Bullous pemphigoid is an autoimmune blistering skin disease characterized by the presence of circulating autoantibodies which recognize specific proteins of the epidermis and dermoepidermal junction. Diagnosis is based on clinical criteria and laboratory investigations, notably histology, direct and indirect immunofluorescence, and ELISA. This study describes a new immunofluorescence assay for parallel determination of anti-BP180 and anti-BP230 based on recombinant antigenic substrates. The aim of the study was to detect BP180 and BP230 autoantibodies by BIOCHIP technology using both a specially designed recombinant BP180-NC16A protein and cells expressing the BP230-gc antigen fragment. 18 patients with bullous pemphigoid were included in the study. Autoantibodies to BP180 were detected by the BIOCHIP technique in 83.33% of patients with clinical, serological, and immunohistological confirmed bullous pemphigoid while autoantibodies against BP230-gC were detected only in 39% of patients. The detection of anti-BP180-NC16A and anti-BP230-gC by a new biochip-based immunoassay is a suitable alternative to indirect immunofluorescence and ELISA. This method has the advantage of easily discriminating the different autoantibody specificities. The BIOCHIP method is faster, cheaper, and easy to use when compared with the ELISA approach. For this reason, the new method could be used as an initial screening test to identify patients with bullous pemphigoid, and doubtful results could then be confirmed by ELISA.

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Abstract. Pemphigus vulgaris (PV) isPemphigus vulgaris (PV) is a rare autoimmune intraepithelial blistering skin disease characterized by the presence of circulating autoantibodies directed against desmoglein 3 (DSG3) and desmoglein 1 (DSG1), resulting in loss of the normal epithelial cell-to-cell adhesion, through a process called acantholysis (1). The diagnosis of PV is generally based on clinical features, histology and immunological tests, notably direct and indirect immunofluorescence and enzyme linked immunoassorbent assay (ELISA) (2). In recent years, a BIOCHIP-based indirect immunofluorescence technique for the determination of anti-DSG3 and anti-DSG1 autoantibodies has been described (3, 4). Today, serum anti-DSG3 and anti-DSG1 ELISA is regularly used for the diagnosis and follow up of pemphigus (5, 6). Saliva may be also used for a non-invasive diagnosis of this autoimmune skin condition. The use of saliva anti-DSG3 and anti-DSG1 ELISA for the diagnosis of PV has been already reported (7,8). In the present pilot study, we performed both ELISA and BIOCHIP using salivary and serum samples from the same patients to investigate if the detection of anti-desmogleins autoantibodies in salivary samples by BIOCHIP could be used as test for the diagnosis of PV. Patients and MethodsThe study comprised 8 caucasian patients with PV, 3 males and 5 females ( Table I). The diagnosis of PV was based on clinical features, histology and immunopathological findings, notably positive direct immunofluorescence, serum detection of anti-DSG3 and anti-DSG1 autoantibodies by ELISA and serum BIOCHIP. Serum and salivary samples from 2 normal healthy individuals, 1 patient with epidermolysis bullosa acquisita and 2 patients with bullous pemphigoid were used for negative controls (Table II).For the detection of autoantibodies, an ELISA assay was employed using recombinant proteins DSG1 and DSG3 (MBL, Nagoya, Japan), made of the entire extracellular domain of DSG1 and DSG3 respectively and produced by Amagai et al. (9) with cut off values of 20 U/ml for DSG1 and DSG3. For the detection of autoantibodies by BIOCHIP Technology recombinant DSG1 and DSG3 expressing human cells (HEK293 cells) have been used as substrates (3). They are applied to thin glass slides, mechanically cut into millimeter-sized fragments (BIOCHIPs) and then applied 97

The results of this study show that patients carrying at least one T allele of the TLR7 promoter polymorphism are associated with an increased probability to be resistant to imiquimod therapy.

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