibodies remains obscure. It has beensuggested that autoantibodies might arise as a result offortuitous cross-reactions exhibited by normal antibodies(28, 29), as a result of chance mutations causing normal B-cellclones elicited by foreign antigen to change specificity andrecognize self components (30, 31), or as antiidiotypeselicited during an immune response against a viral protein (7).Our data are inconsistent with all such models. These modelscould explain an autoimmune response against any singleepitope, but they cannot account for the ACA response, sincemultiple structurally independent epitopes are targeted invirtually every affected individual (requiring multiple chanceevents).Two of our observations presented above suggest that

ACA might arise from an antigen-driven response. (i) ClonedCENP-B [unlike double-stranded DNA (32)] is immunogenicin rabbits and mice (20). [The Sm antigen (a marker antigenfor systemic lupus erythematosus) is also immunogenic inrabbits and mice (33).] (ii) ACA are polyclonal. Serum fromsingle patients recognizes at least four independent epitopeson CENP-B. We have shown here that >90% of ACA+ serarecognize CE1 and CE3 and have shown elsewhere that .95%of the same sera also recognize CE4 and either CE5 or CE6(12). Thus, CENP-B appears to be the target of a diversifiedpolyclonal response. This was postulated earlier to be true foranti-Sm (33, 34) and antiribonucleoprotein (35) autoantibod-ies, but in neither case was the conclusion confirmed byisolating multiple noncross-reactive specificities from patientserum.

In a recent review, Hardin (6) noted that most majorautoantibodies found in systemic lupus erythematosus rec-ognize nucleoprotein structures (small nuclear ribonucleo-proteins, cytoplasmic ribonucleoproteins, and nucleosomes).Our data suggest that ACA fit this pattern and that the ACAresponse may result from self immunization with centro-meres. This is surprising, since centromeres are extremelyminor cellular components and as such seem unlikely can-

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didates for the progenitors of a high-titer immune response(particularly since ACA occur in only a small fraction of theautoimmune patients who make antinuclear antibodies).Monoclonal antibody m-ACA1, which we have shown here tobind to a major autoepitope in CENP-B, should provide animportant tool for future studies that attempt to locate thesource of centromere antigen in affected individuals.

We acknowledge the collaboration ofK. Sullivan in isolation of theoriginal cDNA clone; Carol Cooke and D. Kaiser for isolation ofmonoclonal antibodies; H. Langevin (Baltimore), J.-L. Senecal(Montreal), E. Tan (La Jolla), W. J. van Venrooij (Nijmegan), and H.Guldner (Heidelberg) for the gift of sera; and J. Stobo for helpfuldiscussions. D.W.C. is the recipient ofa National Institutes ofHealthCareer Development Award. This work has been supported byNational Institutes of Health Grant GM 35212 to W.C.E. and N.F.R.,the Arthritis Foundation Devil's Bag Award to W.C.E. and D.W.C.,and National Institutes of Health Grant GM 29513 to D.W.C.

1. Reichlin, M. & Mattioli, M. (1974) Bull. Rheum. Dis. 24,756-760.

2. McDuffie, F. C. & Bunch, T. W. (1977) Bull. Rheum. Dis. 27,900-905.

3. Tan, E. M. (1982) Adv. Immunol. 33, 167-240.4. Shoenfeld, Y. & Schwartz, R. S. (1984) N. Engl. J. Med. 311,

1019-1029.5. Schwartz, R. S. & Stollar, B. D. (1985) J. Clin. Invest. 75,

321-327.6. Hardin, J. A. (1986) Arthritis Rheum. 29, 457-460.7. Plotz, P. H. (1983) Lancet ii, 824-826.8. Grabar, P. (1983) Immunol. Today 4, 337-340.9. Moroi, Y., Peebles, C., Fritzler, M. J., Steigerwald, J. & Tan,

