Normal human skin is superior to monkey oesophagus substrate for detection of circulating BP180-NC16A-specific IgG antibodies in bullous pemphigoid.

BACKGROUND: Bullous pemphigoid (BP) is the most common autoimmune subepidermal blistering skin disease. Two antigens have been identified as targets of circulating autoantibodies (autoAbs) - BP180 and BP230 - with BP180 being a critical transmembrane adhesion protein of basal keratinocytes of the epidermis. The noncollagenous domain 16A (NC16A) of BP180 is the immunodominant epitope in patients with BP, and anti-BP180-NC16A IgG antibodies (Abs) correlate to disease activity. Routine serological testing and follow-up of BP relies on indirect immunofluorescence (IIF) of serum Abs, commonly performed on monkey oesophagus (ME), and/or enzyme-linked immunosorbent assay (ELISA) testing on recombinantly produced fragments of BP180 and BP230 (BP180-NC16A, BP230-C/N). OBJECTIVES: To determine if NC16A epitopes are well represented on ME substrate. METHODS: Sera from different BP cohorts were tested by IIF on ME and normal human skin (NHS). To confirm findings, affinity-purified anti-BP180-NC16A/BP230 polyclonal Abs and recombinant anti-BP180-NC16A/BP230 monoclonal antibodies (mAbs) were used. RESULTS: For sensitive detection of BP180-NC16A-specific IgG Abs, sections of NHS are superior to the widely used ME. Confirmation comes from polyclonal affinity-purified anti-BP180-NC16A/BP230 Abs, and by mAbs cloned from a patient with active BP. CONCLUSIONS: Use of NHS is preferable over ME in routine IIF testing for BP. These results are of clinical relevance because anti-BP180-NC16A IgG titres are correlated to disease activity and detecting them reliably is important for screening, diagnosis and follow-up of patients with BP.

Reduced soluble receptor for advanced glycation end-products (sRAGE) scavenger capacity precedes pre-eclampsia in Type 1 diabetes.

OBJECTIVE: Increased advanced glycation end-products (AGEs) and their soluble receptors (sRAGE) have been implicated in the pathogenesis of pre-eclampsia (PE). However, this association has not been elucidated in pregnancies complicated by diabetes. We aimed to investigate the serum levels of these factors in pregnant women with Type 1 diabetes mellitus (T1DM), a condition associated with a four-fold increase in PE. DESIGN: Prospective study in women with T1DM at 12.2 ± 1.9, 21.6 ± 1.5 and 31.5 ± 1.7 weeks of gestation [mean ± standard deviation (SD); no overlap] before PE onset. SETTING: Antenatal clinics. POPULATION: Pregnant women with T1DM (n = 118; 26 developed PE) and healthy nondiabetic pregnant controls (n = 21). METHODS: Maternal serum levels of sRAGE (total circulating pool), N(ε)-(carboxymethyl)lysine (CML), hydroimidazolone (methylglyoxal-modified proteins) and total AGEs were measured by immunoassays. MAIN OUTCOME MEASURES: Serum sRAGE and AGEs in pregnant women with T1DM who subsequently developed PE (DM PE+) versus those who remained normotensive (DM PE-). RESULTS: In DM PE+ versus DM PE-, sRAGE was significantly lower in the first and second trimesters, prior to the clinical manifestation of PE (P < 0.05). Further, reflecting the net sRAGE scavenger capacity, sRAGE:hydroimidazolone was significantly lower in the second trimester (P < 0.05) and sRAGE:AGE and sRAGE:CML tended to be lower in the first trimester (P < 0.1) in women with T1DM who subsequently developed PE versus those who did not. These conclusions persisted after adjusting for prandial status, glycated haemoglobin (HbA1c), duration of diabetes, parity and mean arterial pressure as covariates. CONCLUSIONS: In the early stages of pregnancy, lower circulating sRAGE levels, and the ratio of sRAGE to AGEs, may be associated with the subsequent development of PE in women with T1DM.

Fetal immunization of baboons induces a fetal-specific antibody response.

