Autoantibodies against the extracellular domain of bullous pemphigoid antigen 2 (BPAG2) are thought to play a key role in the pathogenesis of bullous ...
Detection of IgG Autoantibodies in the Sera of Patients with Bullous and Gestational Pemphigoid: ELISA Studies Utilizing a Baculovirus-Encoded Form of Bullous Pemphigoid Antigen 2 Claudia Haase, Lioba Bu¨dinger, Luca Borradori,*† Carole Yee,* Hans F. Merk, Kim Yancey,* and Michael Hertl Hautklinik der RWTH Aachen, Germany; *Dermatology Branch, National Cancer Institute, Bethesda, Maryland, U.S.A.; and †Clinique de Dermatologie, Hoˆpital Cantonal Universitaire, Geneva, Switzerland
Autoantibodies against the extracellular domain of bullous pemphigoid antigen 2 (BPAG2) are thought to play a key role in the pathogenesis of bullous pemphigoid and their detection may thus be of diagnostic and prognostic value. The aim of this study was to develop a standardized enzyme-linked immunosorbent assay utilizing the baculovirus-derived protein BV13 (extracellular domain of BPAG2 devoid of 68 amino acids at the C terminus linked to glutathione-S-transferase and 6x His tag) to detect BPAG2-specific autoantibodies. For the enzyme-linked immunosorbent assay, nickel agarose affinity-purified BV13 protein was incubated with sera from patients with bullous pemphigoid (n J 39), gestational pemphigoid (n J 10), and pemphigus vulgaris/ pemphigus foliaceus (PV/PF; n J 15), or normal human sera (NHS; n J 18). Nickel affinity-purified proteins from wild-type baculovirus-infected insect cells served
as a control. A positive enzyme-linked immunosorbent assay value was defined as reactivity (ODBV13 – ODWT) > mean reactivity F 1 SD of the negative control sera (PV/ PF; NHS). Thirty-five of 39 bullous pemphigoid sera and 10 of 10 gestational pemphigoid sera were reactive to BPAG2 compared with none of 15 PV/PF sera and one of 18 NHS (sensitivity, 91.8%; specificity, 97%). Of 16 BPAG2-reactive sera in the enzyme-linked immunosorbent assay, only six were BPAG2-reactive in the western blot, whereas 14 sera immunoprecipitated BPAG2 from extracts of epidermal keratinocytes. The enzyme-linked immunosorbent assay utilizing an eukaryotic BPAG2 protein thus seems to be highly sensitive and specific in the detection of BPAG2-specific antibodies and, hence, may be useful in the diagnosis of bullous autoimmune diseases, such as bullous pemphigoid and gestational pemphigoid. Key words: autoimmunity/B lymphocytes/hemidesmosome. J Invest Dermatol 110:282–286, 1998
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cytoplasmatic plaque constituent of these complexes, belongs to a family of intermediate filament binding proteins and has been shown to associate with intermediate filaments in vitro (Green et al, 1992; Yang et al, 1996). In contrast, BPAG2 is a type II transmembrane protein that may serve as a cell matrix receptor for a yet unrecognized ligand within the extracellular matrix (Giudice et al, 1992, 1993, 1994; Hopkinson et al, 1992; Masunaga et al, 1997). The extracellular COOH-terminal half contains 15 interrupted collagenous domains that may form collagen-like triple helices (Giudice et al, 1992; Hirako et al, 1996). Support for the idea that BPAG2 plays an important role in promoting cell adhesion and the assembly of hemidesmosomes derives from transfection studies (Hopkinson et al, 1995; Borradori et al, 1996) and from studies of patients with generalized atrophic benign epidermolysis bullosa, an inherited skin blistering disorder characterized by defective dermo–epidermal adhesion and the formation of rudimentary hemidesmosomes (McGrath et al, 1995; Jonkman et al, 1995; Darling et al, 1997). Although almost all autoantibodies from BP sera target BPAG1, and BPAG2 in approximately half of the cases, their importance in the pathogenesis of the disease has remained unclear. Recent studies have shown that rabbit antibodies raised against a major immunodominant site of the extracellular domain (ECD) of BPAG2 adjacent to the membrane spanning region, are able to induce subepidermal blisters, via complement activation, when injected into neonatal mice (Liu et al, 1993, 1995). These observations strongly suggest that autoantibodies against BPAG2 are pathogenic and are responsible for dermo–epidermal separation in BP. The purpose of this study was to develop an enzyme-linked
