Immunoblot Assay in Differential Diagnosis of Autoimmune Blistering Skin Diseases

HENDRI H. PAS, PhD

ne of the methods to assess the antigen specificity of circulating antibodies in the acquired immunobullous dermatoses is immunoblotting. Many of the targeted antigens known today have initially been discovered by this technique, and this success has made immunoblotting into a widely used diagnostic tool for discriminating between diseases that show up with similar patterns in serum immunofluorescence but in which the actual target antigens may differ. Histology and immunofluorescence usually make the laboratory diagnosis of an acquired bullous disease. Especially the latter is the prime method to establish the presence of an autoimmune process. Based on the autoantibody deposition pattern in skin (as visualized by direct immunofluorescence [DIF]) or the binding pattern of serum autoantibodies to skin or skinequivalent substrates (indirect immunofluorescence [IIF]), a first discrimination can be made between the intraepidermal-directed diseases and the subepidermal epidermal basement membrane zone (EBMZ)-directed diseases. When salt-split skin is used as an additional substrate, the EBMZ group can be further divided into those diseases that the antibodies bind to the epidermal side of the split and those that bind to the dermal side. Since each of these patterns represents more than one single disease (see Table 1) further fine-tuning of the diagnosis is best obtained by actual identification of the targeted antigen or antigens. This can be achieved by either immunoblotting or immunoprecipitation. The advantage of immunoprecipitation is that mild detergents are used for solubilization of the antigens. These preserve the conformation-sensitive epitopes like, for example, those present on the desmogleins or desmocollins, the autoantigens of the various forms of pemphigus. A disadvantage is that not all protein-protein interactions are disrupted, and consequently complexes of proteins are precipitated instead of the sole autoantigen. A good example is laminin-5 in which all three

chains, alpha, beta, and gamma, are visualized irrespective of which subunit is targeted by the immunoglobulin. Another drawback is that no successful immunoprecipitation with immunoglobulin (Ig) A antibodies has yet been reported, restricting the use of immunoprecipitation to IgG-mediated diseases. In immunoblotting sodium dodecyl sulphate (SDS) is used for solubilization of antigens. SDS is a harsh detergent, which, when used at an appropriate concentration, will disrupt all protein-protein interactions enabling the study of free antigens. SDS molecules, however, bind strongly to protein molecules (1.4 g SDS/g protein) and thereby destroy their three-dimensional conformation. For this reason immunoblotting is not suited for detection of conformation-sensitive epitopes. In conclusion, neither immunoblotting nor immunoprecipitation alone will guarantee a 100% positive identification of the suspected autoantigen. Depending on the nature of the epitopes of the antigen in question, the method with the highest chance of success should be chosen. In this chapter I will discuss the method of immunoblotting.

Technical Aspects
In immunoblotting the antigens present in a protein extract are first separated according to molecular size by electrophoresis over a polyacrylamide slab-gel in the presence of sodium dodecyl sulphate (SDS-PAGE). The separated proteins are then electrophoretically transferred to an inert membrane filter; this facilitates further incubation and washing steps by which the patient autoantibodies will bind to and finally visualize the antigen(s) to which they are directed.

Preparation of Antigen Sources

Many tissue and cell extracts can and have been used as antigen sources in immunoblotting. Although these have included animal substrates, the preferred substrate is human. This will prevent misinterpretation due to species differences. Protein extracts can be prepared from either skin or from cultured skin cells. When skin is used as an antigen source, the epidermis and dermis are first separated from each other to expose the EBMZ antigens. This splitting can be achieved by soaking skin in 1 M sodium chloride (NaCl) or 20 mM ethylenediaminetetraacetic acid
0738-081X/01/$see front matter PII S0738-081X(00)00176-0

From the Center for Blistering Skin Diseases, Department of Dermatology, University Hospital, Groningen, The Netherlands. Address correspondence to Hendri H. Pas, PhD, Department of Dermatology, University Hospital, PO Box 30.001, 9700 RB Groningen, The Netherlands. E-mail address: h.h.pas@derm.azg.nl 622 630 by Elsevier Science Inc. All rights reserved. 655 Avenue of the Americas, New York, NY 10010

Clinics in Dermatology

2001;19

AUTOIMMUNE BLISTERING SKIN DISEASES

623

Table 1. Autoantigens in Immune-Mediated Blistering Diseases Antigen

Desmoglein 1 Desmoglein 3 Desmocollin I Pemphaxin Envoplakin Periplakin Desmoplakin 1/II BP230 BP180 LAD-1 Plectin M168 Integrin 4 45 kDa Type VII collagen Laminin 5, alpha3 subunit Laminin 5, beta2 subunit Laminin 5, gamma2 subunit p105 200 kDa Type IV collagen, alpha 5 subunit p84 BP125

IIF Pattern
ICS ICS ICS ICS ICS ICS ICS EBMZ1 EBMZ1 EBMZ1 EBMZ1 EBMZ1 EBMZ1 EBMZ1 EBMZ2 EBMZ2 EBMZ2 EBMZ2 EBMZ2 EBMZ2 EBMZ2 EBMZ2 EBMZ2

Molecular Weight in kDa

160 130 100, 110 75 210 190 250, 210 230 180 120 500 168 205 45 290 200, 165 140 150 105 200 185 84 125

