Journal of Autoimmunity (2002) 18, 299–309doi:10.1006/jaut.2002.0589, available online at http://www.idealibrary.com on

A Highly Sensitive and Simple Assay for the Detection ofCirculating Autoantibodies against Full-Length BullousPemphigoid Antigen 180

Enno Schmidt1, Arno Kromminga2, Saskia Mimietz1, Ulrich Leinfelder1,Cassian Sitaru3, Eva-Bettina Brocker3, Detlef Zillikens3 and Ulrich Zimmermann1

1Department of Biotechnology, Biocenter,University of Wurzburg, Wurzburg,Germany2Institute of Immunology, ClinicalPathology, and Molecular Medicine(IPM), Hamburg, Germany3Department of Dermatology, Universityof Wurzburg, Wurzburg, Germany

Received 19 December 2001Accepted 22 April 2002

Key words: autoantigen,baculovirus, epitope, eukaryotic,immunofluorescence

Bullous pemphigoid antigen 180 (BP180) is the target of autoantibodies invarious subepidermal blistering diseases. The most common one is bullouspemphigoid (BP). The pathological importance of anti-BP180 antibodies hasbeen demonstrated in a passive transfer mouse model. However, sensitiveassays for routinely detecting circulating antibodies directed against bothintra- and extracellular domains of BP180 are only available in specializedlaboratories. In addition, most current assays use prokaryotic recombinantfragments of BP180 that lack conformation-dependent epitopes. A simple andvery sensitive immunofluorescence (IF) assay based on eukaryotic cells isdescribed here. Sf21 insect cells were transfected with full-length (FL) BP180.As revealed by FACS and confocal laser scanning microscopy the protein wasexpressed as type II transmembrane protein as in human keratinocytes. Bytesting serial dilutions of BP180-specific mouse monoclonal antibodies, theeukaryotic IF assay was demonstrated to be more sensitive compared toconventional assays including (1) indirect IF microscopy of human salt-splitskin, (2) Western blotting (WB) of the keratinocyte-derived BP180 ectodomain,(3) WB of recombinant BP180 NC16A, and (4) WB of FL-BP180 extractedfrom Sf21 insect cells. When applied to sera from patients with BP (n=65),pemphigoid gestationis (n=16), and cicatricial pemphigoid (n=7), the novelassay revealed that 58 (89%), 13 (81%), and 6 (84%), respectively, were positive.In contrast, all control sera (pemphigus, n=20; epidermolysis bullosa acquisita,n=5; anti-laminin 5 cicatricial pemphigoid, n=5; systemic lupus erythemato-sus, n=5; atopic dermatitis, n=7; contact dermatitis, n=3; normal human sera,n=30) were negative indicating that the assay is highly specific. In addition,reactivity of the assay was conserved to a large extent when the cells had beenstored at −20°C for 3 months. Thus, this assay meets the demands of a simpleand effective diagnostic tool for detecting circulating antibodies againstFL-BP180 and may also be used in laboratories without access to molecularbiological technology. © 2002 Elsevier Science Ltd. All rights reserved.

Correspondence to: U. Zimmermann, Ph.D., Department ofBiotechnology, University of Wurzburg, Biocenter, Am Hubland,97074 Wurzburg, Germany. Tel: + +49-931-888-4508; Fax: + +49-931-888-4509; E-mail: zimmermann@biozentrum.uni-wuerzburg.de

Introduction

Bullous pemphigoid (BP) is the most common autoim-mune bullous skin disorder [1, 2]. It is characterizedby tissue-bound and circulating autoantibodies to thehemidesmosomal proteins BP180 and BP230 [3, 4].BP230 is localized intracellularly to the hemidesmo-somal plaque and major antigenic epitopes map to thecarboxy (C)-terminal end of the protein [5, 6]. In

2990896–8411/02/$-see front matter

contrast, BP180 is a type II transmembrane glyco-protein with a C-terminal ectodomain that contains15 interrupted collagenous domains and spans thelamina lucida of the dermal-epidermal junction (DEJ)[reviewed in 7]. BP180 interacts with other structuralproteins of the DEJ, such as �6�4 integrin, BP230, andlaminin 5. The protein is essential for the integrity ofthe DEJ since mutations in the BP180 gene result in aninherited blistering skin disorder that was character-ized by loss of adhesion within the DEJ [reviewedin 8].

The majority of BP sera exhibits reactivity withthe 16th non-collagenous (NC) domain directlyadjacent to the transmembrane portion (NC16A)[9–11]. Reactivity of BP sera with other intra- and

© 2002 Elsevier Science Ltd. All rights reserved.

300 E. Schmidt et al.

extracellular sites located on BP180 has also beenreported [12–14]. The pathogenic relevance of anti-BP180 antibodies was demonstrated using a passivetransfer mouse model [15]. In addition, in BP patients,levels of circulating antibodies to BP180 NC16A havebeen shown to parallel disease activity [16].

Diagnosis of BP is usually made by the use ofseveral assays, including (1) direct immunofluor-escence (IF) microscopy of a perilesional skin biopsy,(2) histopathology of a lesional biopsy, and (3) indirectIF microscopy of patient sera on 1 M NaCl-splithuman skin, i.e. an assay which can distinguishbetween epidermal and dermal binding of autoanti-bodies [17; reviewed in 18]. These assays, however,have several drawbacks. While direct IF microscopyand histopathology require a skin biopsy, indirect IFon salt-split skin does not distinguish between differ-ent specificities of autoantibodies directed to differentcomponents of the DEJ and depends on the qualityof human split-skin that may vary considerably. Inrecent years, recombinant fragments of BP180 werealso used to confirm the diagnosis of BP byWestern blotting (WB) and enzyme linked immuno-sorbent assay (ELISA) [9–14, 19–21]. The disadvan-tage of these assays is that recombinant fragments aregenerated in prokaryotic cells, thus conformation-dependent epitopes may not be expressed. Further-more, they do not encompass the full-length (FL)BP180 molecule.

In the present study, using FL-BP180-expressingSf21 insect cells, we have developed a sensitive,specific, and simple IF assay for circulating anti-BP180antibodies that can be performed in every routine IFlaboratory (termed Sf21-based IF assay). Among otherthings, the reason for this was that the applicationof FL-BP180 as target antigen by ELISA exhibitedunspecific binding when normal human sera weretested. In contrast, by applying the Sf21-based IF assayto a large panel of BP and control sera as well asto various BP180-specific mouse monoclonal anti-bodies it could be demonstrated that the novelassay is highly specific and more sensitive for thedetection of circulating anti-BP180 antibodies com-pared to the conventional diagnostic tools mentionedabove.