ullous pemphigoid (BP) is an autoimmune blistering disorder characterized by subepidermal bullae and circulating autoantibodies directed against the basement membrane zone of stratified squamous epithelia (Beutner et al, 1968; Stanley et al, 1981). Previous studies have demonstrated that these autoantibodies react predominantly with two components of hemidesmosomes, bullous pemphigoid antigen 1 (BPAG1, also termed BP230) and bullous pemphigoid antigen 2 (BPAG2, also BP180 or type XVII collagen) (Stanley et al, 1981; Labib et al, 1986; Morrison et al, 1988). Hemidesmosomes are multiprotein complexes that mediate adhesion of epithelial cells to the underlying basement membrane and connect elements of the cytoskeleton to the extracellular matrix in skin and other stratified epithelia (Yancey, 1995; Borradori and Sonnenberg, 1996; Green and Jones, 1996). BPAG1, a
Manuscript received July 29, 1997; revised October 28, 1997; accepted for publication November 13, 1997. Reprint requests to: Dr. Michael Hertl, Hautklinik der RWTH, Pauwelsstr. 30, D-52074 Aachen, Germany. Abbreviations: BP, bullous pemphigoid; BPAG1, bullous pemphigoid antigen 1 (BP230); BPAG2, bullous pemphigoid antigen 2 (BP180 or collagen XVII); BV13, baculoprotein containing the ECD of BPAG2 devoid of 68 amino acids at the carboxyl terminus linked to glutathione-S-transferase and 6x His tag; ECD, extracellular domain; GST, glutathione-S-transferase; IP, immunoprecipitation; MP, milk powder; NHS, normal human serum; PF, pemphigus foliaceus; PG, gestational pemphigoid; PV, pemphigus vulgaris; WT, wild-type.
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immunosorbent assay (ELISA) for the detection of BPAG2-specific autoantibodies in patients with BP, utilizing an eukaryotic recombinant protein consisting of the ECD of BPAG2 produced in a baculovirus expression system. This eukaryotic expression system generates recombinant proteins with post-translational modifications that are frequently identical to those of the authentic protein (Kidd and Emery, 1993). To evaluate the sensitivity and specificity of the ELISA using this recombinant eukaryotic BPAG2 protein, the reactivity of a panel of sera from patients with BP was compared with those of sera from patients with gestational pemphigoid (PG), a disease closely related to BP (Morrison et al, 1988; Jenkins et al, 1995), sera from patients with pemphigus vulgaris (PV) and pemphigus foliaceus (PF), two bullous autoimmune skin diseases not related to BP (Amagai et al, 1994), and sera from healthy donors. MATERIALS AND METHODS Patients and controls Serum samples were obtained from adult patients with generalized BP (n 5 39), PG (n 5 10), PV/PF (n 5 15), and healthy volunteers (n 5 18). The clinical diagnosis of BP and PG was confirmed by histopathology (subepidermal blisters) and direct immunofluorescence microscopy (IgG deposits at the dermal–epidermal junction of involved skin). In addition, these sera contained circulating anti-basement membrane zone IgG upon analysis in one of the following in vitro tests: (i) indirect immunofluorescence microscopy (IgG binding to the epidermal site of 1 M NaCl separated normal human skin) (BP, n 5 17; PG, n 5 4), (ii) western blots with extracts from epidermal keratinocytes (BP, n 5 6), or (iii) immunoprecipitation with biosynthetically radiolabeled extracts from epidermal keratinocytes (BP, n 5 16; PG, n 5 6). All of the 15 PV and PF patients with typical histopathology and positive direct immunofluorescence of involved skin had circulating autoantibodies reacting with the surface of epithelial cells of monkey esophagus. Generation of recombinant BV13 (baculoprotein containing the ECD of BPAG2 devoid of 68 amino acids at the carboxyl terminus linked to glutathione-S-transferase and 6x His tag) baculovirus and expression and purification of recombinant human BPAG2 protein cDNA clones corresponding to the ECD of BPAG2 were generated by reverse transcriptasepolymerase chain reaction using total RNA from cultured human keratinocytes (Masunaga et al, 1997) with primers based on the published sequence of human BPAG2 (Giudice et al, 1992). A cDNA fragment corresponding to BPAG2 nucleotides 1561–2562 