IgG-mediated Disease
PF65, PV8 PV66 PF12 PV13 PNP72 PNP73 PNP14, EM74 BP67, PNP14 BP68, HG27, CP32, OCP41 BP18 PNP71, BP22 CP38 OCP75 EBA49, BSLE50 ECP56 ECP58 ECP57 anti-p10570 anti-200 kDa62 anti-5(IV)64 combined dermal/epidermal76 BP77

IgA-Mediated Disease
IEN17 SPD16

LAD45 CP69, LAD45, OCP41 LAD42

OCP39 LAD51

The substrates for the indirect immunofluorescence patterns are either monkey esophagus (for intercellular substances, ICS) or salt-split human skin (EMBZ1: roof of the split, EMBZ2: floor of the split). Although many antigens have been studied extensively others are less well-defined including p84, BP125, M168, the 45 kDa OCP-antigen and the 200 kDa antigen. Furthermore data on some antigens are limited and rely on one report only, like p84, BP125 and M168 or have so far only be identified by one research group like the proposed OCP antigens 4-integrin and the 45 kDa antigen. EM: Erythema Multiforme

(EDTA) for several days or preferably by heating it at 56C for several minutes.1 Because there is a serious risk of degradation of sensitive antigens as the bullous pemphigoid antigen BP180 (BPAG2 or type XVII collagen), a mix of protease inhibitors should be added to prevent proteolytic degradation. My lab uses the 56C heating method not only to minimize proteolysis, but also for convenient reasons since it is a very fast and reliable method. The skin used is obtained from healthy persons undergoing cosmetic surgery. The tissue, usually from mamma reduction, is washed and then put in phosphate-buffered saline (PBS) containing 10 mM EDTA and a mix of proteolytic inhibitors. The skin is stored at 80C before use, and this freezing of the skin has a positive effect on the ease of the final splitting. Fresh skin appears much more difficult to separate at the dermal-epidermal junction; this has been observed with the above heating method but also with the 1 M NaCl soaking method. After thawing the skin is cut in pieces approximately 3 to 5 cm wide and then heated for 2 to 4 minutes at 56C in PBS buffer containing 10 mM EDTA. Next the pieces are stretched by pinning them on a flat polystyrene surface with the epidermal side facing upward. This way the epidermis can easily be removed with a forceps. The pieces of epidermis are put into a small volume (1 mL per 25 cm2 skin) of an extraction buffer of 62.5 mM Tris-HCl pH 6.8, containing 2% SDS (w/v), 50 mM dithiothreitol (DTT), 2 mM EDTA, and 1 mM 4-(2aminoethyl)-benzenesulfonylfluoride (AEBSF). After 2 minutes of vortexing, the epidermal fragments are re-

moved by centrifugation and the supernatant is stored at 80C. The remaining dermis can be used for preparing dermal extracts. The epidermal side of the dermis is layed onto hot buffer (1 mL per 2.5 cm2 skin, temperature 95C) composed of 62.5 mM Tris-HCl pH 6.8, 2% SDS, 8 M urea, 100 mM DTT, and a mixture of protease inhibitors.2 After 1 minute of incubation, the dermis is removed and the resulting extract is dialyzed to remove the urea. The extract can then be stored at 80C and treated as a normal protein sample for SDS-PAGE.3 Another frequently used substrate is the protein extract of cultured keratinocytes. Serum-free, low-calcium cultured keratinocytes are a good source of the bullous pemphigoid antigens BP230 and BP180 and of laminin 5. Also the characteristic paraneoplastic pemphigus (PNP) antigens of envoplakin and periplakin are easily observed with this substrate (Fig 1). For routine immunoblotting the extract is best prepared by pipetting 600 uL of SDS-PAGE sample buffer directly on a subconfluent layer of cells in a 25 cm2 culture flask. This gives immediate solubilization of the cells and also stops proteolytic breakdown. The synthesis level of other antigens, like type VII collagen and the desmosomal proteins, unfortunately is too low to allow for reliable routine diagnosis. Sufficient amounts of these antigens, however, can be induced by growing the cells on mitomycinetreated fibroblast feeder layers at higher calcium levels.4 Occasionally more exotic substrates like tumor-derived cells have been used because these may produce increased

624 PAS

Clinics in Dermatology

2001;19

uses so-called minigels, 0.75 mm thick, with a final separation gel of approximately 5 by 8 cm; this will allow the simultaneous analysis of 12 different sera. During casting a preparative 2D-gelcomb is used to obtain one 75 mm wide loading slot accompanied by a 3 mm marker slot. From the substrates as described above, 200 L is loaded onto each gel. For visual control of the final separation and successful transfer to the membrane filter, prestained marker proteins are loaded in the small marker slot. For membrane filter nitrocellulose is preferred. Although it is more brittle than PVDF, it produces less background in the colorimetric development system.