E. M. (1980) Proc. Nati. Acad. Sci. USA 77, 1627-1631.10. Fritzler, M. J., Kinsella, T. D. & Garbutt, E. (1980) Am. J.

Med. 69, 520-526.11. Tan, E. M., Rodnan, G. P., Garcia, I., Moroi, Y., Fritzler,

M. J. & Peebles, C. (1980) Arthritis Rheum. 23, 617-625.12. Earnshaw, W. C., Bordwell, B. J., Marino, C. & Rothfield,

N. F. (1986) J. Clin. Invest. 77, 426-430.13. Earnshaw, W. C. & Rothfield, N. F. (1985) Chromosoma

(Berlin) 91, 313-321.14. Cox, J. V., Schenk, E. A. & Olmsted, J. B. (1983) Cell 35,

331-339.

15. Guldner, H. H., Lakomek, H.-J. & Bautz, F. A. (1985) Clin.Exp. Immunol. 58, 13-20.

16. van Venrooij, W. J., Stapel, S. O., Houben, H., Habets,W. J., Kallenberg, C. G. M., Penner, E. & van de Putte, L. B.(1985) J. Clin. Invest. 75, 1053-1060.

17. Valdivia, M. M. & Brinkley, B. R. (1985) J. Cell Biol. 101,1124-1134.

18. Spowart, G., Forster, P., Dunn, N. & Cohen, B. B. (1985) Dis.Markers 3, 103-112.

19. Nyman, Y., Hallman, H., Hadlaczky, G., Pettersson, I.,Sharp, G. & Ringertz, N. R. (1986) J. Cell Biol. 102, 137-141.

20. Earnshaw, W. C., Sullivan, K. F., Machlin, P. S., Cooke,C. A., Kaiser, D. A., Pollard, T. D., Rothfield, N. F. &Cleveland, D. W. (1987) J. Cell Biol. 104, 817-829.

21. Lewis, C. D. & Laemmli, U. K. (1982) Cell 17, 849-858.22. Towbin, H., Staehelin, T. & Gordon, J. (1979) Proc. Natl.

Acad. Sci. USA 76, 4350-4354.23. Young, R. A. & Davis, R. B. (1983) Proc. Natl. Acad. Sci.

USA 80, 1194-1198.24. Young, R. A. & Davis, R. B. (1983) Science 222, 778-782.25. Shero, J. H., Bordwell, B., Rothfield, N. F. & Earnshaw,

W. C. (1986) Science 231, 737-740.26. Budman, D. R., Merchant, E. B., Steinberg, A. D., Doft, B.,

Gershwin, M. E., lizzio, E. & Reeves, J. P. (1977) ArthritisRheum. 20, 829-833.

27. Izui, S., McConahey, P. J. & Dixon, F. J. (1978) J. Immunol.121, 2213-2219.

28. Cooke, A., Lydyard, P. M. & Roitt, I. M. (1983) Immunol.Today 4, 170-175.

29. Klinman, D. M. & Steinberg, A. D. (1986) Arthritis Rheum.29, 697-705.

30. Eilat, D., Hochberg, M., Pomphrey, J. & Rudikoff, S. (1984) J.Immunol. 133, 489-494.

31. Diamond, B. & Scharff, M. D. (1984) Proc. Natl. Acad. Sci.USA 81, 5841-5844.

32. Madaio, M. P., Hodder, S., Schwartz, R. S. & Stollar, B. D.(1984) J. Immunol. 132, 872-876.

33. Shores, E. W., Eisenberg, R. A. & Cohen, P. L. (1986) J.Immunol. 136, 3662-3667.

34. Eisenberg, R. A., Dyer, K., Craven, S. Y., Fuller, C. R. &Yount, W. J. (1985) J. Clin. Invest. 75, 1270-1277.

35. Maddison, P. J. & Reichlin, M. (1977) Arthritis Rheum. 20,819-824.

36. Rothfield, N. F., Whitaker, D., Bordwell, B., Weiner, E., Sene-cal, J.-L. & Earnshaw, W. (1987) Arthritis Rheum., in press.

Immunology: Earnshaw et al.

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