and another to nucleotides 2518–4630 were digested with Cla1, subcloned into pBluescript II SK1 (Stratagene, La Jolla, CA) via a three way ligation and sequenced by the dideoxynucleotide method of Sanger to ensure that the BPAG2 cDNA did not contain any errors. The BPAG2 insert (nucleotides 1561–4630) was subcloned into the baculovirus transfer vector pAcGHLT-A (Pharmingen, San Diego, CA). This vector expresses the glutathione S-transferase (GST) affinity tag and the 6x His tag. Due to a single base substitution (C→T) at nucleotide 4396 resulting at a stop codon at this site near the 39 end of this cDNA, the baculovirus-encoded recombinant of the BPAG2 ectodomain used in these ELISA studies, called BV13, lacked 68 amino acids at its carboxyl terminus. Infectious virus was made using the BaculoGold expression system (Pharmingen). Recombinant BV13 baculovirus was amplified in spodoptera frugiperda insect cells using a previously described protocol (Amagai et al, 1994). For the production of BV13 protein, 30 3 106 spodoptera frugiperda insect cells were inoculated in 175 cm2 culture flasks (Falcon, Oxnard, CA) with culture supernatants containing recombinant baculovirus at a multiplicity of infection greater than 10. Baculovirus-infected insect cells were harvested after 3 d and were lysed for the isolation of intracellular BV13 protein. In brief, 10 3 106 insect cells were suspended in 1 ml of icecold lysis buffer (Pharmingen) supplemented with 6 M urea for 1 h. After centrifugation of the lyzed insect cells (2000 3 g, 10 min), supernatants with solubilized BV13 protein were removed from the pellets. The lysis buffer was then exchanged with binding buffer of a Nickel-NTA agarose column (Quiagen, Hilden, Germany) by centrifugation in centriprep-30 vials (Amicon, Beverly, MA). BV13 protein was purified over nickel-NTA agarose following the manufacturer’s instructions and was finally gradually dialyzed against phosphatebuffered saline (PBS) at 4°C for 48 h. The amount of BV13 protein was analyzed by a commercial kit (Bio-Rad, Munich, Germany). The molecular weight and immunoreactivity of BV13 was determined by western blot with a recently described polyclonal rabbit anti-human BPAG2 antibody (Masunaga et al, 1997) and sera from representative patients with BP, and normal rabbit and human sera served as negative controls. Western blot of recombinant BPAG2 protein Purified boiled BV13 protein was run on a 10% tris-glycine gel (Bio-Rad) by sodium dodecyl sulfate polyacrylamide gel electrophoresis and was then blotted onto nitrocellulose
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(Bio-Rad). The nitrocellulose membrane was incubated with BP sera diluted at 1:20–1:50 in PBS with 5% fat-free milk powder (MP) and 0.05% Tween 20 (all from Bio-Rad) overnight at room temperature. After three washes with PBS/0.05% Tween, the blotted BV13 protein was incubated with a second, alkaline phophatase-labeled goat anti-human IgG (H 1 L) antibody (Zymed, San Francisco, CA) at 1:1000 in PBS/5% MP/0.05% Tween for 2 h at room temperature. Binding of the second antibody was visualized by incubation of the nitrocellulose membrane with 5-bromo-4-chloro-3-indolyl-phosphate and nitroblue tetrazolium as a substrate (Kirkegaard and Perry, Gaithersburg, MD) for 20 min. ELISA with recombinant BPAG2 protein Nickel agarose-purified BV13 and wild-type (WT) baculoproteins (2 µg per well) were immobilized on 96 well polystyrene plates (Poly Sorb, Nunc Intermed, Wiesbaden, Germany) by overnight incubation at 4°C. Wells were blocked with PBS/10% MP/0.05% Tween for 2 h at 37°C and were then washed three times with PBS/0.05% Tween. Sera from BP patients and controls (uniformly diluted at 1:20 in PBS/ 10% MP) were added in triplicate and were incubated overnight at 4°C. After three washes with PBS/0.05% Tween, a polyclonal alkaline phosphatase-labeled goat anti-human IgG (H 1 L) antibody (Zymed) diluted in PBS/5% MP at 1:5000 was added to each well for 2 h at 37°C. After washing again as above, each well received 100 µl of a substrate solution (p-nitrophenylphosphate; Kirkegaard and Perry). The optical density (OD) was determined at 405 nm after 15 min using a Spectramax 250 microplate reader (Molecular Device, Sunnydale, CA). All extinction data were