Immunostaining Procedure
Before incubation with patient serum, the remaining protein binding sites of the filter should be blocked to prevent nonspecific binding of primary antibodies. There is a wide range of blocking agents but the choice of a certain blocking agent depends on the final staining conditions. When 5-bromo-4-chloro-3-indolyl phosphate (BCIP)/nitro blue tetrazolium (NBT) alkaline phosphatase is used for staining, blocking for 2 hours with 2% low fat milk powder (w/v) in 20 mM Tris-HCl pH 7.5 containing 500 mM NaCl works fine. The actual assay conditions (ie, the binding of the autoantibodies to the filter-bound antigens) and their detection by secondary anti-human immunoglobulin conjugates widely differ between laboratories. Screening the literature reveals large differences in incubation time, dilution of the patient serum, buffer compositions, secondary antibodies, and conjugates. Here, I will restrict myself to the methods used in my own laboratory. First, contrary to most other laboratories, a three-step, or triple-layer, is used instead of a two-step detection system. This system has been found to be of superior sensitivity compared to the ordinary two-step detection. The primary binding step is the incubation with patient serum, which is routinely diluted 1:300. Overnight incubation was found to give much better results than incubating for 2 hours. The secondary step consists of 1 hour of incubation with either polyclonal mouse anti-human IgG or goat antihuman IgA. Finally, alkaline phosphatase-conjugated goat anti-mouse IgG or rabbit anti-goat IgG is used again for 1 hour. All incubations and washings are performed in 20 mM Tris-HCl buffer, pH 7.5, containing 500 mm NaCl and 0.05% (v/v) Tween-20. This protocol works for both the cut strips method and the multiblotter method. In the cut strips method, serial strips are cut from the blotted membrane, and each strip is separately incubated with a specific patient serum. The multiblotter method allows the simultaneous incubation of various sera next to each other on the same blot.6 This is achieved by placing a perspex mould over the filter that creates a series of individual incubation

Figure 1. Antigens in extracts of cultured keratinocytes. Keratinocytes were cultured serum-free in the presence of low calcium, and the cellular extract was used as substrate in immunoblotting. Antigens present in the extract were visualized by incubation with sera from three different patients diagnosed respectively as having paraneoplastic pemphigus (PNP), anti-epiligrin cicatricial pemphigoid (ECP), and bullous pemphigoid (BP). The visualized antigens are indicated by and are, top down, for the PNP serum desmoplakin I, desmoplakin II/envoplakin, and periplakin; for the ECP serum the processed and unprocessed form of the laminin 5 alpha chain and for the BP serum the 230 kDa BP230 and the 180 kDa BP180.

amounts of antigen. An example is the synthesis level of type VII collagen by cylindroma cells.5 The medium in which the cells have been cultured is another rich source of antigens. Adhesion molecules secreted by the cells, like laminin 5 and the lamina lucida autoantigen in linear IgA dermatosis, the 120 kDa LAD-1, but also the p105 and the 200 kDa dermal antigen, concentrate in the culture medium. When conditioned medium is replaced it should routinely be collected and stored frozen until used for substrate preparation. After thawing, 10 mM EDTA and proteolytic inhibitors are added to prevent proteolytic breakdown of antigens, especially of LAD-1. Dead cells and cellular debris are removed by 0.22 m filtration, and next the medium is concentrated about 100-fold by ultrafiltration. At this point the concentrate may contain some precipitate that can be removed by centrifugation. The resulting supernatant is then stored at 80C until used.

SDS-PAGE and Protein Transfer

The percentage of the acrylamide-bisacrylamide (37.51 ratio) gel should be consistent with the molecular weight of the suspected autoantigens. Since most autoantigens are large molecules well over 100 kDa, 5% gels will be perfect in most cases. For antigens less than 100 kDa the percentage should be increased. My lab

Clinics in Dermatology

2001;19

AUTOIMMUNE BLISTERING SKIN DISEASES

625

Figure 2. Calibration of immunoblot antigen signals by use of biotinylated molecular marker proteins. Before electrophoresis the SDS-PAGE substrate was mixed with biotinylated marker proteins. After electrophoresis and transfer to a nitrocellulose filter, the filter was mounted into a multiblotter system and subjected to incubation and development as described in the text. (A) Primary staining of antigen bands by immunobullous patient sera. Note that every other lane contains a different patient serum and that some overnight diffusion may be observed to adjacent empty lanes. (B) The same blot as in (A) but after reincubation with alkaline phosphatase-conjugated streptavidin and redevelopment. The biotinylated molecular marker proteins now appear as horizontal lines over the whole blot, enabling close comparison of antibody specificity profiles for every incubation lane on the blot.

lanes. I use the Immunetics Miniblotter 28 system (Immunetics, Cambridge, MA, USA), although more brands are on the market. The Miniblotter 28 system has 28 lanes of which, in combination with the minigel system, about 25 lanes will be in direct contact with the protein pattern of the blot. Although there is no crosscontamination between adjacent lanes during short incubations, it is observed that during overnight incubations, patient immunoglobulins may diffuse to the neighboring lane, probably through the pores of the membrane filter. Therefore it is advisable to use alternating lanes. Finally, to establish the correct position of the antigen on the blot, and to detect small variations in molecular weight of recognized antigens, a calibration procedure based on biotin-streptavidin interaction is used. Briefly, before electrophoresis the substrate is mixed with biotinylated molecular marker proteins. When the immunoglobulin recognition patterns have been developed and digitally recorded, the blot is reincubated in a 1:1000 dilution of alkaline phosphatase-

conjugated streptavidin. After standard washing procedures the blot is then redeveloped, showing the biotinylated marker proteins as horizontal bands extending over the whole of the blot. Figure 2 shows that individual lanes can now accurately be compared for recognized antigens, since small aberrations in running distance are easily noted.

