A New Method For Detecting Aβ Oligomers Of Different Sizes

The prevalence of Alzheimer’s disease（AD）has increased rapidly worldwide,and the cause of AD is currently unclear and incurable,but there is evidence that dementia can be prevented,so it is of great significance for AD to find suitable diagnostic biomarkers for early diagnosis and intervention timely.Recently,most studies have shown that Aβoligomers are highly correlated with the cognitive impairment of AD patients and are suitable as a biomarker for the diagnosis of AD,but there is no effective method for Aβoligomer detection.This paper established a new method detecting of Aβoligomers of different sizes,which is based on the site-specific immobilization of his-tag nanoantibody through Co³⁺-NTA,then improved immunoprecipitation and combined with the covalent cross-linking of protein complex.The specific research results are as follows:（1）In this thesis,recombinant SDP6 nanobody containing his-tag was expressed and purified,and its activity was measured,and its interaction with Aβoligomer was simulated by computer.The results showed that the purity of the recombinant SDP6 nanobody after one time of immobilized metal ion affinity chromatography reached more than 92%,and the results of ELISA and Th T-F determination showed that the recombinant SDP6 nanobody has the binding activity of Aβ_12-35.The molecular docking software was used to simulate the interaction and site of the recombinant SDP6 nanobody with the Aβ_12-35oligomer,the results showed that the main force between them is hydrogen bonding and hydrophobic interaction,and the interaction sites are Ile31,Ile32,Gly33 and Leu34 in Aβ.The above results indicated that the anti-Aβnanobody SDP6 prepared in this paper has the biological activity of binding to Aβoligomer and can be used for Aβoligomer detection.（2）In this thesis,a new method for the detection of Aβoligomers of different sizes was established with a combination of nanobody fixed-site immobilization,immunoadsorption,protein complex covalent cross-linking,and electrophoresis,then the detection process was also optimized.We first obtained stable Aβ_12-35monomers,dimers and trimers through the aggregation in vitro and photo-induced crosslinking of unmodified proteins（PICUP）;the results of the optimization of the immobilized amount of nanobody and its concentration ratio to Aβoligomer indicated that when 30～60μg nanobodies were immobilized per 10μL of dextran gel and the molar ratio of nanobodies to oligomers was in the range of 1:4～1:8,the detection result was good,and the nanobody-dimer complex could be clearly detected.The application of the detection method to the simulation system and the research on non-specific adsorption showed that the method had less non-specific adsorption to albumin and it did not interfere with the detection of oligomers.In short,this paper initially established a new method for the detection of Aβoligomers,but the adsorption capacity of nanobodies needs to be improved to further improve the sensitivity of detection.