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Título:
CONSTRUCTION OF IMMUNE VHH LIBRARIES AND SELECTION OF SPECIFIC NANOBODIES AGAINST HUMAN IGG4 FOR DIAGNOSTIC APPLICATION IN SYSTEMIC LUPUS ERYTHEMATOSUS
Autor/es:
FLORENCIA MARCHESE; JOAQUIN BOZZO; NORA MATTION; VANINA GRIPPO
Lugar:
CABA
Reunión:
Congreso; JOINT MEETING OF BIOSCIENCE SOCIETIES; 2017
Institución organizadora:
LXII ANNUAL MEETING OF ARGENTINE SOCIETY OF CLINICAL INVESTIGATION (SAIC)
Resumen:
Systemic Lupus Erythematosus (SLE) is a chronic and inflammatory autoimmmune disease. The prevalence of SLE in Argentina is 58.6 per 100 thousands inhabitants. Due to the heterogeneity of the clinical manifestations and the wide profile of autoantibodies developed by the affected individuals, it is a challenge to diagnose specifically the disease. On one hand, it has been reported a correlation between IgG4 levels and severity of the disease. For this reason, IgG4 has been proposed as a putative biomarker for SLE diagnosis. On the other hand, camelids present heavy chain only antibodies, so the variable domain consists of a single domain (called VHH or Nanoantibody). The use of VHH has revolutionized the field of monoclonal antibodies due to their unique and superior properties compared to conventional antibodies. They have a great potential for the development of more sensitive diagnosis methods. For this reason, our goal was to construct two VHH libraries and obtain specific nanobodies against human IgG4 for their application in SLE diagnosis.In this work, two llamas were immunized with human IgG4 obtaining a high immune response measured by ELISA. Starting from llama peripheral blood, the heavy chain variable regions of conventional antibodies (VH) and only heavy chain antibodies (VHH) were amplified. In a Nested-PCR, only the VHH fragment were amplified and subsequently cloned in the pHEN4 phagemid vector. Then, TG1 E. coli bacteria were electroporated to obtain two Nanobody libraries. Phages expressing VHH that specifically recognized IgG4 biomarker were selected by Phage Display methodology. Even more, reactivity of selected Nanobodies was confirmed by Phage-ELISA.In conclusion, two immune VHH libraries were obtained with the expected size and high percentage of full length clones. Furthermore, specific Nanobodies against human IgG4 were selected by Phage display. Thus, these VHH constitutes an innovative tool for potential application in SLE diagnosis.