Differential Diagnosis by Immunoblot Antigens in Antiepidermal Intercellular Substances Deposition Patterns

Pemphigus vulgaris (PV), pemphigus foliaceus (PF), and paraneoplastic pemphigus (PNP) all are characterized by intercellular deposition of IgG. Immunoblot is not a suitable technique to differentiate between PV and PF. While PF patients target desmoglein 1, PV patients may also target desmoglein 1 in addition to desmoglein 3.79 The majority of these desmoglein autoimmune epitopes appear to be conformation sensitive, and

626 PAS

Clinics in Dermatology

2001;19

therefore immunoblot may lead to misinterpretation; except that most patients would be missed anyway, false-negative anti-desmoglein 3 and positive anti-desmoglein 1 would classify PV as PF. A far more reliable alternative is the recently developed Dsg1/Dsg3 enzyme-linked immunosorbent assay (ELISA) test. This kit utilizes recombinant extracellular domains of Dsg1 and Dsg3 for measuring antibody titers and appears to be very sensitive and specific.10 Although some other antigens have been identified by immunoblotting, like the desmocollins I and II and a 190 kDa antigen, their diagnostic value remains elusive.11,12 Recently a 75 kDa acetylcholine binding annexin-like protein, which was named pemphaxin, was shown to be involved in pemphigus blister formation.13 In paraneoplastic pemphigus, immunofluorescence shows ICS staining sometimes accompanied by linear EBMZ staining. PNP-sera immunoprecipitate a characteristic set of antigens including desmoplakin I, BP230, periplakin, envoplakin, and a 170 kDa, not yet identified, polypeptide.14 Although immunoprecipitation is the gold standard for diagnosis, in a study of six PNP patients, all six sera bound the 210/190 kDa periplakin/envoplakin bands by immunoblot.15 My own lab so far has the same experience with four PNP patients sera. Immunoblot cannot diagnose the subcorneal pustular dermatosis (SPD) type of IgA pemphigus.16 The human autoantigen desmocollin 1 fails to be visualized by patient serum IgA, probably since the epitopes are conformationdependent. For the other type of IgA pemphigus, intraepidermal neutrophilic IgA dermatosis (IEN), no large series of patient sera have so far been blotted for reaction with desmoglein 3.17

Figure 3. Immunoblot showing bullous pemphigoid patient sera having IgG antibodies against the 120 kDa linear IgA dermatosis antigen LAD-1. Before electrophoresis cellular and conditioned medium extracts were mixed to obtain a substrate containing all three antigens: BP230, BP180, and LAD-1. Three patient sera (lanes 4, 5, and 6) have specificity to LAD-1 only but not to the classical antigens BP230 and/or BP180, in contrast to the patient sera in lanes 1, 2, and 3. Over the years we have observed that individual patient sera may recognize any possible combination of these three antigens.

EBMZ Antigens in the Roof of Split-Skin

Most sera that in serum immunofluorescence show linear EBMZ staining when assayed on split skin will label the epidermal side. For these epidermal-staining sera, I found a very high correlation between immunofluorescence and immunoblot. In a series of 200 IIF-positive sera, over 90% bound on immunoblot to one or more of the bullous pemphigoid antigens, thereby also showing these to be by far the most targeted proteins. The other sera were either negative or reacted with unknown proteins (unpublished observation). Historically BP230 (BPAG1) and BP180 (BPAG2) are considered the two target antigens of bullous pemphigoid. In 1997 LAD-1 was shown to be the third antigen in bullous pemphigoid, an observation which since then has been confirmed by others.18 21 LAD-1 probably originates as the cleaved-off extracellular part of BP180, which explains the cross-reactivity displayed by many BP patients sera between BP180 and LAD-1.18 The IgG of some BP patients, however, is directed uniquely to LAD-1, and as such these patients seem the IgG counterpart of the

lamina lucida form of linear IgA disease (see Fig 3). Figure 4 shows immunoblot reactions of BP sera with another hemidesmosomal molecule, plectin/HD1. Occasionally I find sera that bind this antigen. In the literature only two patients so far have been described, and both these patients were reported to have vesicular eruptions.22,23 Initially, based on the frequency with which it was encountered in immunoblot and immunoprecipitation, BP230 was considered the most significant and therefore named the major antigen.24 More recent studies, however, strongly suggest BP180 to be the prime antigen. It has been reported that the serum anti-BP180 titer correlated with the disease activity in bullous pemphigoid25 and that anti-BP180 autoantibodies, but not anti-BP230 autoantibodies, are a marker of poor prognosis in bullous pemphigoid.26 Other diseases in which serum antibodies may bind the epidermal side of split-skin include herpes gestationes (HG), cicatricial pemphigoid (CP), and linear IgA disease (LAD). BP180 is the preferential herpes gestationes target antigen,27,28 although reactions to BP230 have also been reported.29,30 As in bullous pemphigoid, most cicatricial pemphigoid patients have antibodies toward BP230 and BP180, but here BP180 is the more frequently observed major antigen.31,32 Furthermore, not only IgG but also IgA antibodies may bind BP180,