Material and Methods

Human and rabbit sera

Serum samples were taken from 65 BP patients beforetherapy was initiated. The mean age of BP patientswas 75 years; 27 were male and 38 female. All patientsexhibited (1) blister formation on the skin that healedwithout scarring, (2) subepidermal splits as revealedby histopathology of lesional skin biopsies, and (3)titres ≥1:10 of circulating IgG antibodies that boundalong the epidermal side of 1 M NaCl-split humanskin as indicated by indirect IF microscopy [22]. In

63 BP patients, linear deposits of IgG and/or C3 weredetected by direct IF microscopy of perilesional skinbiopsies. In two patients, direct IF was negative,although a subepidermal split was seen by histo-pathology of lesional skin biopsies and circulating IgGantibodies could be detected by indirect IF on salt-split skin. In these two patients, direct IF microscopywas regarded as false negative. False negative resultsderived from direct IF microscopy were previouslyshown to result from biopsies from either clinicallyinvolved areas or from skin being too far away fromthe lesion [23].

Furthermore, sera from 16 patients with pemphig-oid gestations (PG) were tested. Direct IF microscopyof perilesional skin biopsies showed linear depositionof IgG and/or C3 along the DEJ in the PG patients. Inall PG sera, linear C3 deposits were present at the DEJas revealed by IF microscopy on normal human skin.In addition, sera from seven patients with cicatricialpemphigoid (CP) were used that showed both linearbinding of C3 and/or IgG along the DEJ (as demon-strated by direct IF microscopy of perilesional skinbiopsies) and linear IgG deposits that exclusivelybound to the epidermal side (as revealed by indirectIF microscopy on NaCl-split human skin).

As negative controls, sera from patients with pem-phigus vulgaris (PV; n=15), pemphigus foliaceus (PF;n=5), and epidermolysis bullosa acquisita (EBA; n=5)were used. All pemphigus sera yielded the typicalintercellular staining pattern (as revealed by indirectIF microscopy on monkey esophagus) and recog-nized recombinant desmoglein 1 and/or 3 by ELISA(Medical and Biological Laboratories, Nagoya, Japan).All EBA sera contained circulating antibodies thatexclusively labelled the dermal side of salt-split skin(as seen by indirect IF microscopy) and recognizedcollagen type VII by immunoblotting of dermalextracts [22]. As further controls, 5 CP were employedthat (1) revealed linear binding of C3 and/or IgGalong the DEJ as demonstrated by direct IFmicroscopy of perilesional skin biopsies, (2) showedlinear IgG deposits that stained to the dermal sideby using indirect IF microscopy on NaCl-splithuman skin, and (3) were previously shown to reactwith laminin 5 [24]. In addition, sera from patientswith atopic dermatitis (n=7), patients with contactdermatitis (n=3), and 5 patients with systemic lupuserythematosus that reacted with double strandedDNA (n=5) as well as sera from healthy volunteers(n=30) were included in the study.

Three polyclonal rabbit sera, R2296, R8009, andR136, were used in this study. R2296 and R8009 wereraised against a GST fusion protein containing a42 amino acid portion of human BP180 NC16A(NC16A2-4; amino acids 507 to 548). R136 was gener-ated against a GST fusion protein encompassing a49 amino acid stretch of a C-terminal portion ofhuman BP180 (BP180 4575; amino acids 1365–1413)[25] (Figure 1). Preimmune rabbit sera and seraraised against the GST moiety (Sigma, Deisenhofen,Germany) were used as controls. For comparison ofthe sensitivity of the eukaryotic IF assay with that ofconventional assays, mouse monoclonal antibodies

Simple and sensitive detection of anti-BP180 antibodies 301

(mAb) against human BP180 were used: mAb1A8c isdirected against an intracellular epitope, mAb233against a C-terminal epitope of the BP180 ectodomain[26], and mAbHD18 against the NC16A domain, i.e.the N-terminus of the BP180 ectodomain [27] (Figure1). As negative control, the mouse mAb1E5 directed tohuman BP230 was used [26].

Figure 1. Schematic diagram of recombinant and cell-derived forms of human BP180. The cytoplasmatic domain of BP180(left side) and the C-terminal ectodomain (right side) consisting of 15 interrupted collagen domains (shaded boxes) areshown. In addition, the extracellular portion of the 16th non-collagenous (NC) domain (NC16A), the C-terminal fragment4575, and the soluble ectodomain of BP180, LAD-1, are presented. Amino acid numbers of the different proteins are depictednext to the proteins. The epitopes recognized by the mouse monoclonal and rabbit polyclonal antibodies to human BP180used in this study are indicated at the top of the panel. TM, transmembrane portion of BP180; 5??, N-terminus of LAD-1, exactamino acid number unknown.

Figure 2. Full-length BP180 is expressed as transmembraneprotein in Sf21 insect cells. Sf21 insect cells were infectedwith baculovirus encoding full-length human BP180 (FL-BP180) and then stained with the mouse monoclonal anti-body 1A8c directed against the intracellular portion ofhuman BP180. Confocal laser scanning microscopic imagesof four different focal planes (A–D) throughout theFL-BP180-expressing Sf21 insect cell are shown. Theapproximate focal plane in which the scanning wasperformed is given on the left lower side of each image.