standardized based on the negative internal standards, which were set to zero OD units. In preliminary experiments, several BP sera (n 5 10) were also incubated with immobilized GST protein as a control to BV13 leading only to minimal binding to GST protein (ODGST , 0.1). To exclude the possibility that the serum reactivity to BV13 protein in the ELISA was influenced by reactivity to proteins other than the GST-tagged BV13 protein expressed by baculovirus-infected insect cells, specific reactivity to BPAG2 was defined as reactivity to BV13 (ODBV13) minus reactivity to nickel affinity-purified proteins from insect cells infected with WT baculovirus (ODWT). The background reactivity of sera with WT protein in the ELISA (ODWT) ranged from 0.1 to 0.35. Twenty-three of 39 sera from patients with BP and ten of 15 sera from normal volunteers were analyzed and scored against known controls in a completely blinded manner to assess the specificity of the ELISA. Immunoprecipitation (IP) with biosynthetically radiolabeled protein extracts from epidermal keratinocytes Subconfluent monolayers of normal human keratinocytes were biosynthetically radiolabeled for 2 h with [35S]methionine (59 µCi per ml; specific activity approximately 1100 Ci per mmol, New England Nuclear, Boston, MA) in methionine-free media. Radiolabeled human keratinocyte extracts were processed as described previously and studied by immunoprecipitation using sera from patients and normal controls as described previously (Stanley et al, 1981; Basset-Seguin et al, 1990). Immunoprecipitation samples were studied by polyacrylamide gel electrophoresis and fluorography. Statistical analysis The plate-to-plate variability of the assay and the dayto-day variability of the assay fell into a range of less than 10% (data not shown). The ELISA results obtained from the two negative control groups of sera [PV/PF, normal human sera (NHS)] were normalized using the Box–Cox transformation (Box and Cox, 1964). The transformed data were used to compute the specificity of the ELISA. Significant differences between the extinction data with BP sera and NHS in the ELISA were calculated with the Wilcoxon test (SAS system).
RESULTS BPAG2-specific autoantibodies are detected in the sera of BP and PG patients by ELISA Sera from patients with BP (n 5 39), PG (n 5 10), PV/PF (n 5 15), and NHS (n 5 18) were incubated (at a dilution of 1:20) with BV13 protein (ECD of BPAG2) immobilized onto ELISA plates. Binding of BPAG2-specific antibodies was visualized by the formation of a yellow dye catalyzed by a second, AP-labeled goat anti-human antibody. Proteins from WT baculovirus-infected insect cells that were also purified over nickel agarose served as a control. The BPAG2-specific serum reactivity of each sample was determined by subtracting the reactivity against the WT baculovirus from its reactivity versus BV13. The results of these analyses are shown in Fig 1. By the Wilcoxon test, differences in the BPAG2 reactivities were highly significant (p , 0.0001) between sera from BP/PG patients and control sera (including all of the NHS and PV/PF sera). The cutoff value of the ELISA was set at an ODBV13-WT value of 0.11 that was equal to the mean value of the negative control sera 1 1 SD.
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Table II. Comparison of BPAG2-specific serum reactivities (ELISA) and severity of skin disease in patients with BP
Figure 1. Scatter blot of reactivities of sera from patients with BP, PG, PV/PF, and NHS in the ELISA with BV13 protein (BPAG2). Immobilized BV13 protein (ECD of BPAG2) was incubated with sera (diluted at 1:20) from adult patients with BP, PG, PV/PF, and NHS. Binding of serum antibodies to immobilized BV13 was visualized by incubation with nitroblue tetrazolium and 5-bromo-4-chloro-3-indolyl-phosphate after incubation with a second, APconjugated goat anti-human IgG (1:5000) and was expressed as OD at 405 nm. By the Wilcoxon test, differences in the BPAG2 reactivities were highly significant (p , 0.0001) between sera from BP/PG patients and control sera (NHS and PV/PF sera). The cut-off of the ELISA was set at an ODBV13–WT value of 0.11 that was equal to the mean value of the negative control sera 1 1 SD. This cut-off is indicated by the spaced line.