Clinics in Dermatology

2001;19

AUTOIMMUNE BLISTERING SKIN DISEASES

627

EBMZ Antigens in the Floor of Split-Skin

Dermal staining in indirect IF may result from several antigens. The first identified dermal antigen was type VII collagen. IgG from patients with epidermolysis bullosa aquisita (EBA) immunoblotted a 290 kDa protein present in a dermal extract.49 IgG antibodies against type VII collagen are not unique for EBA since they are also found in bullous systemic lupus erythematosis (BSLE)50. Also IgA can target type VII collagen. The dermal staining subgroup in linear IgA disease has been shown to contain IgA antibodies, which will bind in immunoblot to type VII collagen.51,52 It is questionable how sensitive immunoblotting for type VII collagen is. An ELISA for anti-type VII antibodies has been developed and compared to immunoblotting.53 Although there was a good correlation between ELISA and immunoblot, the latter was less sensitive. Out of 27 ELISA-positive sera, two (7%) could not label the same substrate, recombinant NC-1 domain, by immunoblot. Immunoblotting with full-length type VII collagen from WISH cells53 (a human amniotic epithelial cell line) resulted in even lower sensitivity since no clear signal could be obtained with 35% of the sera. Although this was caused in part by heavy background staining and low antigen levels, especially with low-titer sera, some problems with loss of conformation-dependent epitopes may also have been involved. Another blistering disease with dermal staining is anti-epiligrin cicatricial pemphigoid, which may manifest as EBA but in which the target antigen is laminin 5.54,55 Although the alpha3 chain is the main target antigen, the other chains, beta3 and gamma2, may also act as autoantigens.56 59 Of 20 patient sera that immunoprecipitated laminin-5, 18 sera (90%) also reacted with laminin 5 subunits by immunoblotting. The remaining two were probably directed against conformational epitopes.57 Two more lower lamina lucida antigens that can be distinguished by immunoblotting are a 105 kDa and a 200 kDa antigen. The 105 kDa antigen, or p105, so far has only been described in two patients.60,61 The clinical features of both patients were very different, one resembling toxic epidermal necrolysis and the other more like bullous pemphigoid, although the 105 kDa antigen in this second patient was accompanied by epidermal staining due to BP230. Antibodies against the 200 kDa antigen have been found in nine patients, of which five had BP-like features. In four cases an association with psoriasis was found.62,63 All these sera were positive by immunoblotting. The most recent addition to the list of dermal antigens is the alpha5 chain of type IV collagen. Two patients that, in addition to subepidermal blisters, also

Figure 4. Immunoblot showing plectin/HD1 as a possible autoantigen in pemphigoid patients. Occasionally we have encountered pemphigoid patient sera that react with a high molecular weight protein also recognized by monoclonal antibody HD121 (specific to HD1/plectin).78,79 Lanes 1 and 2, pemphigoid patient sera; lane 3, HD121 (gift of Dr. K. Owaribe, Nagoya University, Japan). Note that serum 1 also contains antibodies to BP230, while serum 2 binds both BP230 and BP180. As substrate for immunoblot, a cellular extract of cultured keratinocytes was used.

which suggests a pathogenic role for IgA in CP.33 The presence of additional IgA antibodies may result in a more severe and persistent disease.34 Occasionally other immunoblot reactions in CP have been described, like a 120 kDa antigen, an uncein complex, and a 168 kDa antigen found in a mucosal extracts, but these data await confirmation by other laboratories.3538 Several hypothetical autoantigens have been reported in immunoblot studies of ocular cicatricial pemphigoid (OCP) patient sera, but no consensus has been reached yet. IgA binding to a 45 kDa keratin-like molecule and IgG binding to 4-integrin have been described in studies of small patient groups.39,40 In one case immunoblot showed both IgA and IgG against BP180.41 The initial antigen reported in LAD was a 97 kDa protein found in skin extracts.42 In a later study this appeared to be a degradation product of a 120 kDa antigen,43 which itself appeared to be the cleaved-off external part of BP180.18,44 Given that BP180 and BP230 may also be targeted by serum IgA,45,46 linear IgA disease and bullous pemphigoid seem to share the same antigens; also, here occasional reactions with other antigens have been described but these have not further been characterized.47,48

628 PAS

Clinics in Dermatology

2001;19

suffered from renal insufficiency are described in the literature.64

11.

Conclusions
Immunoblot is a valuable contribution to the laboratory diagnosis of immunobullous diseases. Identification of the targeted antigen or antigens can be useful for confirmation of weak or atypical fluorescence patterns; furthermore, it enables discrimination between diseases that cannot be differentiated on basis of their serum immunofluorescence binding patterns. When antibodies against conformation-sensitive epitopes dominate the immune response, like in pemphigus, immunoblot is of less value and other techniques should be used. Although immunoblotting seems a relatively simple technique with easy results, care should be taken in controlling variables as substrate preparation and incubation conditions, since these may influence the final result.

12.

13.

14.

15.

16.

References
1. Meyer LJ, Taylor TB, Kadunce DP, et al. Bullous pemphigoid antigens: extraction and degradation of antigens during epidermal preparation. J Invest Dermatol 1991;96: 9913. 2. Bruckner-Tuderman L, Schnyder UW, Winterhalter KH, et al. Tissue form of type VII collagen from human skin and dermal fibroblasts in culture. Eur J Biochem 1987;165: 60711. 3. Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 1970; 227:680 5. 4. Batteux F, Franck N, Jaffray P, et al. An extract from cultured human keratinocytes that contains the major autoantigens related to autoimmune bullous skin diseases. J Clin Immunol 1997;17:228 33. 5. Bruckner-Tuderman L, Pfaltz M, Schnyder UW. Cylindroma overexpresses collagen VII, the major anchoring fibril protein. J Invest Dermatol 1991;96:729 34. 6. Pas HH, de Jong MC, Jonkman MF, et al. Bullous pemphigoid: Serum antibody titre and antigen specificity. Exp Dermatol 1995;4:372 6. 7. Hashimoto T, Amagai M, Garrod DR, et al. Immunofluorescence and immunoblot studies on the reactivity of pemphigus vulgaris and pemphigus foliaceus sera with desmoglein 3 and desmoglein 1. Epithelial Cell Biol 1995; 4:639. 8. Amagai M, Tsunoda K, Zillikens D, et al. The clinical phenotype of pemphigus is defined by the anti-desmoglein autoantibody profile. J Am Acad Dermatol 1999;40: 16770. 9. Mahoney MG, Wang Z, Rothenberger K, et al. Explanations for the clinical and microscopic localization of lesions in pemphigus foliaceus and vulgaris. J Clin Invest 1999;103:461 8. 10. Ishii K, Amagai M, Hall RP, et al. Characterization of autoantibodies in pemphigus using antigen-specific enzyme-linked immunosorbent assays with baculovirus-ex17.