Expression of FL-BP180 and the C-terminal 55 kDfragment of BP230 in Sf21 insect cells

FL-BP180 and a 55 kD fragment of BP230 (BP230–55 kD) were expressed in Spodoptera frugiperda21 (Sf21) insect cells (Invitrogen, Groningen,Netherlands) grown in Grace insect medium(Invitrogen) supplemented with 10% FCS (PAA, Linz,Austria) as previously reported in detail for FL-BP180[28]. Briefly, PCR primer pairs were synthesized byPerkin-Elmer/Applied Biosystems (Foster City, CA,USA). DNA sequence data of BP230–55 kD wereretrieved from EMBL DNA data bank (Cambridge,UK) based on the accession number M69225 [29]. The4516 bp fragment (nt 106–4599) encoding FL-BP180and the 1461 bp fragment (nt 5166–6604) encoding theBP230–55 kD fragment were cloned into linearizedpBHH2a (Invitrogen) resulting in the recombinanttransfer vectors pBHH77-FL and pBBH84-55, respec-tively. Sf21 insect cells were then co-transfected withpBHH77-FL or pBBH84-55 and linearized baculovirusAcMNPV DNA (Bac-N-Bluey, Invitrogen) using theliposome technique. Recombinant clones were puri-fied by plaque assay. The hexa-histidine taggedproteins were expressed by infecting the cells withrecombinant baculovirus for 2 days. As negative con-trol, Sf21 insect cells were co-transfected with therecombinant transfer vector p88H2C encoding a hexa-histidine peptide (negative control construct) and thelinearized baculovirus AcMNPV DNA (Bac-N-Bluey,Invitrogen) resulting in the expression of thehexa-histidine tag only. FL-BP180 expression wasdetected by IF microscopy or flow cytometry analysis.Alternatively, FL-BP180 and BP230–55 kD weredissolved in PBS without addition of denaturingagents, purified using Ni-NTA agarose (Qiagen,Hilden, Germany), and analysed by Western Blotting.

302 E. Schmidt et al.

Preparation of the soluble BP180 ectodomain(LAD-1)

HaCaT cells kindly provided by Dr N. Fusenig(DKFZ, Heidelberg, Germany) were grown in low-calcium keratinocyte growth medium (Clonetics,Wakersville, MD, USA) to 70–80% confluency [30].The 120 kD soluble ectodomain of BP180 (LAD-1) wasisolated from culture medium of HaCaT cells accord-ing to the protocol of Marinkovich et al. [31] withminor modifications [28].

Generation of recombinant GST-BP180 NC16A

BP180 NC16A (amino acids 490–562) was expressed inE. coli DH5� [10] (Figure 1). Expression was inducedby adding 1.5 mM isopropyl �-D-thiogalacto-pyranoside. After a 3-h incubation at 37°C, cells weresonicated and recombinant GST fusion proteinswere purified by glutathione agarose affinitychromatography [10].

Figure 3. The full-length recombinant BP180 has a type IIorientation in the Sf21 insect cells. Sf21 insect cells wereinfected with baculovirus encoding full-length BP180 (FL-BP180) or, as negative control, with baculovirus encoding ahexa-histidine peptide. FL-BP180- (grey columns) and nega-tive control construct-expressing cells (bright columns) werestained with mouse monoclonal antibodies (mAb) againstvarious epitopes on human BP180 and analysed by flowcytometry after visualization with FITC-labelled anti-mouseantibody. Both mAb233 directed against the C-terminalportion of BP180 and mAbHD18 directed against theNC16A domain stained Triton X-100-permeabilized (datanot shown) and intact FL-BP180-expressing Sf21 insect cells.In contrast, mAb1A8c directed against the intracellulardomain labelled permeabilized (+Triton X-100), but notintact FL-BP180-expressing Sf21 insect cells. mAb1E5directed to BP230 did not recognize permeabilized (+TritonX-100) or intact cells.

Figure 4. Full-length BP180 expressed in Sf21 insect cellsis recognized by mouse monoclonal antibodies directedagainst both intra- and extracellular epitopes on humanBP180. When Sf21 insect cells were infected with baculo-virus encoding full-length BP180 at a multiplicity of infec-tion of 4.0, about 30% cells were strongly stained afterfixation with acetone and incubation with various mousemonoclonal antibodies (mAb) to human BP180 (for detailssee Figure 1). Non- or weakly-stained cells served as inter-nal negative control. No staining was seen with mAb1E5directed against human BP230.

Western blotting

Recombinant proteins and conditioned concentratedHaCaT medium were subjected to 15%, 8%, and 6%SDS-PAGE, respectively, and transferred electropho-retically to nitrocellulose [10, 28, 31]. Nitrocellulosewas blocked with 1% (v/v) Tween-20 in PBS (154 mMNaCl, 20 mM KH2PO4, pH 7.6) for 1 h. Patient andrabbit sera were diluted 20- and 200-fold, respectively,in PBS; mAbs were diluted 100-fold if not otherwisestated. After a 2-h incubation at room temperature,blots were washed and stained with alkalinephosphatase-conjugated rabbit anti-human IgG,goat anti-rabbit IgG (both 1:10,000) or rabbit anti-mouse IgG (1:3,000; all Dianova, Hamburg, Germany).Reactivity was then visualized by the NBT/BCIPstaining system.

Simple and sensitive detection of anti-BP180 antibodies 303

Immunofluorescence microscopy of Sf21 insectcells

For IF microscopy, Sf21 insect cells were infected withbaculovirus encoding FL-BP180 and the negative con-trol construct, respectively, for 48 h. Afterwards, cellviability was determined by trypan blue staining.Infected Sf21 insect cells were then washed with coldPBS and centrifuged for 6 min and 600 rpm onto glasscoverslips (30,000 cells/coverslip) using the Cytospin2 centrifuge (Shandon, Runcorn, England). Cells werefixed with acetone (Ferak, Berlin, Germany) and 37%formaldehyde (FAD) plus 2% sucrose (both Merck,Darmstadt, Germany) at room temperature, respec-tively, or with −20°C cold methanol (Roth, Karlsruhe,Germany) at −20°C for 8 min. Subsequently, cellswere washed with PBS and dried. To reduce back-ground staining, human and rabbit sera were diluted1:20 and 1:100, respectively, in PBS and pre-adsorbedwith non-infected Sf21 insect cells at a concen-tration of 10×106 cells/ml between 30 min and 4 h.Pre-adsorbed human and rabbit sera as well as mAbs(diluted in PBS between 1:5 and 1:20,480) were incu-bated for 30 min at room temperature. After washingthree times in PBS, cells were stained with fluorescein-isothiocyanate (FITC)-labelled goat anti-human IgG(1:50; Sanofi Diagnostic Pasteur, Marnes la Coquette,France), swine anti-rabbit IgG, or rabbit anti-mouseIgG (both 1:20; Dako, Hamburg, Germany) for 30 minat room temperature in the dark. After wash-ing, fluorescence of cells was inspected using aZeiss Axiophoty fluorescence microscope (Frankfurt,Germany) or, in some cases, a confocal laser scan-ning microscope (Leica TSC, Leica Microsystems.Bensheim, Germany). Photographic images weretaken with identical exposure times and were docu-mented digitally or with FUJI Sensia 400 colourdiapositives.

