Design of a Dimeric‐nanobody Specific for Aß1‐42 Oligomers: the Dimeric‐DesAb‐O

AbstractBackgroundSmall, soluble oligomers, rather than mature fibrils, are the major neurotoxic agents in Alzheimer’s disease (AD). In the last few years, Aprile and co‐workers designed and purified a single‐domain antibody (sdAb), called DesAb‐O, with high specificity for Aβ1‐42 oligomeric conformers. Recently, Cascella and co‐workers showed that DesAb‐O can selectively detect synthetic Aβ1‐42 oligomers both in vitro and in cultured cells, neutralizing their associated neuronal dysfunction. DesAb‐O can also identify Aβ1‐42 oligomers in the cerebrospinal fluid (CSF) of AD patients, with respect to healthy individuals, preventing cell dysfunction induced by the administration of CSFs to neuronal cells. Given the extraodinary potentialities of this nanobody, we design a dimeric‐structure of DesAb‐O, with the aim to increase its avidity and affinity for toxic Aβ1‐42 oligomers.MethodWe designed the dimeric‐DesAb‐O structure by linking two DesAb‐O monomeric domains with a flexible linker region (GGGGS)3. Once expressed and purified the protein, we characterised its molecular weight by mass spectrometry and its secondary structure by Circular Dichroism. Then, we performed an aggregation assay to monitor its ability to interfere with the Aβ1‐42 aggregation process and a Real‐Time based ELISA assay to study its binding for Aβ1‐42 oligomers.ResultThe Dimeric‐DesAb‐O is able to interfere with the Aβ1‐42 aggregation process to a greater extent than DesAb‐O. Furthermore, the dimeric structure of DesAb‐O showed a higher specificity and affinity for Aβ1‐42 oligomers compared to DesAb‐O.ConclusionThe Dimeric‐DesAb‐O appears to be a promising tool for the future development of sdAbs‐based immunodiagnostic tests for the early diagnosis of AD.