18.

19.

20.

21.

22.

23.

24.

25.

26.

pressed recombinant desmogleins. J Immunol 1997;159: 2010 7. Dmochowski M, Hashimoto T, Garrod DR, et al. Desmocollins I and II are recognized by certain sera from patients with various types of pemphigus, particularly Brazilian pemphigus foliaceus. J Invest Dermatol 1993;100: 380 4. Joly P, Gilbert D, Thomine E, et al. Identification of a new antibody population directed against a desmosomal plaque antigen in pemphigus vulgaris and pemphigus foliaceus. J Invest Dermatol 1997;108:469 75. Nguyen VT, Ndoye A, Grando SA. Pemphigus vulgaris antibody identifies pemphaxinA novel keratinocyte annexin-like molecule binding acetylcholine. J Biol Chem 2000;275:29466 76. Anhalt GJ, Kim SC, Stanley JR, et al. Paraneoplastic pemphigus. An autoimmune mucocutaneous disease associated with neoplasia. N Engl J Med 1990;323:1729 35. Hashimoto T, Amagai M, Watanabe K, et al. Characterization of paraneoplastic pemphigus autoantigens by immunoblot analysis. J Invest Dermatol 1995;104:829 34. Hashimoto T, Kiyokawa C, Mori O, et al. Human desmocollin 1 (Dsc1) is an autoantigen for the subcorneal pustular dermatosis type of IgA pemphigus. J Invest Dermatol 1997;109:12731. Wang J, Kwon J, Ding X, et al. Nonsecretory IgA1 autoantibodies targeting desmosomal component desmoglein 3 in intraepidermal neutrophilic IgA dermatosis. Am J Pathol 1997;150:19017. Pas HH, Kloosterhuis GJ, Heeres K, et al. Bullous pemphigoid and linear IgA dermatosis sera recognize a similar 120-kDa keratinocyte collagenous glycoprotein with antigenic cross-reactivity to BP180. J Invest Dermatol 1997;108:4239. Egan CA, Taylor TB, Meyer LJ, et al. Bullous pemphigoid sera that contain antibodies to BPAg2 also contain antibodies to LABD97 that recognize epitopes distal to the NC16A domain. J Invest Dermatol 1999;112:148 52. Schumann H, Baetge J, Tasanen K, et al. The shed ectodomain of collagen XVII/BP180 is targeted by autoantibodies in different blistering skin diseases. Am J Pathol 2000; 156:68595. Roh JY, Yee C, Lazarova Z, et al. The 120-kDa soluble ectodomain of type XVII collagen is recognized by autoantibodies in patients with pemphigoid and linear IgA dermatosis. Br J Dermatol 2000;143:104 11. Fujiwara S, Shinkai H, Takayasu S, et al. A case of subepidermal blister disease associated with autoantibody against 450 kD protein. J Dermatol 1992;19:610 3. Ohnishi Y, Tajima S, Ishibashi A, et al. A vesicular bullous pemphigoid with an autoantibody against plectin. Br J Dermatol 2000;142:8135. Mueller S, Klaus-Kovtun V, Stanley JR. A 230-kD basic protein is the major bullous pemphigoid antigen. J Invest Dermatol 1989;92:33 8. Schmidt E, Obe K, Brocker EB, et al. Serum levels of autoantibodies to BP180 correlate with disease activity in patients with bullous pemphigoid. Arch Dermatol 2000; 136:174 8. Bernard P, Bedane C, Bonnetblanc JM. Anti-BP180 autoantibodies as a marker of poor prognosis in bullous pem-

Clinics in Dermatology

2001;19

AUTOIMMUNE BLISTERING SKIN DISEASES

629

27.

28.

29.

30.

31.

32.

33.

34.

35.

36.

37.

38.

39.

40.

41.

42.