Flow cytometry analysis

Aliquots of 3–4×105 infected Sf21 insect cells (seeabove) were analysed using the Coulter EPICS XLy

(Beckman Coulter, Krefeld, Germany). Infected Sf21insect cells were washed in ice-cold PBS. In someexperiments, cells were then treated with 0.5% TritonX-100 (Sigma) in PBS for 3 min. Human and rabbitsera were pre-adsorbed by incubation with non-infected Sf21 insect cells and, subsequently, cells werestained using the same dilutions, incubation times,and antibodies as detailed above for IF microscopy.Prior to flow cytometry analysis, propidium-iodide(10 �g/ml) was added to each sample of non-permeabilized cells in order to identify dead cells.

Detection of murine IgG

The IgG concentration of mouse mAbs in the hybri-doma supernatants was determined by conventionalenzyme-linked immunosorbent assay (ELISA). Briefly,

96-well round bottom plates (Greiner, Nurtingen,Germany) were coated with rabbit anti-mouse IgG(1:1,000; Dako). After over-night incubation at 4°Cand blocking with RPMI 1640 plus 10% FCS (PAA),hybridoma supernatant and mouse IgG standards(between 4,000 and 62.5 pg/ml; Sigma) were incu-bated in quadruplicate. As secondary antibody, horse-radish peroxidase-labelled rabbit anti-mouse IgG(1:2,500; Dako) was used. Bound antibodies werevisualized by staining with 1,2-o-phenylenediaminedihydrochloride (Dako) and by subsequent measure-ment of the optical density at 490 nm.

Results

Immunolocalization of FL-BP180 in Sf21 insectcells

Sf21 insect cells were infected by recombinant baculo-virus encoding FL-BP180, fixed with acetone, andlabelled with mouse mAb1A8c directed to the intra-cellular domain of human BP180 (see schematic dia-gram in Figure 1). Typical staining patterns taken atdifferent focal planes of a Sf21 insect cell by confocallaser scanning microscopy are shown in Figure 2. Inthe focal plane at the top of the cell, FL-BP180 expres-sion appeared as a central spot (Figure 2A). In the nextlower plane, BP180 expression was distributed homo-geneously over the cell surface except of a slightlyweaker staining in the central area (Figure 2B). In thefocal planes located closer to the glass coverslip, acircular staining pattern was observed (Figure 2C, D).These data can be taken as evidence that FL-BP180 isexpressed uniformly within the cell membrane of Sf21insect cells. Identical staining patterns were seen withmAb233 and mAbHD18 directed to the C- and N-terminus of the BP180 ectodomain, respectively (datanot shown).

In order to demonstrate that FL-BP180 is expressedas type II transmembrane protein (like in the humankeratinocyte), Sf21 insect cells were infected with theFL-BP180 construct or, alternatively, with a negativecontrol construct and incubated with mAbs 233,HD18, and 1A8c directed to BP180 (for details seeFigure 1) and 1E5 against BP230, respectively. Stainingwas then analysed by flow cytometry (Figure 3). It isevident that mAbs against the extracellular domain ofBP180 (i.e. 233, HD18) were bound, but not the mAbagainst the intracellular domain (1A8c). In contrast,when the cell membrane of the FL-BP180-expressinginsect cells was permeabilized prior to incubationwith mAb1A8c, the intracellular domain was labelled.Intact and permeabilized control cells did not exhibitany specific staining. Similarly, no labelling was seenwith mAb1E5 (targeting BP230) in both intact andpermeabilized FL-BP180-expressing Sf21 cells (Figure3). Taking these data together, it is evident thatFL-BP180 is expressed as type II transmembraneprotein in Sf21 insect cells.

304 E. Schmidt et al.

Detection limits of the eukaryoticimmunofluorescence assay

In order to quantitatively determine the sensitivity ofthe IF assay using FL-BP180-expressing Sf21 insectcells, binding of the mouse mAbs 233, HD18, and1A8c was studied at serial dilutions of 1:5 to 1:20,480.These antibodies bind to different intra- and extra-cellular epitopes of BP180 (Figure 1). The IgG concen-tration in the culture supernatants of mAb-producinghybridomas was measured to be 92.0±15.3 �g/ml(mAb233; mean±SD), 10.6±2.2 �g/ml (mAbHD18),and 40.9±6.4 �g/ml (mAb1A8c) by ELISA. Binding ofthe mAbs to FL-BP180-expressing Sf21 cells and tocells infected with the negative control construct wasexamined by indirect IF microscopy. While no stainingwas seen with the control cells (data not shown), themAbs labelled the FL-BP180-expressing Sf21 insectcells (Figure 4). Staining depended on the viabilityof the cells and on the subsequent fixation agent.Unspecific staining increased with decreasing cellviability, i.e. with the multiplicity of infection (MOI).Therefore, MOIs ranging from 0.125, 0.25, 0.5, 1.0, 2.0,4.0, and 8.0 and different fixation agents (−20°C coldmethanol, acetone, and formaldehyde) were tested.When a MOI of 4.0 was selected approximately 90%cells survived. After fixation with acetone about 30%of these cells exhibited a pronounced staining uponincubation with mAbs 233, HD18, and 1A8c (Figure4). With a MOI of 8.0, cell viability decreased to75–80%, but about 50% of the cells were labelled bythe mAbs. Using a MOI of 4.0, replacement of acetoneby methanol or formaldehyde did not further increasethe percentage of stained cells together with a lowerdetection limit as seen by serial dilutions of the mAbs(Table 1; data for methanol and formaldehyde notshown). Therefore, in subsequent experiments, cellsinfected with a MOI of 4.0 and fixed with acetonewere used.

In the following set of experiments, we comparedthe sensitivity of the eukaryotic IF assay with (1)indirect IF microscopy on salt-split skin, (2) WB ofFL-BP180 generated in Sf21 insect cells, and (3) WB ofrecombinant BP180 NC16A. The results are listed inTable 1. It is evident that the eukaryotic IF assay wasmore sensitive than the conventional assays. Based onthe IgG concentration in mAb preparations and thedata in Table 1, the detection limit of the eukaryotic IF

assay was calculated to be 72 ng/ml (mAb233), 17 ng/ml (mAbHD18), and 16 ng/ml (mAb1A8c).