Neonates face a high risk of infection because of the immaturity of their immune systems. Although the transplacental transfer of maternal antibodies to the fetus may convey improved postnatal immunity, this transfer occurs late in gestation and may fail to prevent in utero infection. Both fetal immunization and in utero exposure to antigen can result in a state of immunologic tolerance in the neonate. Tolerance induction of fetal and premature infant lymphocytes has become a paradigm for neonatal responsiveness. However, fetal IgM responses have been demonstrated to maternal immunization with tetanus toxoid and to congenital infections such as rubella, toxoplasma, cytomegalovirus and human immunodeficiency virus. Moreover, 1-week-old infants can respond to standard pediatric vaccination, and neonates immunized with polysaccharide antigens do not develop immunologic tolerance. Here, direct immunization of the baboon fetus with recombinant hepatitis B surface antigen produced a specific fetal IgG antibody response. No specific maternal antibody response was detected, eliminating the possibility of vertical antibody transmission to the fetus. Some infants also responded to later vaccinations with hepatitis B surface antigen, indicating that no immunological tolerance was induced by prior fetal immunization. These results characterize the ability of the fetal immune system to respond to in utero vaccination. We demonstrate that active fetal immunization can serve as a safe and efficient vaccination strategy for the fetus and neonate.

Toxin in bullous impetigo and staphylococcal scalded-skin syndrome targets desmoglein 1.

Exfoliative toxin A, produced by Staphylococcus aureus, causes blisters in bullous impetigo and its more generalized form, staphylococcal scalded-skin syndrome. The toxin shows exquisite specificity in causing loss of cell adhesion only in the superficial epidermis. Although exfoliative toxin A has the structure of a serine protease, a target protein has not been identified. Desmoglein (Dsg) 1, a desmosomal cadherin that mediates cell-cell adhesion, may be the target of exfoliative toxin A, because it is the target of autoantibodies in pemphigus foliaceus, in which blisters form with identical tissue specificity and histology. We show here that exfoliative toxin A cleaved mouse and human Dsg1, but not closely related cadherins such as Dsg3. We demonstrate this specific cleavage in cell culture, in neonatal mouse skin and with recombinant Dsg1, and conclude that Dsg1 is the specific receptor for exfoliative toxin A cleavage. This unique proteolytic attack on the desmosome causes a blister just below the stratum corneum, which forms the epidermal barrier, presumably allowing the bacteria in bullous impetigo to proliferate and spread beneath this barrier.

Human autoantibodies against a desmosomal protein complex with a calcium-sensitive epitope are characteristic of pemphigus foliaceus patients.

Pemphigus foliaceus (PF) patients have antibodies against a tightly, but noncovalently bound complex of polypeptides, which consists of desmoglein I (DGI) and other, possibly desmosomal, proteins. Most PF antibodies bind a calcium-sensitive epitope on this complex and chelation of calcium destroys the reactivity of these sera with the complex, but not the complex itself. The PF sera that do bind the desmosomal complex in the absence of calcium are those sera capable of binding denatured DGI on immunoblotting, and these same sera also immunoprecipitate only DGI when the desmosomal complex is dissociated with SDS. These findings demonstrate that autoantibodies against a complex of desmosome-associated proteins are characteristic of PF and define a calcium-sensitive conformational epitope on this complex.

Human autoantibodies against a desmosomal core protein in pemphigus foliaceus.

Pemphigus foliaceus (PF) is a human autoimmune disease in which antibodies are directed against the cell surface of epidermal cells with resultant blister formation. The histopathology of these blisters indicates that cells have detached from each other, and electron microscopy of early blisters shows diminished numbers, to complete loss, of desmosomes as well as abnormalities of the tonofilament-desmosome complex. In this study we demonstrate that autoantibodies from certain PF patients bind to a desmosomal core glycoprotein called desmoglein (DG) I. Proteins in extracts of normal human epidermis were separated by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis (PAGE), then transferred to nitrocellulose or 2-aminophenylthioether paper for immunoperoxidase staining. Results of these immunoblots indicated that sera from 6 of 13 PF patients specifically and intensely stained an approximately 160,000 mol wt polypeptide, "PF antigen". Such staining was not seen with normal human sera or sera from patients with pemphigus vulgaris or bullous pemphigoid, two autoimmune blistering skin diseases that are clinically, histologically, and immunochemically distinct from PF. However, rabbit antiserum directed against DGI, that was isolated from bovine muzzle desmosomes, stained a polypeptide band which co-migrated with PF antigen. Furthermore, when proteins from extracts of normal human epidermis were electrophoresed in two dimensions (isoelectric focusing, then SDS-PAGE) before transfer to nitrocellulose for immunoperoxidase staining, PF antibodies and antibodies to DGI stained identical spots. Finally, PF sera as well as PF IgG that was affinity purified with PF antigen from normal human epidermis, both selectively bound to DGI extracted from bovine muzzle desmosomes. These studies demonstrate that the human autoantibodies from certain patients with PF, a disease of epidermal cell adhesion, are directed against a desmosomal core protein.