phigoid: A cohort analysis of 94 elderly patients. Br J Dermatol 1997;136:694 8. Morrison LH, Labib RS, Zone JJ, et al. Herpes gestationis autoantibodies recognize a 180-kD human epidermal antigen. J Clin Invest 1988;81:2023 6. Diaz LA, Ratrie H III, Saunders WS, et al. Isolation of a human epidermal cDNA corresponding to the 180-kD autoantigen recognized by bullous pemphigoid and herpes gestationis sera. Immunolocalization of this protein to the hemidesmosome. J Clin Invest 1990;86:1088 94. Kelly SE, Bhogal BS, Wojnarowska F, et al. Western blot analysis of the antigen in pemphigoid gestationis. Br J Dermatol 1990;122:4459. Murakami H, Amagai M, Higashiyama M, et al. Analysis of antigens recognized by autoantibodies in herpes gestationis. Usefulness of immunoblotting using a fusion protein representing an extracellular domain of the 180 kD bullous pemphigoid antigen. J Dermatol Sci 1996;13: 1127. Bernard P, Prost C, Durepaire N, et al. The major cicatricial pemphigoid antigen is a 180-kD protein that shows immunologic cross-reactivities with the bullous pemphigoid antigen. J Invest Dermatol 1992;99:174 9. Balding SD, Prost C, Diaz LA, et al. Cicatricial pemphigoid autoantibodies react with multiple sites on the BP180 extracellular domain. J Invest Dermatol 1996;106:141 6. Murakami H, Nishioka S, Setterfield J, et al. Analysis of antigens targeted by circulating IgG and IgA autoantibodies in 50 patients with cicatricial pemphigoid. J Dermatol Sci 1998;17:39 44. Setterfield J, Shirlaw PJ, Kerr MM, et al. Mucous membrane pemphigoid: A dual circulating antibody response with IgG and IgA signifies a more severe and persistent disease. Br J Dermatol 1998;138:60210. Sarret Y, Reano A, Nicolas JF, et al. Bullous pemphigoid and cicatricial pemphigoid: Immunoblotting detection of involved autoantigens. Autoimmunity 1989;2:14553. Petit A, Perrin P, Bernard P, et al. A case of cicatricial pemphigoid with circulating IgA and IgG antibodies directed against 280 kD, 165 kD and 120 130 kD epidermal antigens. Acta Derm Venereol 1990;70:236 8. Horiguchi Y, Ueda M, Shimizu H, et al. An acquired bullous dermatosis due to an autoimmune reaction against uncein. Br J Dermatol 1996;134:934 8. Ghohestani RF, Nicolas JF, Rousselle P, et al. Identification of a 168-kDa mucosal antigen in a subset of patients with cicatricial pemphigoid. J Invest Dermatol 1996;107: 136 9. Smith EP, Taylor TB, Meyer LJ, et al. Identification of a basement membrane zone antigen reactive with circulating IgA antibody in ocular cicatricial pemphigoid. J Invest Dermatol 1993;101:619 23. Mohimen A, Neumann R, Foster CS, et al. Detection and partial characterization of ocular cicatricial pemphigoid antigens on COLO and SCaBER tumor cell lines. Curr Eye Res 1993;12:74152. Matsuzaki T, Mashima Y, Idei T, et al. Unilateral ocular cicatricial pemphigoid with circulating IgA and IgG autoantibodies reactive with the 180 kD bullous pemphigoid antigen. Br J Ophthalmol 1996;80:769. Zone JJ, Taylor TB, Kadunce DP, et al. Identification of the

43.

44.

45.

46.

47.

48.

49.

50.

51.

52.

53.

54.

55.

56.

57.

cutaneous basement membrane zone antigen and isolation of antibody in linear immunoglobulin A bullous dermatosis. J Clin Invest 1990;85:81220. Marinkovich MP, Taylor TB, Keene DR, et al. LAD-1, the linear IgA bullous dermatosis autoantigen, is a novel 120-kDa anchoring filament protein synthesized by epidermal cells. J Invest Dermatol 1996;106:734 8. Zone JJ, Taylor TB, Meyer LJ, et al. The 97 kDa linear IgA bullous disease antigen is identical to a portion of the extracellular domain of the 180 kDa bullous pemphigoid antigen, BPAg2. J Invest Dermatol 1998;110:20710. Ghohestani RF, Nicolas JF, Kanitakis J, et al. Linear IgA bullous dermatosis with IgA antibodies exclusively directed against the 180- or 230-kDa epidermal antigens. J Invest Dermatol 1997;108:854 8. Zillikens D, Herzele K, Georgi M, et al. Autoantibodies in a subgroup of patients with linear IgA disease react with the NC16A domain of BP180. J Invest Dermatol 1999;113: 94753. Yamane Y, Sato H, Higashi K, et al. Linear immunoglobulin A (IgA) bullous dermatosis of childhood: Identification of the target antigens and study of the cellular sources. Br J Dermatol 1996;135:78590. Zhou S, Ferguson DJ, Allen J, et al. The localization of target antigens and autoantibodies in linear IgA disease is variable: Correlation of immunogold electron microscopy and immunoblotting. Br J Dermatol 1998;139:5917. Woodley DT, Briggaman RA, OKeefe EJ, et al. Identification of the skin basement-membrane autoantigen in epidermolysis bullosa acquisita. N Engl J Med 1984;310:1007 13. Barton DD, Fine JD, Gammon WR, et al. Bullous systemic lupus erythematosus: An unusual clinical course and detectable circulating autoantibodies to the epidermolysis bullosa acquisita antigen. J Am Acad Dermatol 1986;15: 369 73. Zambruno G, Manca V, Kanitakis J, et al. Linear IgA bullous dermatosis with autoantibodies to a 290 kd antigen of anchoring fibrils. J Am Acad Dermatol 1994;31: 884 8. Hashimoto T, Ishiko A, Shimizu H, et al. A case of linear IgA bullous dermatosis with IgA anti-type VII collagen antibodies. Br J Dermatol 1995;134:336 9. Chen M, Chan LS, Cai X, et al. Development of an ELISA for rapid detection of anti-type VII collagen autoantibodies in epidermolysis bullosa acquisita. J Invest Dermatol 1997;108:68 72. Domloge-Hultsch N, Gammon WR, Briggaman RA, et al. Epiligrin, the major human keratinocyte integrin ligand, is a target in both an acquired autoimmune and an inherited subepidermal blistering skin disease. J Clin Invest 1992; 90:1628 33. Domloge-Hultsch N, Anhalt GJ, Gammon WR, et al. Antiepiligrin cicatricial pemphigoid. A subepithelial bullous disorder. Arch Dermatol 1994;130:15219. Kirtschig G, Marinkovich MP, Burgeson RE, et al. Antibasement membrane autoantibodies in patients with antiepiligrin cicatricial pemphigoid bind the alpha subunit of laminin 5. J Invest Dermatol 1995;105:543 8. Lazarova Z, Hsu R, Yee C, et al. Antiepiligrin cicatricial pemphigoid represents an autoimmune response to sub-

630

PAS

Clinics in Dermatology

2001;19

58.

