Establishment Of Amyloid-β Oligomer Detection Method

Cognitive dysfunction syndrome（CDS）is a neurodegenerative disease characterized by the accumulation of amyloid-β（Aβ）and the phosphorylation of microtubule-binding protein tau in the brains of animals such as cats and dogs,which leads to cognitive decline in cats and dogs.There is currently little research on the disease and no effective treatment available,but if they can be detected earlier,drugs and nutrition can be treated to improve their brain function.Therefore,early diagnosis is particularly important for improving CDS symptoms.There is currently no method for early diagnosis of CDS.Amyloid-βoligomers were found in cats at the age of 9,and they developed dementia symptoms at age of 10 or older.We speculate that Aβoligomers are the major cause of CDS as Alzheimer’s disease（AD）.However,the number of Aβoligomers in blood is very low,and a highly sensitive detection method is required for detection of Aβoligomers.Compared with the enzyme linked immunosorbent assay（ELISA）,the Single Molecular Array（Simoa）immunoassay platform and Meso Scale Discovery（MSD）electrochemiluminescence immunoassay platform both have a thousand-fold higher sensitivity and a wider linear range.In this study,we established detection methods for Aβoligomers for the diagnosis of early CDS based on electrochemiluminescence and single-molecule immunoassays.In this study,an Aβoligomer-specific single-chain antibody 3F,which was previously screened in our laboratory by phage display,was paired with anti-Aβmonoclonal antibodies A16（prepared in our laboratory）and 102（commercialized antibody）.Aβoligomers detection methods have been established on the Simoa and MSD platform,the specific research work is as follows:In this study,a detection method for Aβoligomers was established using the Simoa platform.First,A16 was coated on the magnetic beads as a coating antibody,and biotinylated3F was used as a detection antibody to optimize the reaction conditions.When the detection conditions were the number of magnetic beads 1.0×10~7 beads/m L,the concentration of biotinylated 3F was 0.3μg/m L,and the concentration of SBG was 50 p M,the sensitivity is the highest（0.35 pg/m L）.Further research found that when 3F was used as the coating antibody to coat the magnetic beads,and the biotinylated A16 was used as the detection antibody,the sensitivity was higher,about 0.16 pg/m L.Then the reaction conditions of 3F-Aβ-A16 were optimized.The optimal detection conditions were that the number of magnetic beads was1.0×10~7 beads/m L,the concentration of biotinylated A16 was 0.5μg/m L,the concentration of SBG was 150 p M,and the sample volume was 200μL,but the sample recovery rate was low.AD is caused by Aβaggregation as CDS in cats and dogs,this study detected the Aβoligomers in the sera of AD（positive samples）and healthy control（negative samples）by the above method,the results showed that the method did not distinguish positive samples and negative samples.Then,an antibody 102 with high affinity for Aβwas screened from many commercial antibodies,and the reaction conditions were optimized.The optimal detection conditions were that the number of magnetic beads was 1.0×10~7 beads/m L,and the concentration of biotinylated 102 was 0.1μg/m L,the SBG concentration was 50 p M,and the sample volume was 100μL,and the sensitivity was 0.056 pg/m L.This study detected the purified Aβoligomers in the sera of AD,mild cognitive impairment（MCI,weak positive samples）and healthy control by the optimal method.The results showed that the method could distinguish positive samples,weak positive samples and negative samples,suggesting that the method could detect Aβoligomers with high sensitivity.Subsequently,this study used MSD to establish a detection method for Aβoligomers.3F was used as the coating antibody,and ruthenium-labeled A16 and 102 were used as the detection antibodies,respectively,and the reaction conditions were optimized.The study found that 3F-Aβ-102 was more sensitive than 3F-Aβ-A16.Using ruthenium-labeled 102 as the detection antibody,when the concentration of 3F was 4μg/m L,the concentration of ruthenium-labeled 102 antibody was 1μg/m L,and the sample volume was 50μL,the sensitivity was the highest（0.26 pg/m L）.The biotin-streptomycin system has a further amplification effect on electrochemiluminescence,and the sensitivity of the kit was further detected by streptomycin-labeled 96-well plate.The highest sensitivity was 0.13 pg/m L when the 102 concentration was0.5μg/m L and the sample volume was 50μL.The purified samples were then tested under this condition,and the results showed that the method could clearly distinguish positive samples from negative samples,but not weakly positive samples.In conclusion,this study successfully established detection methods of Aβoligomers based on Simoa and MSD platform,and these methods could distinguish positive samples,and/or weak positive samples from negative samples,which provide new methods for the early diagnosis of CDS in cats and dogs.