Table I. Sensitivities of ELISA and the western blot of BV13 (BPAG2) protein and immunoprecipitation with epidermal keratinocyte extracts in the detection of BPAG2-specific autoantibodies in sera of patients with BPa Patient
ELISA (BV13)
Western blot (BV13)
IP (BPAG2)
BP2 BP3 BP8 BP10 BP11 BP18 BP20 BP21 BP22 BP23 BP25 BP26 BP27 BP28 BP29 BP30
1:400* 1:400 1:400 1:200 1:200 1:50 1:20 1:20 1:20 1:20 1:20 1:20 1:100 1:200 1:200 1:200
pos. pos. pos. neg. pos. pos. neg. neg. neg. neg. neg. neg. neg. pos. neg. neg.
BPAG2 BPAG1, BPAG2 BPAG1, BPAG2 BPAG2 BPAG2 BPAG1, BPAG2 BPAG2 BPAG2 BPAG1, BPAG2 BPAG1, BPAG2 BPAG2 BPAG1, BPAG2 BPAG2 BPAG2 BPAG1 BPAG1
aBV13,
extracellular portion of BPAG2; IP, immunoprecipitation with biosynthetically radiolabeled BPAG1/BPAG2 antigen from epidermal keratinocyte extracts; *, antibody titers.
Using this cut-off value, 35 of 39 BP sera (89.7%) and 10 of 10 PG sera (100%) reacted specifically with BV13 protein (BPAG2) and only one of 18 NHS and none of 15 PV/PF sera reacted in the ELISA. A higher cut-off value (i.e., mean of negative sera 1 2 SD) led to false negative results (i.e., exclusion of three BP and one PG sera, which definitively contained BPAG2-specific autoantibodies, as determined by IP with epidermal extracts) without much improvement of the specificity, and was therefore not applied. Differential sensitivities of BV13-ELISA, western blot of BV13 protein, and IP with extracts from epidermal keratinocytes in the detection of BPAG2-specific antibodies Sera of 16 adult patients with BP that were reactive in the ELISA with BV13 (BPAG2) were also tested in the western blot of BV13 protein and the IP with biosynthetically radiolabeled epidermal keratinocyte extracts to compare the sensitivities of these in vitro tests (Table I). Fourteen of 16 (87.5%)
Patient
Disease activitya
BP1 BP2 BP3 BP4 BP5 BP6 BP7 BP8 BP9 BP10 BP11 BP12 BP13 BP14 BP15 BP16 BP17 BP18 BP19 BP20 BP21 BP22 BP23 BP24 BP25 BP26 BP27 BP28
1 1 1 1 1 1 1 1 1 – 1 – 1 – 1 1 1 1 1 1 1 1 1 1 – 1 1 1
1 1 1 1 1 1 1 1 1
1 1 1 1 1 1 1 1 1
BPAG2-antibody titer
(trunk) (trunk, arms) (abdomen, arms) (trunk) (trunk, extremities) (abdomen, arms) (trunk, extremities) (trunk) (trunk, extemities)
1 (upper trunk) 1 1 (trunk, extremities) (arms) (trunk) 1 1 (trunk) 1 (trunk) (trunk) 1 1 (trunk) (oral erosions, neck) (arms) 1 1 (trunk, legs) 1 1 (trunk) 1 (trunk, legs) 1 1 (trunk, extremities) 1 (trunk, extremities)
.1:400 1:400 .1:400 1:400 .1:400 1:400 1:400 1:400 1:200 1:200 1:200 1:200 1:200 1:100 1:50 1:50 1:50 1:50 1:50 1:20 1:20 1:20 1:20 1:20 1:20 1:20 1:100 1:200
a1 1 1, disseminated bullae; 1 1, bullae at limited areas; 1, few bullae or erosions; –, no skin lesions.