59.

60.

61.

62.

63.

64.

65.

66.

67.

68.

69.

units present in laminin 5 (alpha3beta3gamma2). Br J Dermatol 1998;139:7917. Fujimoto W, Toi Y, Okazaki F, et al. Anti-epiligrin cicatricial pemphigoid with IgG autoantibodies to the beta and gamma subunits of laminin 5. J Am Acad Dermatol 1999; 40:6379. Nousari HC, Rencic A, Hsu R, et al. Anti-epiligrin cicatricial pemphigoid with antibodies against the gamma2 subunit of laminin 5. Arch Dermatol 1999;135:173 6. Chan LS, Wang XS, Lapiere JC, et al. A newly identified 105-kD lower lamina lucida autoantigen is an acidic protein distinct from the 105-kD gamma 2 chain of laminin-5. J Invest Dermatol 1995;105:759. Cotell SL, Lapiere JC, Chen JD, et al. A novel 105-kDa lamina lucida autoantigen: Association with bullous pemphigoid. J Invest Dermatol 1994;103:78 83. Zillikens D, Kawahara Y, Ishiko A, et al. A novel subepidermal blistering disease with autoantibodies to a 200kDa antigen of the basement membrane zone. J Invest Dermatol 1996;106:46570. Kawahara Y, Zillikens D, Yancey KB, et al. Subepidermal blistering disease with autoantibodies against a novel dermal 200-kDa antigen. J Dermatol Sci 2000;23:93102. Ghohestani RF, Hudson BG, Claudy A, et al. The alpha 5 chain of type IV collagen is the target of IgG autoantibodies in a novel autoimmune disease with subepidermal blisters and renal insufficiency. J Biol Chem 2000;275: 16002 6. Stanley JR, Koulu L, Klaus-Kovtun V, et al. A monoclonal antibody to the desmosomal glycoprotein desmoglein I binds the same polypeptide as human autoantibodies in pemphigus foliaceus. J Immunol 1986;136:122730. Amagai M, Klaus-Kovtun V, Stanley JR. Autoantibodies against a novel epithelial cadherin in pemphigus vulgaris, a disease of cell adhesion. Cell 1991;67:869 77. Stanley JR, Tanaka T, Mueller S, et al. Isolation of complementary DNA for bullous pemphigoid antigen by use of patients autoantibodies. J Clin Invest 1988;82:1864 70. Labib RS, Anhalt GJ, Patel HP, et al. Molecular heterogeneity of the bullous pemphigoid antigens as detected by immunoblotting. J Immunol 1986;136:12315. Hashimoto T, Han Yaku H, Higashiyama M, et al. Four

70.

71.

72.

73.

74.

75.

76.

77.

78.

79.

Japanese patients with cicatricial pemphigoid showing both IgG and IgA antibodies against the 180 kDa bullous pemphigoid antigen. Br J Dermatol 1997;137:305 6. Chan LS, Fine JD, Briggaman RA, et al. Identification and partial characterization of a novel 105-kDalton lower lamina lucida autoantigen associated with a novel immunemediated subepidermal blistering disease. J Invest Dermatol 1993;101:2627. Proby C, Fujii Y, Owaribe K, et al. Human autoantibodies against HD1/plectin in paraneoplastic pemphigus. J Invest Dermatol 1999;112:153 6. Kim SC, Kwon YD, Lee IJ, et al. cDNA cloning of the 210-kDa paraneoplastic pemphigus antigen reveals that envoplakin is a component of the antigen complex. J Invest Dermatol 1997;109:3659. Mahoney MG, Aho S, Uitto J, et al. The members of the plakin family of proteins recognized by paraneoplastic pemphigus antibodies include periplakin. J Invest Dermatol 1998;111:308 13. Foedinger D, Elbe BA, Sterniczky B, et al. Erythema multiforme associated human autoantibodies against desmoplakin I and II: biochemical characterization and passive transfer studies into newborn mice. J Invest Dermatol 1998;111:50310. Tyagi S, Bhol K, Natarajan K, et al. Ocular cicatricial pemphigoid antigen: partial sequence and biochemical characterization. Proc Natl Acad Sci USA 1996;93:14714 9. Gao SQ, Bystryn JC. Identification of a novel basement membrane antigen (p84) defined by sera with antibodies to both the epidermal and dermal side of split skin. J Invest Dermatol 1994;102:236 40. Gao SQ, Bystryn JC. A novel bullous pemphigoid antigen (BP125) located in the deeper layers of the basement membrane zone. Arch Dermatol 1994;130:873 8. Hieda Y, Nishizawa Y, Uematsu J, et al. Identification of a new hemidesmosomal protein, HD1: A major, high molecular mass component of isolated hemidesmosomes. J Cell Biol 1992;116:1497506. Gache Y, Chavanas S, Lacour JP, et al. Defective expression of plectin/HD1 in epidermolysis bullosa simplex with muscular dystrophy. J Clin Invest 1996;97:2289 98.