Initial Test Of Immobilization And Application Of Anti-A? Nanobody

Alzheimer's disease is a chronic and progressive development of central nervous system degenerative diseases,accounting for 60%-70%of the total dementia type.It is important to study effective Alzheimer's treatment and prevention strategies.The amyloid cascade is widely accepted.It is believed that A?,the main component of senile plaques in the brain of patients,is the main pathogenic factor of Alzheimer's disease.Soluble aggregates formed by A?are more toxic to the nervous system than monomer A?.Antibodies against A?soluble aggregates are important for the treatment of AD.Nanobodies have small molecular weight,high specificity and stability,strong hydrophilicity and easy modification.They are good adsorbent ligands.Because of the structural characteristics of Nanobodies,it is necessary to adopt suitable immobilization methods to ensure that their activities are as free from loss.Co³⁺can mediate the interaction between His-tag protein and NTA.The complex of Co³⁺-Pr-NTA has thermodynamic stability and is weakly reacted with a chelating agent or a reducing agent,and can be used for immobilization of a Nanobody.This study explored the immobilization conditions of Nanobody,and immobilized Nanobodies were used to detect,adsorpt and remove A?,and initially explored the removal effect of A?in cerebrospinal fluid.?1?In this study,two anti-A?Nanobodies,SDP6 and SH2 were purified and assayed for activity.The results showed that the purity of SDP6 after immobilized metal ion affinity chromatography was above 90%,and the Biacore measurement showed that the affinity between SDP6 and A?was 4.6×10^-7M.The purity of SH2 after metal ion affinity chromatography and gel chromatography was 85%or more in turn,and the Biacore measurement showed that the affinity between SH2 and A?was 4.0×10^-10M.All of these results indicate that we have successfully obtained antibodies with higher A?binding capacity.?2?The His-tagged Nanobody was immobilized on an agarose beads coupled with cobalt ions,and the stability of the adsorbent after the immobilization process and the immobilization was evaluated.Increasing the concentration of Nanobodies while controlling the oxidation time of H₂O₂ can achieve higher immobilization efficiency at 60 min.The parameters of Nanobodies after immobilization were tested.The immobilized Nanobodies showed higher tolerance to imidazole and EDTA.Storage stability was good and kept high activity.?3?The immobilized Nanobody was used for A?detection,which showed good A?detection ability.Nanobody was used as a ligand for affinity chromatography,and micro-dialysis probe was used to microdialysis the simulated cerebrospinal fluid to achieve the removal of A?in cerebrospinal fluid.Simulation experiments of the adsorbent in vitro showed that it can achieve90%A?removal in the simulated cerebrospinal fluid system.In order to investigate the A?removal effect of sorbent in cerebrospinal fluid,we established an APP/PS1 double transgenic mouse model and evaluated its behavior.Through genetic identification and behavioral testing,we have successfully established an 8-month-old AD mouse model.