Detection of circulating rabbit antibodies againstBP180 using the eukaryotic immunofluorescenceassay

Sera of rabbits immunized against recombinanthuman BP180 NC16A2-4 (R2296, R8009) and againstthe recombinant C-terminal fragment BP180 4575(R136) were tested for their reactivity with FL-BP180-expressing Sf21 insect cells. As shown in Figure 5,strong labelling of FL-BP180-expressing Sf21 insectcells was observed after incubation with R8009 andR136. No difference in the binding pattern was seenbetween the two rabbit sera (Figure 5). Non- orweakly-infected Sf21 insect cells in these cell prepar-ations, that represented about 70% of the fixed cells,were used as internal negative controls. In addition,the specificity of the antibody binding was tested by(1) incubation of FL-BP180-infected Sf21 cells withnormal rabbit serum (NRS) and serum of rabbitsimmunized against the GST moiety (RGST) and (2)incubation of negative control construct-infected cellswith the same serum samples (Figure 5). These find-ings obtained by indirect IF microscopy could beconfirmed by subjecting FL-BP180-expressing Sf21insect cells and negative control construct-infectedcells to flow cytometry analysis after incubation withrabbit sera. In contrast to the control cells, strongreactivity of FL-BP180-expressing Sf21 insect cells wasfound when incubated with R8009 and R136, but notwith RGST serum or NRS (Figure 5).

Table 1. Sensitivity of immunofluorescence (IF) microscopy of full-length (FL)-BP180-expressing Sf21insect cells compared with the sensitivity of current assays using serial dilutions of mouse monoclonalantibodies directed to various intra- and extracellular epitopes on BP180

Designation Immunofluorescence ImmunoblottingFL-BP1801 Salt-split skin FL-BP180 BP180 NC16A

233 1,280 1,280 20 naHD18 640 20 5 6401A8c 2,560 640 10,240 na

1Expressed in Sf21 insect cells fixed with acetone; na, not applicable; NC16A, extracellular portion of the 16thnon-collagenous domain of BP180.

Detection of circulating anti-BP180 antibodies inpatients with bullous pemphigoid, cicatricialpemphigoid, and pemphigoid gestationis

To further characterize the sensitivity and specificityof the Sf21-based IF assay 65 sera of BP patients withcirculating antibodies to the DEJ as detected byindirect IF microscopy on salt-split human skin wereexamined. As shown by screening experiments,unspecific staining occurred in about a third of humansera derived from both patients and healthy controls

Figure 5. Rabbit polyclonal antibodies against various epitopes on human BP180 and sera from patients with bullouspemphigoid recognized full-length BP180 (FL-BP180) using the Sf21-based immunofluorescence (IF) assay and flowcytometry. Sf21 insect cells were infected with baculovirus encoding FL-BP180 or with baculovirus containing a negativecontrol construct. Rabbit sera generated against BP180 NC16A (R8009) and BP180 4575 (R136) as well as sera from patientswith bullous pemphigoid (BP-1, BP-2) stained FL-BP180-expressing Sf21 insect cells as revealed by indirect IF microscopy (leftside of the IF panels) and flow cytometry analysis (grey columns). In contrast, staining was not observed with rabbit serumagainst the GST moiety (RGST) and normal human serum (NHS). Similarly, Sf21 insect cells infected with the negative controlconstruct (right side of the IF panels and bright columns, respectively) did not exhibit any staining.

306 E. Schmidt et al.

and was also seen with R2296 (directed against BP180NC16A). Unspecific IF reactivity varied considerablybetween the different sera and was present in bothFL-BP180-expressing Sf21 insect cells and cellsinfected with the control construct. However, whenthe sera were pre-incubated with non-infected Sf21insect cells, the unspecific background staining couldgreatly be reduced, particularly when the incubationtime was adjusted to 2 h (Figure 6). After pre-adsorption 58 out of 65 BP sera (89%) were reactive.When seven CP sera with circulating antibodies to theepidermal side of the artificial as observed by indirectIF on human salt-split skin and 16 PG sera were testedby the Sf21-based IF assay, six (86%) and 13 (81%)were reactive, respectively. In contrast, no reactivitywas observed with sera from patients with pemphigusvulgaris (PV; n=15), pemphigus foliaceus (PF; n=5),epidermolysis bullosa acquisita (EBA; n=5), atopicdermatitis (n=7), contact dermatitis (n=3), systemiclupus erythematosus (n=5) as well as with normalhuman sera (n=30).

Of the seven BP sera that were non-reactive in theSf21-based IF assay, five showed strong binding withthe 55 kD fragment of BP230 and two with the cell-derived soluble ectodomain of BP180 (LAD-1) asrevealed by immunoblotting. The 65 BP sera were alsoanalysed by immunoblotting with recombinant frag-ments of BP180 that have been previously used todetect circulating autoantibodies in BP patients (seefurther above). In addition, all sera were probed witha recombinant 55 kD C-terminal stretch of BP230.The data of these experiments are summarized inTable 2. Comparison of the data shows that indirect IFmicroscopy of FL-BP180-expressing Sf21 insect cellsappears to be the most sensitive tool for the detectionof circulating antibodies against BP180.

Figure 6. Pre-adsorption of BP sera with non-infected Sf21 insect cells reduced unspecific background immunofluorescence(IF) staining of negative control construct-infected Sf21 insect cells. Sf21 insect cells were infected with baculovirus encodingfull-length BP180 (FL-BP180) or a hexa-histidine peptide (negative control construct). Without pre-adsorption, the bullouspemphigoid (BP) serum strongly labelled FL-BP180-expressing Sf21 insect cells. However, unspecific staining is seen in cellsinfected with the negative control construct. After pre-adsorption of the BP serum with non-infected Sf21 insect cells, theserum still labelled FL-BP180-expressing insect cells but unspecific staining of negative control construct-infected cells wasgreatly reduced.

Table 2. Detection of antibodies to BP180 in 65 bullous pemphigoid sera that contained antibodies againstcomponents of the dermal-epidermal junction determined by the eukaryotic immunofluorescence assay andconventional assays

FL-BP180 NC16A1 LAD-11 BP230–55 kD1

Sf21 Insect cells2 Immunoblot

58/65 39/65 49/65 38/65 38/6589% 60% 75% 58% 58%

1Analysed by immunoblotting; 2detected by indirect immunofluorescence microscopy; FL-BP180, full-lengthBP180; NC16A, extracellular portion of the 16th non-collagenous domain of BP180; LAD-1, linear IgA diseaseantigen (cell-derived ectodomain of BP180); BP230–55 kD, 479 amino acid C-terminal stretch of BP230.