Anti-angiogenic factors and pre-eclampsia in type 1 diabetic women.

AIMS/HYPOTHESIS: Elevated anti-angiogenic factors such as soluble fms-like tyrosine kinase 1 (sFlt1), a soluble form of vascular endothelial growth factor receptor, and endoglin, a co-receptor for TGFbeta1, confer high risk of pre-eclampsia in healthy pregnant women. In this multicentre prospective study, we determined levels of these and related factors in pregnant women with type 1 diabetes, a condition associated with a fourfold increase in pre-eclampsia. METHODS: Maternal serum sFlt1, endoglin, placental growth factor (PlGF) and pigment epithelial derived factor were measured in 151 type 1 diabetic and 24 healthy non-diabetic women at each trimester and at term. RESULTS: Approximately 22% of the diabetic women developed pre-eclampsia, primarily after their third trimester visit. In women with pre-eclampsia (diabetic pre-eclampsia, n = 26) vs those without hypertensive complications (diabetic normotensive, n = 95), significant changes in angiogenic factors were observed, predominantly in the early third trimester and prior to clinical manifestation of pre-eclampsia. Serum sFlt1 levels were increased approximately twofold in type 1 diabetic pre-eclampsia vs type 1 diabetic normotensive women at the third trimester visit (p < 0.05) and the normal rise of PlGF during pregnancy was blunted (p < 0.05). Among type 1 diabetic women, third trimester sFlt1 and PlGF were inversely related (r(2) = 42%, p < 0.0001). Endoglin levels were increased significantly in the diabetic group as a whole vs the non-diabetic group (p < 0.0001). CONCLUSIONS/INTERPRETATION: Higher sFlt1 levels, a blunted PlGF rise and an elevated sFlt1/PlGF ratio are predictive of pre-eclampsia in pregnant women with type 1 diabetes. Elevated endoglin levels in women with type 1 diabetes may confer a predisposition to pre-eclampsia and may contribute to the high incidence of pre-eclampsia in this patient group.

Intracellular domain of desmoglein 3 (pemphigus vulgaris antigen) confers adhesive function on the extracellular domain of E-cadherin without binding catenins.

For the extracellular (EC) domain of E-cadherin to function in homophilic adhesion it is thought that its intracytoplasmic (IC) domain must bind alpha- and beta-catenins, which link it to the actin cytoskeleton. However, the IC domain of pemphigus vulgaris antigen (PVA or Dsg3), which is in the desmoglein subfamily of the cadherin gene superfamily, does not bind alpha- or beta-catenins. Because desmogleins have also been predicted to function in the cell adhesion of desmosomes, we speculated that the PVA IC domain might be able to act in a novel way in conferring adhesive function on the EC domain of cadherins. To test this hypothesis we studied aggregation of mouse fibroblast L cell clones that expressed chimeric cDNAs encoding the EC domain of E-cadherin with various IC domains. We show here that the full IC domain of PVA as well as an IC subdomain containing only 40 amino acids of the PVA intracellular anchor (IA) region confer adhesive function on the E-cadherin EC domain without catenin-like associations with cytoplasmic molecules or fractionation with the cell cytoskeleton. This IA region subdomain is evolutionarily conserved in desmogleins, but not classical cadherins. These findings suggest an important cell biologic function for the IA region of desmogleins and demonstrate that strong cytoplasmic interactions are not absolutely necessary for E-cadherin-mediated adhesion.

Specific epidermal protein markers are modulated during calcium-induced terminal differentiation.

