Development of monoclonal antibodies for detecting hemidesmosomal proteins, bullous pemphigoid antigen 2 (BPAG2) and &alpha6 integrin: Expression of BPAG2 in epidermal malignancies

MD Marczinovits PhD^a, T Krenacs PhD^d, E Stelkovics^d, MD Korom^b, M Kiss PhD^b, S Husz MD DSc^b, G Toth DSc^c, K Rasky PhD^e, J Molnar MD DSc^a

^aUniversity of Szeged, Szeged, Hungary, ^bDermatology, Szeged, Hungary, ^cMedical Chemistry, Szeged, Hungary, ^dBay Zoltan Foundation for Applied Research, Szeged, Hungary, ^eDiagnosticum Co, Budapest, Hungary

AIM: Our purpose was to produce monoclonal antibodies specific for hemidesmosomal proteins, the BPAG2 and α6 integrin, in order to reveal their expression pattern with immunohistochemistry in normal and neoplastic tissues. METHODS: Mice were immunized with synthetic peptides and/or those of recombinant fusion variants. Hybridomas were developed with the standard methods. Supernatants of the hybridomas were then screened for immune reactivity with a sensitive polymer-immunoperoxidase technique (EnVision+Dako, Glostrup, Denmark) in frozen normal skin sections. A few antibodies could detect their epitopes in archived tissues following heat induced epitope retrieval (HIER), which were used in a retrospective study of skin malignancies. RESULTS: (a) We developed monoclonal antibodies against RSAA (542-566) peptide which represents a part of the BPAG2 NC16A domain designated as 6D1, 9G2, 7E5 and against SPDA/GFAT (685-695/1040-1053) and NKDA (1045-1073) of integrin α6 designated as 3G9 and 8G11. NKDA is the splice variant A of α6 integrin. (b) Two antibody clones, 6D1 and 9G2, were used to study BPAG2 expression in 12 solar keratosis, 6 incipient invasive squamous cell carcinomas developed from solar keratosis, 6 carcinoma basocellulare, 5 carcinoma epidermoides cornescence and 1 basosquamous carcinoma. In normal skin BPAG2 expression was restricted to the basal and basolateral membranes of basal epidermal cells. Desmogleins were parallely immunostained. In squamous cell carcinomas we observed the upregulation and delocalization to the upper cell layers of BPAG2 indicating dedifferenciation, while desmoglein reaction was either weak or missing. CONCLUSIONS: Our monoclonal antibodies are suitable for the immunohistochemical detection of BPAG2, α6 and αA integrins in formaldehyde-fixed and paraffin-embedded tissue sections. Immunodetection of formaldehyde resistant epitopes of BPAG2 with our monoclonal antibodies may have diagnostic value for indicating malignant transformation in the epidermis.

Paper presented at the International Symposium on Predictive Oncology and Intervention Strategies; Nice, France; February 7 - 10, 2004; in poster session 893 (Molecular pathology).