Reactivity of stored FL-BP180-expressing Sf21insect cells

FL-BP180-expressing Sf21 insect cells and cellsinfected with the negative control construct were fixed

Simple and sensitive detection of anti-BP180 antibodies 307

with acetone, −20°C methanol or FAD and stored at4–8°C or −20°C for 3 months. After thawing, cellswere incubated with mouse mAbs 233, HD18, and1A8c to BP180 (for details see Figure 1) as well as withrabbit antibody 8009 against BP180 NC16A at dilu-tions of 1:320 and 1:640. Reactivity was comparedwith cells that were prepared on the same day andfixed with acetone. IF reactivity was scored from‘+ + +’ for bright staining to ‘+/−’ for faint staining(Table 3). When cells were stored at 4–8°C, no or onlyweak staining was observed after incubation withmAbs or R8009. In contrast, reactivity to BP180 wasconserved in FL-BP180-expressing cells when storedat −20°C. Highest reactivity was seen after fixationwith acetone. However, the reactivity of acetone-fixedcells stored at −20°C was about one titre steplower compared with freshly prepared cells (Table 3).Similar results were obtained by the use of BP sera(data not shown).

Table 3. BP180 expression is conserved upon storage of full-length BP180-expressing Sf21 insect cells at−20°C for 3 months

Designation1 Fresh2 −20°C3 4–8°C3

Acetone Methanol FAD Acetone Methanol FAD

mAb233 + + +4 + + +/− +/− +/− − −mAbHD18 + +/− − − − − −mAb1A8c + + + + + + + − − −R8009 + + + + + + + − + − −

1Mouse monoclonal antibodies (mAb) 233, HD18, and 1A8c directed against various epitopes on human BP180(for details see Figure 1) and rabbit serum R8009 generated against the immunodominant 16thnon-collagenous domain (NC16A) of BP180 were tested at dilutions of 1:320 and 1:640; 2prepared, fixed withacetone, and stained on the same day; 3stored for 3 months after fixation with acetone, methanol, andformaldehyde (FAD), respectively; 4immunofluorescence reactivity was scored from ‘+ + +’ for bright to ‘+/−’for faint staining. When no staining was seen, the score was ‘−’.

Discussion

The data presented here show that the indirect IFmicroscopy of full-length BP180-expressing Sf21 insectcells meets the requirements of a highly sensitive,specific, and simple assay for the detection of circulat-ing anti-BP180 antibodies that may be used in anyroutine IF laboratory. This assay is more sensitive indetecting anti-BP180 antibodies than conventionalassays, such as indirect IF microscopy on humansalt-split skin, Western blotting of recombinant andcell-derived fragments of BP180, as well as Westernblotting of FL-BP180 produced in Sf21 insect cells.This conclusion can be drawn from the low detectionlimit of the eukaryotic IF assay as determined by serialdilutions of BP180-specific mouse monoclonal anti-bodies and from the reactivity with a large panel of BPand negative control sera. About 90% of BP sera, 86%of CP, and 81% of PG sera were found to be positiveprovided that unspecific background reactivity wasremoved by pre-incubation of the sera with non-infected Sf21 insect cells. Without pre-adsorption,unspecific IF reactivity was seen in about one-third ofhuman (both patients and healthy volunteers) as well

as in 1 of 3 rabbit sera and varied considerablybetween the different sera. Furthermore, backgroundIF staining increased with decreasing viability of Sf21insect cells that was observed when in screen-ing experiments increasing MOIs were used. Thisphenomenon may be due to the unspecific bind-ing of antibodies to proteins involved in cell death.Background reactivity could be greatly reduced by (1)using a MOI of 4.0, (2) pre-adsorption of sera withnon-infected Sf21 insect cells, and (3) controlling thepre-adsorption procedure by incubation of sera withcontrol construct-infected Sf21 insect cells.

In contrast to the Sf21-based IF assay, in the case ofimmunoblotting of recombinant BP180 NC16A andkeratinocyte-derived LAD-1, only 75% and 60% of BPsera, respectively, were reactive. Similar reactivity forBP180 NC16A and LAD-1 in BP sera was reported byother authors [9, 11, 20, 21, 28, 32]. The discrepancybetween the higher reactivity of BP sera with BP180NC16A (91%) described previously [11] and the BP180NC16A reactivity presented here, may be explained bythe different patient collective. This view is supportedby the finding that in two other studies (employingthe same recombinant protein and secondary anti-bodies than in the present study) 81% and 62% of BPsera were found to be reactive with BP180 NC16A [28,32]. Hata et al., using the recombinant ectodomain ofBP180 generated in insect cells, found about 85% of BPsera positive [33]. With the same technique, Haaseet al. found even 92% of sera positive [34]. However,the protein was extracted under strongly denaturingconditions; thus it is unlikely that the physiologicalconformation of human BP180 was maintained. Inaddition, only sera of patients with generalized BPwere tested [34]. It is clear that the BP180 ectodomaincan not cover the entire spectrum of anti-BP180 anti-bodies present in these patients since reactivity withthe intracellular domain of BP180 was demonstratedin about a third of BP patients [14, 28].

It is interesting to note that in five of seven BPpatients where the Sf21-based IF assay failed, the seracontained antibodies against BP230. This findingcould indicate that BP230-specific antibodies or anti-bodies against unknown antigens may cause separ-ation of the DEJ in some BP patients. Alternatively, the

308 E. Schmidt et al.

expression conditions of the insect cells may have tobe further improved in order to detect anti-BP180antibodies even in BP sera which were apparentlynon-reactive at present. This seems to be supported bythe finding that three of 15 PG sera were non-reactivein our assay. The remaining two BP sera that werenegative in our system lacked BP230-specific anti-bodies, but strongly reacted with LAD-1. This couldpoint to (some) imperfections in the authentic expres-sion of the FL-BP180 molecule. However, the highpercentage of reactive BP sera indicate that FL-BP180exhibits post-translational modifications and confor-mation, similar to those of the authentic protein. Thisis also corroborated by the finding that the Sf21 insectcells express FL-BP180 as type II transmembrane pro-tein as in human keratinocytes. The importance ofconformational epitopes has been demonstrated forthe binding of antibodies against desmoglein 3, thetarget antigen of pemphigus vulgaris, another auto-immune blistering skin disease [35, 36]. Moreover,proteins produced in eukaryotic expression systemshave been successfully employed for the detection ofantibodies in other autoimmune diseases such asinsulin-dependent diabetes mellitus, and rheumatoidarthritis [37].

