Establishment Of A Method For Determination Of Aβ Antibodies In Plasma And Preliminary Analysis Of Aβ Antibodies Level In Plasma Of Donors

Objective:Establish an accurate enzyme linked immunosorbent assay(ELISA)method for determination of amyloid beta 42(Aβ42)oligomers antibodies in plasma and use it to examine Aβ42 antibodies level in plasma of donors,so as to lay the foundation for preparation of specific Intravenous Immunoglobulin(IVIg)with rich Aβ42 antibodies.Methods:1)Aβ42 oligomers were prepared from synthetic Aβ42 peptide at different initial aggregation concentrations of Aβ42 and through addition of 0.05%sodium dodecyl sulfate(SDS)to phosphate buffer saline(PBS)system,then oligomeric preparations were analyzed by SDS-polyacrylamide gel electrophoresis(SDS-PAGE)and western blot(WB);finally the best synthetic method was chosen.2)In preliminary experiment,factors having greater impact on determination of Aβ42 oligomers antibodies in plasma by ELISA were screened out;the orthogonal experiment wasdesigned to establish an accurate determination system;and the performance of this system was evaluated.3)The levels of Aβ42 oligomers antibodies were tested in plasma samples collected from different sources,and their distribution characteristics were analyzed.Results:1)The amount of Aβ42 oligomers rose with the increase of the initial aggregation concentration.Trimers and dispersion zones of medium-and high-molecular-weight oligomers were generated from Aβ42 at 250,125 and 62.5 μmol/L when there was no SDS in PBS;trimers and medium-molecular-weight oligomers were generated at the same concentrations when SDS was mixed with PBS.2)The orthogonal analysis showed that the detection system consisting of Aβ42 oligomers coating concentration(0.2μg/hole),coating pH(6.0),coating ion concentration(0.01mol/L),blocking buffer(15%BSA),primary antibody(4 ℃/over night)and secondary antibody(1/2000,25℃/3h)conditions was the best.The correlation coefficient was 0.994;the relative deviation of stability was 3.5%;the relative deviation of precision was 4.77%;and the accuracy error was 10.16%;all of which met the standards of Clinical and Laboratory Standards Institute(CLSI)and other standards.3)The testing of plasma samples from Sichuan and Guizhou revealed that Aβ42 oligomers antibodies in plasma of female donors were 10.1%higher than those in male plasma and that the level of A《42 oligomers antibodies increased with age.The level of antibodies in samples from Guizhou was 25.0%higher than that in those from Sichuan.There was no obvious difference between A》42 oligomers antibodies of different ABO blood types.The proportion of high value samples from Guizhou is higher than that from Sichuan.Conclusion:1)Appropriate initial aggregation concentrations contribute to the generation of Aβ42 oligomers.Addition of 0.05%SDS to PBS is conducive to formation of stable Aβ42 oligomers.2)The ELISA determination system obtained from orthogonal experiment is stable and can be used for testing of plasma samples.3)The distribution of Aβ42 oligomers antibodies varies with gender,age and region.Plasma with high levels of Aβ42 oligomers antibodies can be screened out.