P1-365: A method for measuring anti-beta amyloid (Aβ) oligomer antibodies in human plasma and intravenous iimmunoglobulin

Abstract

Background: We have found natural human antibodies in plasma and intravenous immunoglobulin (IVIg) that bind to soluble A oligomers but not monomers and exhibit conformational specificity. These antibodies have a strong affinity for A oligomers 56kD in size and may be part of an innate immune response to toxic peptide oligomers. To further explore the properties of these antibodies, we have developed a sensitive assay for quantifying anti-oligomer antibodies in plasma and other fluids. Methods: There is a sub-population of natural antibodies and other proteins in humans that bind to polystyrene and other matrixes and yield signals nearly comparable to anti-A oligomer antibodies. This background binding interferes with standard ELISAs in which with A oligomers are bound to the plate. To address this issue, we mixed antibodies with biotinylated A 12mer in solution and captured the resulting antigen/antibody complexes using pre-coated streptavidin plates. The A 12-mer oligomer was made in the presence of 0.2% SDS, which promotes the formation of a globular A 12mer [Barghorn, et al. J.Neurochem. 95:834-47]. SEC and SDS:PAGE analysis demonstrated that the bulk of the A in this preparation was a stable 12mer. Biotin was coupled to the A 12mer using EZ-Link NHS-PEO12-biotin [Pierce] as specified by the manufacturer. Approximately 10 g/ml of this biotinylated 12mer was incubated with decreasing amounts of either IVIg or IgG, purified from human plasma by Protein G chromatography, for 1hr at RT and then bound to streptavidin plates for 2 hrs at RT. The amount of human antibody bound was determined using HRP-conjugated anti-human IgG secondary antibodies. Results: Although there was still detectable binding of human antibodies to the streptavidin plates in control reactions lacking the biotinylated 12mer, the signal to noise ratio was greatly improved over standard ELISA allowing meaningful comparisons of antibody levels. Signal to noise was further improved by pre-absorbing samples on glass/agarose/polystyrene beads. Conclusions: Using this newly developed method, we are now in the process of determining whether the level of anti-A 12mer antibodies varies among individuals as a function of age and AD disease state.