For routine application of the eukaryotic assay inlaboratories without access to molecular biologicaltechnology it is interesting to note that in principle,FL-BP180 expression can be conserved (at least to alarge extent) when infected Sf21 insect cells were fixedwith acetone and stored at −20°C. Further improve-ment of the storage conditions is certainly requiredand should be addressed in the nearest future. In thelight of the data reported here, work in this directionseems to be justified because of the superior proper-ties of the Sf21-based IF assay in comparison toconventional assays using BP180 fragments. Certainly,application of FL-BP180 as target antigen by ELISAoffers several advantages (such as higher antigenconcentration, more convenient screening, and exactquantification of anti-BP180 antibodies during thecourse of the disease) compared to the Sf21-based IFassay. However, the FL-BP180 ELISA requires delicateextraction and purification procedures that maychange the conformation of FL-BP180. Furthermore,as noted above, whereas in the IF assay FL-BP180 isexpressed in its native position (like in the humankeratinocyte), the molecule is most likely uncoordinat-edly presented to the antibody in ELISA which candecrease the sensitivity as mentioned above.

Acknowledgements

This work was supported by grants from the Bundes-ministerium fur Bildung und Forschung (BMBF-16SV1329/0 to U.Z.) and the Interdisciplinary Centerfor Clinical Research at the University of Wurzburg,Germany (IZKF-01KS9603 to E.S.). We thank DrKatshushi Owaribe, Nagoya, Japan for providing uswith monoclonal antibodies 233 and 1A8c to BP180and 1E5 to BP230. We also thank Dr George J. Giudice,

Milwaukee, USA that kindly provided us with rabbitantibody 136 to GST-BP180 4575.

References1. Bernard P., Vaillant L., Labeille B., Bedane C., Arbeille

B., Denoeux J.P., Lorette G., Bonnetblanc J.M., Prost C.1995. Incidence and distribution of subepidermalautoimmune bullous skin diseases in three Frenchregions. Arch. Dermatol. 131: 48–52

2. Zillikens D., Wever S., Roth A., Hashimoto T., BrockerE.B. 1995. Incidence of autoimmune subepidermalblistering dermatoses in a region of central Germany.Arch. Dermatol. 131: 957–958

3. Stanley J.R., Hawley-Nelson P., Yuspa S.H., ShevachE.M., Katz S.I. 1981. Characterization of bullouspemphigoid antigen: a unique basement membraneprotein of stratified squamous epithelia. Cell 24:897–903

4. Labib R.S., Anhalt G.J., Patel H.P., Mutasim D.F., DiazL.A. 1986. Molecular heterogeneity of the bullouspemphigoid antigens as detected by immunoblotting.J. Immunol. 136: 1231–1235

5. Gaucherand M., Nicolas J.F., Paranhos Baccala G.,Rouault J.P., Reano A., Magaud J.P., Thivolet J., JolivetM., Schmitt D. 1995. Major antigenic epitopes ofbullous pemphigoid 230 kDa antigen map within theC-terminal end of the protein. Evidence using a55 kDa recombinant protein. Br. J. Dermatol. 132:190–196

6. Skaria M., Jaunin F., Hunziker T., Riou S., SchumannH., Bruckner-Tuderman L., Hertl M., Bernard P., SauratJ.H., Favre B., Borradori L. 2000. IgG autoantibodiesfrom bullous pemphigoid patients recognize multipleantigenic reactive sites located predominantly withinthe B and C subdomains of the COOH-terminus ofBP230. J. Invest. Dermatol. 114: 998–1004

7. Zillikens D., Giudice G.J. 1999. BP180/type XVIIcollagen: its role in acquired and inherited disorders orthe dermal-epidermal junction. Arch. Dermatol. Res.291: 187–194

8. Borradori L., Sonnenberg A. 1999. Structure andfunction of hemidesmosomes: more than simpleadhesion complexes. J. Invest. Dermatol. 112: 411–418

9. Matsumura K., Amagai M., Nishikawa T., HashimotoT. 1996. The majority of bullous pemphigoid andherpes gestationis serum samples react with theNC16a domain of the 180-kDa bullous pemphigoidantigen. Arch. Dermatol. Res. 288: 507–509

10. Zillikens D., Rose P.A., Balding S.D., Liu Z.,Olague-Marchan M., Diaz L.A., Giudice G.J. 1997.Tight clustering of extracellular BP180 epitopesrecognized by bullous pemphigoid autoantibodies.J. Invest. Dermatol. 109: 573–579

11. Zillikens D., Mascaro J.M., Rose P.A., Liu Z., EwingS.M., Caux F., Hoffmann R.G., Diaz L.A., Giudice G.J.1997. A highly sensitive enzyme-linkedimmunosorbent assay for the detection of circulatinganti-BP180 autoantibodies in patients with bullouspemphigoid. J. Invest. Dermatol. 109: 679–683

12. Giudice G.J., Emery D.J., Zelickson B.D., Anhalt G.J.,Liu Z., Diaz L.A. 1993. Bullous pemphigoid andherpes gestationis autoantibodies recognize a commonnon-collagenous site on the BP180 ectodomain.J. Immunol. 151: 5742–5750

Simple and sensitive detection of anti-BP180 antibodies 309

13. Egan C.A., Taylor T.B., Meyer L.J., Petersen M.J., ZoneJ.J. 1999. Bullous pemphigoid sera that containantibodies to BPAg2 also contain antibodies toLABD97 that recognize epitopes distal to the NC16Adomain. J. Invest. Dermatol. 112: 148–152

14. Perriard J., Jaunin F., Favre B., Budinger L., Hertl M.,Saurat J.H., Borradori L. 1999. IgG autoantibodiesfrom bullous pemphigoid (BP) patients bind antigenicsites on both the extracellular and the intracellulardomains of the BP antigen 180. J. Invest. Dermatol. 112:141–147

15. Liu Z., Diaz L.A., Troy J.L., Taylor A.F., Emery D.J.,Fairley J.A., Giudice G.J. 1993. A passive transfermodel of the organ-specific autoimmune disease,bullous pemphigoid, using antibodies generatedagainst the hemidesmosomal antigen, BP180. J. Clin.Invest. 92: 2480–2488