BP sera reactive in the ELISA of BV13 (BPAG2) also showed reactivity for BPAG2 in the IP. In contrast, only six of these 16 (37.5%) BP sera reacted in the western blot of BV13. To investigate whether the lower sensitivity of the western blot was caused by further denaturation (upon boiling) of the recombinant protein, nine BPAG2-reactive BP sera were analyzed in the ELISA with nonboiled versus boiled immobilized BV13 protein (2 µg per well). In the ELISA with boiled BV13 protein, reactivities of five BP sera with BPAG2-specific antibody titers of 1:400 (as determined in the ELISA with nonboiled BV13) were uniformly reduced to titers of ,1:100, whereas BPAG2-specific reactivities of four additional BP sera (antibody titers of 1:100 in the ELISA with nonboiled BV13) could not be detected at a dilution of 1:20. Together, these results suggest a correlation between the results of ELISA and IP, and indicate that the western blot of BV13 is less sensitive than the ELISA in detecting BPAG2-specific autoantibodies. Titration of BPAG2-specific antibodies in the sera of BP and PG patients The ELISA with BV13 protein was also performed with serial dilutions (1:20–1:400) of BP sera to quantitatively determine serum reactivity to BPAG2. Titers of BPAG2-specific antibodies were compared with the extent of skin disease in 28 adult BP patients whose clinical course of disease was well characterized (Table II). Twelve of 19 BP patients with extensive skin disease had high titers of antibodies against BPAG2 (1:200–ù1:400). In contrast, six of 19 BP patients with extensive skin disease had low BPAG2-specific antibody titers (1:20– 1:50); one severely affected BP patient had BPAG2-specific antibody titers of 1:100. Six of nine sera from BP patients with few or no skin symptoms had low titers (1:20–1:50) of BPAG2-specific antibodies whereas three BP patients with no skin symptoms had BPAG2-specific antibody titers of 1:100–1:200. DISCUSSION In this study, we have developed an ELISA utilizing a baculovirusencoded recombinant protein consisting of the ECD of human BPAG2 to detect autoantibodies against BPAG2, one of the two major autoantigens of BP. The eukaryotic baculovirus expression system was
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chosen because it generates recombinant proteins that frequently exhibit post-translational modifications and conformation similar to those of the authentic protein (Kidd and Emery, 1993). Using this ELISA, antibody reactivity to BPAG2 was detected in the majority (89.7%) of sera from adult patients with BP and in 100% of the sera from patients with PG, a bullous autoimmune disease closely related to BP (Morrison et al, 1988; Jenkins et al, 1995). In addition, our results indicate that this ELISA is also highly specific in that only one of 33 negative control sera (PV/PF sera; NHS) fell within the positive range in this test. Previous studies using western blot of protein extracts from normal human epidermis (Labib et al, 1986; Bernard et al, 1989; Robledo et al, 1990; Machado et al, 1992) showed that 35–50% of the sera from BP patients contain autoantibodies against BPAG2. A recent large survey study detected autoantibodies against BPAG2 in 129 of 263 (49%) of BP sera by western blot using epidermal proteins (Ghohestani et al, 1996). The sensitivities of ELISA, western blot, and/or IP cannot be directly compared mainly because the antigen and secondary antibodies used for the detection of BPAG2-specific autoantibodies differed between the tests. In this study, the ELISA was more sensitive than the western blot of the identical BPAG2 recombinant in the detection of autoantibodies specific for BPAG2 (Table I). Most likely, further denaturation of the recombinant BPAG2 protein led to a loss of epitopes recognized by BPAG2-reactive antibodies. This contention is supported by the finding that the ELISA with boiled BV13 protein was less sensitive than the ELISA with nonboiled BV13 protein in the detection of BPAG2-specific autoantibodies. Consistent with our findings with BP sera, a recent study detected autoantibodies against type-VII collagen in sera of patients with epidermolysis bullosa acquisita more readily by ELISA with a recombinant eukaryotic type-VII collagen than by western blot of the identical recombinant protein (Chen et al, 1997). The potential diagnostic value of the ELISA is further supported by our IP studies using extracts of epidermal keratinocytes (Table I). Our findings suggest that the ELISA has a sensitivity comparable with the IP in which antigen-antibody reaction occurs prior to denaturation by sodium dodecyl sulfate. For the clinical routine, this ELISA with an eukaryotic protein containing the entire ECD of BPAG2 may be very useful because: (i) it is easy to perform, (ii) it is suitable for the rapid and simultaneous testing of many sera, (iii) it allows for the quantitative analysis of BPAG2-specific antibody titers, and (iv) it appears to be highly sensitive and specific. Recently, Giudice et al established