Extracellular calcium concentration has been shown to be an important determinant of proliferation rate in a number of cell culture models. Recently, the role of calcium as a regulator of cellular differentiation has also become apparent. This effect of calcium was exemplified by the discovery that keratinocytes of mouse or human origin grew as a proliferating monolayer in medium with a calcium concentration of 0.02-0.09 mM but that proliferation ceased and cells stratified and cornified when calcium was increased greater than 0.1 mM. While the morphological and biological effects of changes in calcium concentration are dramatic in keratinocyte cultures, it has been difficult to identify specific protein changes associated with the modulation of maturation. In vivo, however, several proteins that are markers for stratified squamous epithelia have been identified by specific autoimmune sera. Pemphigoid antigen is a 220-kdalton protein found in the basement membrane and closely associated with the plasma membrane of the basal cell. Pemphigus antigen is a 130-kdalton glycoprotein found on the cell surface of stratifying epithelial cells. Immunofluorescence staining of cells cultured in low Ca2+ or cells switched to high Ca2+ for 48 h before staining demonstrated that pemphigoid antigen was detected in low Ca2+ cultures but was diminished or absent in high Ca2+ cultures and that pemphigus antigen was seen only in high Ca2+ cultures. The synthesis of each antigen was studied in immunoprecipitates of cell lysates radiolabeled with 14C-amino acids or D-[1-14C]glucosamine. Pemphigoid antigen was synthesized mainly by proliferating cells in low Ca2+ medium and its synthesis was decreased by greater than 90% in cells switched to high Ca2+ medium. In contrast, synthesis of pemphigus antigen was detected only in stratifying cells cultured in high Ca2+ medium. These studies indicate that extracellular calcium concentrations which modulate the transition between proliferating and stratifying epidermal cells also modulate, in parallel, the synthesis of specific marker proteins for these cell types.

Pemphigus vulgaris antigen, a desmoglein type of cadherin, is localized within keratinocyte desmosomes.

Pemphigus vulgaris antigen (PVA) is a member of the desmoglein subfamily of cadherin cell adhesion molecules. Because autoantibodies in this disease cause blisters due to loss of epidermal cell adhesion, and because desmoglein is found in the desmosome cell adhesion junction, we wanted to determine if PVA is also found in desmosomes. By immunofluorescence, PV IgG bound, in a dotted pattern, to the cell surface of cultured human keratinocytes induced to differentiate with calcium, suggesting junctional staining. However, by preembedding, immunogold electron microscopic studies only slight labeling could be detected in desmosomes, presumably because of difficulty in gold penetration of intact desmosomes. We therefore treated the keratinocytes with 0.01% trypsin in 1 mM calcium, conditions known to preserve cadherin antigenicity but that caused slight separation of desmosomes, before immunogold staining. In this case there was extensive labeling of the extracellular part of desmosomes but not of the interdesmosomal cell membrane which was stained with anti-beta 2-microglobulin antibodies. To confirm the specificity of this binding we showed that antibodies raised in rabbits against the extracellular portions of PVA also bound desmosomes in these cultures. In intact mouse epidermis we could also show slight, but specific, immunogold desmosomal labeling with PV IgG. Furthermore, neonatal mice injected with PV IgG affinity purified on PVA showed desmosomal separation with the IgG localized to desmosomal cores. These results indicate that PVA is organized and concentrated within the desmosome where it presumably functions to maintain the integrity of stratifying epithelia.

Desmoglein isoform distribution affects stratum corneum structure and function.

Desmogleins are desmosomal cadherins that mediate cell-cell adhesion. In stratified squamous epithelia there are two major isoforms of desmoglein, 1 and 3, with different distributions in epidermis and mucous membrane. Since either desmoglein isoform alone can mediate adhesion, the reason for their differential distribution is not known. To address this issue, we engineered transgenic mice with desmoglein 3 under the control of the involucrin promoter. These mice expressed desmoglein 3 with the same distribution in epidermis as found in normal oral mucous membranes, while expression of other major differentiation molecules was unchanged. Although the nucleated epidermis appeared normal, the epidermal stratum corneum was abnormal with gross scaling, and a lamellar histology resembling that of normal mucous membrane. The mice died shortly after birth with severe dehydration, suggesting excessive transepidermal water loss, which was confirmed by in vitro and in vivo measurement. Ultrastructure of the stratum corneum showed premature loss of cohesion of corneocytes. This dysadhesion of corneocytes and its contribution to increased transepidermal water loss was confirmed by tape stripping. These data demonstrate that differential expression of desmoglein isoforms affects the major function of epidermis, the permeability barrier, by altering the structure of the stratum corneum.

Targeted disruption of the pemphigus vulgaris antigen (desmoglein 3) gene in mice causes loss of keratinocyte cell adhesion with a phenotype similar to pemphigus vulgaris.

