Single chain variable fragment against aß expressed in baculovirus inhibits abeta fibril elongation and promotes its disaggregation.

ABSTRACT

Alzheimer's disease (AD) is the most common form of age-related dementia, and the most urgent problem is that it is currently incurable. Amyloid-ß (Aß) peptide is believed to play a major role in the pathogenesis of AD. We previously reported that an Aß N-terminal amino acid targeting monoclonal antibody (MAb), A8, inhibits Aß fibril formation and has potential as an immunotherapy for AD based on a mouse model. To further study the underlying mechanisms, we tested our hypothesis that the single chain fragment variable (scFv) without the Fc fragment is capable of regulating either Aß aggregation or disaggregation in vitro. Here, a model of cell-free Aß "on-pathway" aggregation was established and identified using PCR, Western blot, ELISA, transmission electron microscopy (TEM) and thioflavin T (ThT) binding analyses. His-tagged A8 scFvs was cloned and solubly expressed in baculovirus. Our data demonstrated that the Ni-NTA agarose affinity-purified A8 scFv inhibited the forward reaction of "on-pathway" aggregation and Aß fibril maturation. The effect of A8 scFv on Aß fibrillogenesis was markedly more significant when administered at the start of the Aß folding reaction. Furthermore, the results also showed that pre-formed Aß fibrils could be disaggregated via incubation with purified A8 scFv, which suggested that A8 scFv is involved in the reverse reaction of Aß aggregation. Therefore, A8 scFv was capable of both inhibiting fibrillogenesis and disaggregating matured fibrils. Our present study provides valuable insight into the regulators of ultrastructural dynamics of cell-free "on-pathway" Aß aggregation and will assist in the development of therapeutic strategies for AD.