an ELISA for the detection of BPAG2-specific antibodies using a prokaryotic (Escherichia coli-derived) recombinant protein consisting of a noncollagenous 42 amino acid long stretch of the ECD of BPAG2 (Giudice et al, 1994). Previous studies indicated that this region adjacent to the membrane spanning domain harbors the major immunodominant sites for BP sera (Giudice et al, 1993). In their ELISA study, 53% of the BP sera and 71% of the PG sera showed reactivity to BPAG2 (Giudice et al, 1994). This is in contrast to a reactivity of 89.7% of BP sera and 100% of PG sera in the ELISA of this study. Although one cannot directly compare these two studies, it is likely that the ELISA of Giudice et al with a bacterial recombinant protein may be less sensitive for the following reasons. (i) In contrast to eukaryotic proteins, prokaryotic proteins frequently lack post-translational modifications that may be critical for folding and thus for conformation of the protein. We have no precise data on the exact conformation of the recombinant BPAG2 protein (BV13) used in this ELISA study. There are definitively imperfections in the use of the baculovirus system with regard to the expression of collagenous proteins (low prolyl hydoxylase activity). Currently, efforts are being made to optimize the expression and assembly of recombinant BPAG2 protein by coexpression of baculovirus-encoded prolyl hydroxylase in the identical insect cell system. Because we did see specific immunoreactivity of most BP and PG sera with BV13 protein, the major B cell epitopes must be exposed on this recombinant. The importance of conformational epitopes has been recently demonstrated for the binding of pathogenic antibodies specific for desmoglein 3, the autoantigen of PV (Amagai et al, 1994); in this study, a baculovirusderived desmoglein 3 protein, but not an E. coli-derived recombinant protein, was able to remove all pathogenic autoantibodies from PV sera. (ii) The MCW-1 region encompasses only a small portion of the
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ECD of BPAG2 and may thus not contain all of the epitopes recognized by BPAG2-specific circulating autoantibodies. This assumption is supported by a recent study demonstrating that sera of patients with cicatricial pemphigoid, a bullous autoimmune disease related to BP, contain autoantibodies that react with at least two distinct regions within the ECD of BPAG2 (Balding et al, 1996). An ELISA utilizing the ECD of BPAG2 protein may thus allow for the detection of BPAG2reactive antibodies of heterogeneous specificity. The C terminus of the ECD of BPAG2 (final 68 amino acids) that was not included in the recombinant BV13 protein utilized in this study, does not harbor major antibody epitopes of BPAG2 because (i) the majority of the tested BP sera were reactive in the ELISA with BV13 and (ii) a recent study by Zillikens et al demonstrated that the major antibody epitopes of BPAG2 were clustered around the NC16 domain that is adjacent to the membrane spanning region (Zillikens et al, 1997). Recent observations suggested that the presence of BPAG2-specific but not BPAG1-specific autoantibodies was a poor prognostic marker in a cohort of 94 adult BP patients (Bernard et al, 1997). In addition, the exclusive presence of BPAG2-specific autoantibodies in BP sera appeared to be associated with an unusually long course of disease (Venning et al, 1991). Together, these data suggest that quantitative assessment of antibody reactivity to BPAG2 provides a prognostic parameter of disease activity in BP. We thus compared the titers of BPAG2-specific antibodies in the sera of BP patients with the extent of their skin involvement (Table II). Our results demonstrate that sera of 64% of the BP patients with extensive skin disease had high titers of anti-BPAG2 antibodies, whereas 67% of the sera from BP patients with few or no skin symptoms had low titers of BPAG2-specific antibodies. Even though end point titration of BP and PG sera provides only a semiquantitative analysis of BPAG2-specific autoantibodies, these preliminary observations suggest that the concentration of antiBPAG2 IgG was not always related to the extent of skin disease in BP patients. The novel ELISA described here that utilizes an eukaryotic form of the ECD of BPAG2 is thus a sensitive and specific technique for the detection of BPAG2-reactive antibodies in the sera of patients with BP and PG. The detection of BPAG2-specific autoantibodies in the majority of BP and PG sera lends additional support to the assumption that BPAG2 is a primary target molecule in the pathogenesis of BP and PG.
We thank Profs. Dr. H. Pullmann and E. Haneke and Drs. A. Schattenkirchner, A. Mira, and T. Zollner for providing BP sera for this study. This study was supported by grant He 1604/5–1 from the Deutsche Forschungsgemeinschaft, Bonn, Germany. L.B. has been supported by a grant from the Swiss National Foundation for Scientific Research.
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