Desarrollo de plataforma tecnológica de obtención de nanoanticuerpos para diagnóstico de la enfermedad de Alzheimer

GRIPPO V; BOZZO J; DE NICOLA J; MATTION N

LA HABANA

Congreso; 5to Seminario Internacional de Nanociencias y Nanotecnologías; 2015

Oficina del Asesor Científico, Consejo de Estado, Cuba

DEVELOPMENT OF A TECHNOLOGICAL PLATFORM TO OBTAIN NANOBODIES FOR DIAGNOSIS OF ALZHEIMER´S DISEASEIntroduction. Alzheimer´s Disease (AD) is an incurable neurodegenerative disease. Due to the lack or misdiagnosis, it is important to identify easily measured biomarkers for diagnosis as Alpha-2-macroglobulin (A2M) and Alpha-1-antitrypsin (A1AT). Due to its unique properties, Nanobodies revolutionized the field of monoclonal antibodies and have great potential in the development of new diagnostic methods more sensitive and specific. Purpose of the study. Obtain and produce Nanobodies against A2M and A1AT for direct measurement in patients allowing the development of a simple diagnostic method for EA. Materials and methods. Two llamas were immunized with A2M and A1AT obtaining a high immune response. From 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 phagemid vector pHEN4. TG1 E.coli bacteria were electroporated to obtain two Nanobody libraries. Phage expressing VHH that specifically recognized biomarkers in Phage-ELISA polyclonal and monoclonal were selected by Phage display. Soluble expression of the selected Nanobodies was induced and specific reactivity was confirmed by ELISA and Western Blot. Results and conclusions. Two immune VHH libraries were obtained with size > 107 ufc /ug DNA and high percentage of full length clones (92% and 100%) determined by colony-PCR. Specific Nanobodies against the biomarkers of AD, A2M and A1AT, were selected and characterized. This platform constitutes an innovative tool in the development of a new diagnostic method for AD and could be used for more biomarkers and/or diseases.