In patients with pemphigus vulgaris (PV), autoantibodies against desmoglein 3 (Dsg3) cause loss of cell-cell adhesion of keratinocytes in the basal and immediate suprabasal layers of stratified squamous epithelia. The pathology, at least partially, may depend on protease release from keratinocytes, but might also result from antibodies interfering with an adhesion function of Dsg3. However, a direct role of desmogleins in cell adhesion has not been shown. To test whether Dsg3 mediates adhesion, we genetically engineered mice with a targeted disruption of the DSG3 gene. DSG3 -/- mice had no DSG3 mRNA by RNase protection assay and no Dsg3 protein by immunofluorescence (IF) and immunoblots. These mice were normal at birth, but by 8-10 d weighed less than DSG3 +/- or +/+ littermates, and at around day 18 were grossly runted. We speculated that oral lesions (typical in PV patients) might be inhibiting food intake, causing this runting. Indeed, oropharyngeal biopsies showed erosions with histology typical of PV, including suprabasilar acantholysis and "tombstoning" of basal cells. EM showed separation of desmosomes. Traumatized skin also had crusting and suprabasilar acantholysis. Runted mice showed hair loss at weaning. The runting and hair loss phenotype of DSG3 -/- mice is identical to that of a previously reported mouse mutant, balding (bal). Breeding indicated that bal is coallelic with the targeted mutation. We also showed that bal mice lack Dsg3 by IF, have typical PV oral lesions, and have a DSG3 gene mutation. These results demonstrate the critical importance of Dsg3 for adhesion in deep stratified squamous epithelia and suggest that pemphigus autoantibodies might interfere directly with such a function.

Identification of pemphigus vulgaris antigen extracted from normal human epidermis and comparison with pemphigus foliaceus antigen.

Immunoprecipitations of cultured keratinocyte extracts have shown that pemphigus vulgaris (PV) sera bind a polypeptide of 210,000 mol wt with disulfide-linked chains of 130,000 and 85,000 mol wt. To identify proteins in normal human skin recognized by PV antibodies, we performed immunoprecipitations of normal human epidermal extracts. All 22 PV sera tested immunoprecipitated a complex of polypeptides (PV complex) of 210,000, 130,000, and 85,000 mol wt, after reduction. One- and two-dimensional gel electrophoresis showed that the 130,000- and 85,000-mol-wt polypeptides of the PV antigen from both cultured keratinocytes and epidermis have identical charges and sizes. In addition to precipitating the PV complex, 14 of 22 PV sera also have antibodies to a calcium-sensitive epitope on a different complex of polypeptides (PF complex) which has previously been shown to be precipitated by all pemphigus foliaceus (PF) sera. The PF complex consists of polypeptides of 260,000, 160,000, 110,000, and 85,000 mol wt. Although the majority of PV sera also precipitate the PF complex, no PF sera precipitate the PV complex. Thus, PV and PF can be absolutely distinguished on a molecular level using the patients' autoantibodies. The PV and PF complexes, although distinct, have certain similarities. The 85,000-mol-wt polypeptide of each is identical. The 160,000-mol wt-peptide of the PF complex and the 130,000-mol-wt peptide of the PV complex have the same isoelectric point and both are capable of disulfide linkage to the 85,000-mol-wt polypeptide. The PV and PF complexes are closely related and may prove important in cell adhesion.

Distinction between epidermal antigens binding pemphigus vulgaris and pemphigus foliaceus autoantibodies.

Pemphigus vulgaris (PV) and pemphigus foliaceus (PF) are autoimmune blistering diseases in which antibodies develop to the cell surface of epidermal cells. In this study we sought to determine the antigenic specificity of antibodies in the sera of patients with PV and PF. Sera from 12 patients with PV were used to immunoprecipitate extracts of cultured human epidermal cells that were radiolabeled with 14C-amino acids. Immunoprecipitates were identified by SDS polyacrylamide gel electrophoresis (PAGE) and fluorography. All 12 PV sera precipitated a protein which, when reduced, displayed chains of 130,000 and 80,000 mol wt on SDS-PAGE. Electrophoresis under nonreducing conditions identified a 210,000-mol wt molecule, which was presumably formed by disulfide crosslinking of the 130,000 and 80,000-mol wt chains. Immunoprecipitates of epidermal cell extracts that were labeled with 14C-glucosamine indicated that the 130,000-mol wt chain. Seven of eight PF sera, which were run concurrently with the PV sera in this immunoprecipitation assay, did not precipitate this glycoprotein, nor did they specifically precipitate any protein. To determine if a specific molecule which reacted with antibodies in PF sera could be identified, we used immunoblot analysis of extracts of normal human epidermis. The proteins in these extracts were reduced, separated by SDS-PAGE, and electrophoretically transferred to nitrocellulose sheets or to 2-aminophenylthioether paper. Immunoperoxidase staining of the transferred proteins with PF sera indicated that four of eight PF sera contained antibodies that stained a protein band of 160,000 mol wt. Indirect immunofluorescence, using normal human skin as the substrate, indicated that IgG that was eluted from this protein band stained the epidermis in a cell surface pattern. PV sera did not specifically recognize any bands by immunoblot analysis. Immunoblots performed with PV antigen that was immunoprecipitated from cell culture extracts suggested that, once denatured for SDS-PAGE, PV antigen is no longer immunoreactive. Taken together, these data indicate that: autoantibodies contained in PV sera from various patients have a unique molecular specificity; autoantibodies from most PF sera have a specificity different from that of PV autoantibodies; and autoantibodies from various PF patients may not have identical antigenic specificities. These differences in antigenic specificity between PV and PF sera may account for the clinical and histologic differences between these diseases.

