An ultra-sensitive immunoassay detects and quantifies soluble Aβ oligomers in human plasma

Abstract

Introduction: Evidence strongly suggests that soluble oligomers of amyloid beta protein (oAβ) help initiate the pathogenic cascade of Alzheimer's disease (AD). To date, there have been no validated assays specific for detecting and quantifying oAβ in human blood.

Methods: We developed an ultrasensitive oAβ immunoassay using a novel capture antibody (71A1) with N-terminal antibody 3D6 for detection that specifically quantifies soluble oAβ in the human brain, cerebrospinal fluid (CSF), and plasma.

Results: Two new antibodies (71A1; 1G5) are oAβ-selective, label Aβ plaques in non-fixed AD brain sections, and potently neutralize the synaptotoxicity of AD brain-derived oAβ. The 71A1/3D6 assay showed excellent dilution linearity in CSF and plasma without matrix effects, good spike recovery, and specific immunodepletion.

Discussion: We have created a sensitive, high throughput, and inexpensive method to quantify synaptotoxic oAβ in human plasma for analyzing large cohorts of aged and AD subjects to assess the dynamics of this key pathogenic species and response to therapy.

Keywords: Alzheimer's disease; amyloid beta protein; oligomeric amyloid beta; plasma biomarkers.

Figure 1. 1G5 and 71A1 pull down Aβ from human brain soaking extract.

(A) Schematic illustration of preparation of human brain soaking extracts. (B) immunoblots of immunoprecipitation with protein G by different antibodies from human brain soaking extract, detected by 2 antibodies against middle region and N-terminal of Aβ. 7.5 μL of the extract was used for the input lane and IP was from 800 μL of extract. (C-D) Aβ x-40 and x-42 measured by ELISA of immunoprecipitation or post-immunoprecipitation supernatant (all denatured in 8M GnCl) by the specified antibodies from human brain soaking extracts, n = 3, means ± SD.

Figure 2. 1G5 and 71A1 recognize Aβ in human brain in situ.

(A) Immunohistochemistry using 1C22, 1G5, and 71A1 on human brain cryo-sections; scale bar = 200 μm. (B) Immunohistochemistry using 1C22 or 71A1 on human brain cryo-sections, with double labeling by Aβ monomer antibody D54D2; bar = 200 μm for left two panels and 100 μm for right panel. (C) LTP induction after treatment with aCSF (n = 6), human brain soaking extract (n = 5), 2.12 ug/mL 71A1 (n = 4), or human brain soaking extract premixed with 2.12 ug/mL 71A1 (n = 4), means ± SD. (D) LTP induction after treatment with aCSF (n = 6) or 71A1 affinity purified oAβ (n=4), mean ± SD.

Figure 3. 1G5 and 71A1 recognize Aβ from human CSF.

(A) Aβ x-40 and x-42 measured by ELISA of immunoprecipitates or post-immunoprecipitation supernatants (all denatured by 8M GnCl) by different antibodies from 3 human CSFs, with technical replicates = 3, means ± SD. (B) Aβ x-40 and x-42 measured by ELISA of immunoprecipitatees or post-immunoprecipitation supernatants (all denatured by 8M GnCl) by 71A1 from 19 individual human CSFs, technical replicates = 3, means ± SD. (C) correlation between Aβ x-40 and x-42 measured by ELISA of immunoprecipiates (all denatured by 8M GnCl) by 71A1 and the ADmark Aβ 1–42 measurements from the same CSFs. Pearson correlation is used.

Figure 4. Development of 71A1/3D6 immunoassay specific for oAβ.

(A) Schematic illustration of the SMCxPRO beads-based immunoassay. (B-C) 1G5/3D6 and 71A1/3D6 assay performance on ADDLs as calibrator; (D) average CV and recovery of ADDLs signals using 1G5/3D6 and 71A1/3D6 assays. (E) 1C22/3D6 and 71A1/3D6 assay performance on ADDLs as calibrator. (F) CBB staining of PAGE analysis of Aβ1–40 S26 dimer without (−) or with (+) reduction by DTT. (G-H) 1C22/3D6 and 71A1/3D6 assay performance on Aβ1–40 S26 dimer without (native) or with reduction by DTT. (I) 71A1/3D6 assay performance on human brain soaking extract: left panel: raw value of 71A1/3D6 signals from serial diluted brain extract calibrated by ADDLs; middle panel: calculated value adjusted by dilution factors; right panel; recovery of each dilution normalized to 1:1K; n = 3, mean ± SD.

Figure 5. 71A1/3D6 immunoassay recognizes high molecular weight oAβ from human brain.

(A-B) Chromatogram (UV280nm absorbance) of molecular weight calibrators and human brain soluble extract fractioned by Superdex 200 increase SEC column. (C) oAβ measured by 71A1/3D6 immunoassay from SEC fractions natively; n = 3, mean ± SD. (D) monomeric Aβ x-42 measured by MSD ELISA from SEC fractions natively (red bars) or denatured (blue bars), n = 2, mean ± SD.

Figure 6. 71A1/3D6 immunoreactive oAβ in CSF correlates with t-Tau and p-Tau levels.

(A) 71A1/3D6 assay performance on human CSF: left panel: raw values of 71A1/3D6 signals from serially diluted CSF (against ADDLs calibrator); middle panel: calculated value adjusted by dilution factors; right panel: recovery of each dilution normalized to 1:2; n = 3, means ± SD. (B-D) Correlations between 71A1/3D6 reactive oAβ levels in CSF and ADmark measurements of Aβ 1–42, t-Tau and p-Tau; n = 29. Pearson correlation is used. (E) Chromatogram (UV280nm absorbance) of human CSF fractioned on a Superdex 200 Increase SEC column. (F) oAβ measured by the 71A1/3D6 immunoassay in SEC fractions natively; n = 3, mean ± SD.

Figure 7. 71A1/3D6 immunoassay accurately quantifies oAβ from human plasma.

(A) 71A1/3D6 assay performance on human plasma: left panel: raw value of 71A1/3D6 signals from serially diluted plasma against ADDLs calibrator; middle panel: calculated values adjusted by dilution factors; right panel: recovery of each dilution normalized to 1:4; n = 3, means ± SD. (B) spike-in recovery test by spiking human brain homogenate (Brain H), human brain soaking extract (Brain S) or human CSF into 8-fold diluted individual plasma; n = 3, means ± SD. (C) 71A1/3D6 assay of oAβ from 8-fold diluted individual plasmas immunodepleted by different antibodies, compared to the input plasma; n = 4. (D) 71A1/3D6 assay of oAβ from human brain soaking extract, CSF and plasma denatured by 8M GnCl (or not); n = 3, means ± SD. (E) 71A1/3D6 assay of oAβ from 73 human plasmas (Mayo Aging cohort), n = 4.