Detection of complement activation on antigen microarrays generates functional antibody profiles and helps characterization of disease-associated changes of the antibody repertoire.

Humoral immune responses are traditionally characterized by determining the presence and quality of Abs specific for certain Ags. Arraying of large numbers of Ags allows the parallel measurement of Abs, generating patterns called Ab profiles. Functional characterization of these Abs could help draw an even more informative map of an immune response. To generate functional Ab profiles we simultaneously tested not only IgM, IgG, and IgA binding to, but also complement activation by, a panel of endogenous and exogenous Ags printed as microarrays, using normal and autoimmune human sera. We show that complement activation by a particular Ag in a given individual cannot be predicted by the measurement of Ag-specific Abs, despite a general correlation between the amount of Ag-bound Ab and the deposited C3 fragments. This is due to both differences in the isotypes that dominate in the recognition of an Ag and individual variations for a given isotype, resulting in altered complement activation potential. Thus, Ag-specific C3 deposition can be used as an additional parameter in immune response monitoring. This is exemplified by comparing the coordinates of Ags, used for the diagnosis of systemic lupus erythematosus, of normal and autoimmune serum samples in a two-dimensional space derived from C3 deposition and Ab binding. Since cleavage fragments of C3 mediate important immunological processes, we propose that measurement of their deposition on Ag microarrays, in addition to Ab profiling, can provide useful functional signature about the tested serum.

Collagen XVII/BP180 protein expression in squamous cell carcinoma of the skin detected with novel monoclonal antibodies in archived tissues using tissue microarrays and digital microscopy.

Collagen XVII/BP180, a hemidesmosomal adhesion protein, is lost during normal keratinocyte maturation; however, it may be reexpressed upon malignant transformation. In this work, highly sensitive monoclonal antibodies 6D1 and 9G2 were produced, characterized, and used for the detection of collagen XVII in a tissue microarray series of archived samples of nonmelanocytic epithelial neoplasias, including 5 verruca vulgaris, 14 seborrheic keratosis, 38 actinic keratosis, 38 basal cell carcinoma (BCC), 15 basosquamous carcinoma, 58 squamous cell carcinoma (SCC), and 9 normal skin. Digital microscopy and a new tissue microarray software linking image and patient data allowed easy and validated evaluation and quality archiving of stained samples. In normal skin and benign epidermal lesions, collagen XVII protein was restricted to basal keratinocytes. However, possibly as a sign of undifferentiated/transformed state, it was widely expressed in SCC showing elevated levels around invasive tumor fronts with some staining in tumor adjacent stroma, endothelium, and histiocytes. Collagen XVII immunostaining of atypical keratinocytes in most actinic/solar keratosis supports the view of their malignancy and common origin with SCC. Squamous component of basosquamous carcinoma showed moderate reaction, whereas islets of BCC were mainly negative reflecting the diverse genotype and phenotype, and pathogenesis of SCC and BCC. These results suggest that collagen XVII neoexpression may be associated with early atypia/malignant transformation of keratinocytes. Further investigations are under way to analyze the potential of these antibodies for tracing progression and metastatic potential of skin tumors.