INVESTIGADORES
GRIPPO vanina
congresos y reuniones científicas
Título:
OBTENTION OF SPECIFIC SINGLE DOMAIN ANTIBODIES DERIVED FROM CAMELIDS (VHH OR NANOBODIES) AGAINST PLASMA BIOMARKERS OF ALZHEIMER?S DISEASE FOR DIAGNOSTIC APPLICATION
Autor/es:
JOAQUIN IGNACIO BOZZO; MARINA GALLO CALDERON; NORA MARTA MATTION; VANINA GRIPPO
Lugar:
Mar del Plata
Reunión:
Congreso; LXI REUNIÓN ANUAL DE LA SOCIEDAD ARGENTINA DE INVESTIGACIÓN CLÍNICA (SAIC); 2016
Institución organizadora:
SAIC, SAI, SAFE, NANOMEDAR, AACYTAL
Resumen:
Alzheimer?s disease (AD) affects Nervous Central System before clinical symptoms are fully expressed. Currently AD diagnosis depends on clinical evaluations, and there is no reliable early diagnostic test. Direct measure of plasma biomarkers for AD, such as alpha-1-antitrypsin (A1AT) and alpha-2-macroglobulin (A2M), could lead to an earlier and more sensitive diagnosis. Nowadays, single domain antibodies derived from camelids (VHH or nanobodies) represent a very useful tool with unique characteristics, making VHH more suitable for sensitive and specific diagnosis. For this reason, we aimed to obtain and produce specific VHH against blood-based protein biomarkers for AD, A1AT and A2M, with potential application in the development of a simple and early diagnostic test.In this work, two llamas were immunized with A2M and A1AT proteins obtaining a high humoral response in both animals. Two VHH libraries were constructed starting from peripheral blood lymphocytes from both llamas. Library quality controls were performed to corroborate full-length VHH insert and repertoire variability. A high percentage of full-length clones (97.6 % and 100%) and at least 35 different fingerprinting profiles were determined for each library. Then, phages that expressed in its surface specific VHH against both biomarkers were selected by Phage Display methodology. After two-round selection, specific reactivity of 45 and 10 selected clones for A2M and A1AT respectively, was confirmed by ELISA. Four most reactive nanobodies were sub-cloned into pHEN6 vector and expressed as soluble protein in E. coli. VHH expression was confirmed by Western Blot detecting a unique band of approximately 15 kD as expected. Finally, specific biomarker recognition by expressed VHH was confirmed by ELISA and Western Blot. In conclusion, specific Nanobodies against blood-based protein biomarkers of AD, A2M and A1AT, were obtained, expressed and preliminarily characterized for potential application in AD diagnosis.