Pemphigus antibodies identify a cell surface glycoprotein synthesized by human and mouse keratinocytes.

Pemphigus is an antibody-mediated autoimmune skin disease in which loss of cell-to-cell contacts in the epidermis results in blister formation. Patients with pemphigus develop antibodies that bind to the keratinocyte cell surface, the site of primary pathology. The purpose of this study was to characterize the antigen(s) to which pemphigus antibodies bind. Because we could detect pemphigus antigen by indirect immunofluorescence on the surface of multiply-passaged cells in cultures of both a spontaneously transformed mouse keratinocyte cell line (Pam) and normal human epidermal cells, we used these cells as a source of antigen. In order to demonstrate biosynthesis of antigen and to characterize the antigen(s), we radiolabeled cell cultures with [(14)C]glucosamine or d-[2-(3)H]mannose and used different pemphigus sera to immunoprecipitate antigen from nonionic detergent extracts of these labeled cells. Specifically precipitated radiolabeled molecules were identified using sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis (PAGE) and fluorography. Sera from five of seven pemphigus patients specifically precipitated (from extracts of both Pam cells and human epidermal cells) a molecule that, when reduced, was approximately 130 kD, whereas seven normal human sera and two pemphigoid sera did not precipitate this molecule. The findings that (a) these precipitated molecules comigrated on SDS-PAGE and that (b) the 130-kD molecule could no longer be precipitated from cell extracts that had been previously reacted with a pemphigus serum, indicate that reactive pemphigus sera bind the same molecule. The molecule was not detected in the culture medium of these cells. This finding, along with the cell surface immunofluorescence pattern, suggests that the antigen is bound to the cell surface. Cultured mouse and human fibroblasts do not synthesize the antigen. The antigen contains protein because it was degraded by V8 protease and chymotrypsin, and it could also be labeled with [(14)C]amino acids. It is probably not a sulfated proteoglycan because it did not label with (35)SO(4). Taken together, these data indicate that some, but not all, pemphigus sera bind a specific cell surface glycoprotein that is synthesized by keratinocytes.

Isolation of complementary DNA for bullous pemphigoid antigen by use of patients' autoantibodies.

Autoantibodies from bullous pemphigoid (BP) patients define a 230-kD protein found in the basement membrane of stratified squamous epithelia. The purpose of this study was to isolate and characterize a cDNA clone with coding sequences for BP antigen. Poly(A+) RNA derived from total RNA of cultured keratinocytes was used, with oligo-dT priming, to construct a cDNA library in the lambda gt11 expression vector, which was screened by the immunoperoxidase method with one BP serum. One darkly stained clone, called here the BP clone, was further characterized. 9 of 9 BP sera, but none of 6 normal and 11 pemphigus sera, bound the plaques of this BP clone. Furthermore, BP IgG affinity purified on plaques of this clone, but not unrelated clones, bound the epidermal basement membrane by immunofluorescence and immunoprecipitated the 230-kD BP antigen from extracts of cultured keratinocytes. Eco RI digestion of the BP clone's cDNA insert demonstrated a 680- and 1,500-bp fragment. Northern blots of total keratinocyte RNA showed that complementary riboprobes transcribed from both fragments hybridized to a 9-kb RNA. Dideoxy DNA sequencing from the 5' end of the BP cDNA demonstrated a 1,992-bp open reading frame, encoding a peptide of 76 kD. This BP cDNA clone will be valuable for understanding the protein structure, expression, and gene organization of BP antigen.