16. Schmidt E., Obe K., Brocker E.B., Zillikens D. 2000.Serum levels of autoantibodies to BP180 correlate withdisease activity in patients with bullous pemphigoid.Arch. Dermatol. 136: 174–178

17. Kelly S.E., Wojnarowska F. 1988. The use of chemicallysplit tissue in the detection of circulating anti-basement membrane zone antibodies in bullouspemphigoid and cicatricial pemphigoid. Br. J. Dermatol.118: 31–40

18. Zillikens D. 1999. Acquired skin disease ofhemidesmosomes. J. Dermatol. Sci. 20: 134–154

19. Balding S.D., Prost C., Diaz L.A., Bernard P., BedaneC., Aberdam D., Giudice G.J. 1996. Cicatricialpemphigoid autoantibodies react with multiple siteson the BP180 extracellular domain. J. Invest. Dermatol.106: 141–146

20. Nakatani C., Muramatsu T., Shirai T. 1998.Immunoreactivity of bullous pemphigoid (BP)autoantibodies against the NC16A and C-terminaldomains of the 180 kDa BP antigen (BP180):immunoblot analysis and enzyme-linkedimmunosorbent assay using BP180 recombinantproteins. Br. J. Dermatol. 139: 365–370

21. Husz S., Kiss M., Molnar K., Marczinovits I.,Molnar J., Toth G.K., Dobozy A. 2000. Developmentof a system for detection of circulating antibodiesagainst hemidesmosomal proteins in patients withbullous pemphigoid. Arch. Dermatol. Res. 292:217–224

22. Zillikens D., Kawahara Y., Ishiko A., Shimizu H.,Mayer J., Rank C.V., Liu Z., Giudice G.J., Tran H.H.,Marinkovich M.P., Brocker E.B., Hashimoto T. 1996.A novel subepidermal blistering disease withautoantibodies to a 200-kDa antigen of the basementmembrane zone. J. Invest. Dermatol. 106: 1333–1338

23. Gammon W.R. 1987. In Immunopathology of the skinE.H. Beutner, T.P. Chorzelski, V. Kumar, eds. JohnWiley and Sons, New York, pp. 323–336

24. Leverkus M., Schmidt E., Lazarova Z., Brocker E.B.,Yancey K.B., Zillikens D. 1999. Antiepiligrin cicatricialpemphigoid: an underdiagnosed entity within thespectrum of scarring autoimmune subepidermalbullous diseases? Arch. Dermatol. 135: 1091–1098

25. Balding S.D., Diaz L.A., Giudice G.J. 1997. Arecombinant form of the human BP180 ectodomainforms a collagen-like homotrimeric complex.Biochemistry 36: 8821–8830

26. Hirako Y., Usukura J., Uematsu J., Hashimoto T.,Kitajima Y., Owaribe K. 1998. Cleavage of BP180, a180-kDa bullous pemphigoid antigen, yields a 120-kDacollagenous extracellular polypeptide. J. Biol. Chem.273: 9711–9717

27. Pohla-Gubo G., Lazarova Z., Giudice G.J., Liebert M.,Grassegger A., Hintner H., Yancey K.B. 1995.Diminished expression of the extracellular domain ofbullous pemphigoid antigen 2 (BPAG2) in theepidermal basement membrane of patients withgeneralized atrophic benign epidermolysis bullosa.Exp. Dermatol. 4: 199–206

28. Kromminga A., Scheckenbach C., Georgi M., Hagel C.,Arndt R., Christophers E., Brocker E.B., Zillikens D.2000. Patients with bullous pemphigoid and linear IgAdisease show a dual IgA and IgG autoimmuneresponse to BP180. J. Autoimmun. 15: 293–300

29. Sawamura D., Li K., Chu M.L., Uitto J. 1991. Humanbullous pemphigoid antigen (BPAG1). Amino acidsequences deduced from cloned cDNAs predictbiologically important peptide segments and proteindomains. J. Biol. Chem. 266: 17784–17790

30. Boukamp P., Petrussevska R., Breitkreuz D., HornungJ., Markham A., Fusenig N.E. 1988. Normalkeratinization in a spontaneously immortalizedaneuploid human keratinocyte cell line. J. Cell. Biol.106: 761–771

31. Marinkovich M.P., Taylor T.B., Keene D.R., BurgesonR.E., Zone J.J. 1996. LAD-1, the linear IgA bullousdermatosis autoantigen, is a novel 120-kDa anchoringfilament protein synthesized by epidermal cells.J. Invest. Dermatol. 106: 734–738

32. Schumann H., Baetge J., Tasanen K., Wojnarowska F.,Schacke H., Zillikens D., Bruckner-Tuderman L. 2000.The shed ectodomain of collagen XVII/BP180 istargeted by autoantibodies in different blistering skindiseases. Am. J. Pathol. 156: 685–695

33. Hata Y., Fujii Y., Tsunoda K., Amagai M. 2000.Production of the entire extracellular domain of BP180(type XVII collagen) by baculovirus expression.Dermatol. Sci. 23: 183–190

34. Haase C., Budinger L., Borradori L., Yee C., Merk H.F.,Yancey K., Hertl M. 1998. Detection of IgGautoantibodies in the sera of patients with bullous andgestational pemphigoid: ELISA studies utilizing abaculovirus-encoded form of bullous pemphigoidantigen 2. J. Invest. Dermatol. 110: 282–286

35. Amagai M., Klaus-Kovtum V., Stanley J.R. 1991.Autoantibodies against a novel epithelial cadherin inpemphigus vulgaris, a disease of cell adhesion. Cell 67:869–877

36. Ishii K., Amagai M., Hall R.P., Hashimoto T.,Takayanagi A., Gamou S., Shimizu N., Nishikawa T.1997. Characterization of autoantibodies in pemphigususing antigen-specific enzyme-linked immunosorbentassays with baculovirus-expressed recombinantdesmogleins. J. Immunol. 159: 2010–2017

37. Kerokoski P., Ilonen J., Gaedigk R., Dosch H.M., KnipM., Hakala M., Hinkkanen A. 1999. Production of theislet cell antigen ICA69 (p69) with baculovirusexpression system: analysis with a solid-phasetime-resolved fluorescence method of sera frompatients with IDDM and rheumatoid arthritis.Autoimmunity 29: 281–289