We performed several tests of the sensitivity and specificity of the HJ3.4-based, 384 well plate single photon counting immunoassay. The number of single photon detected events rose monotonically with increasing concentrations of synthetic Aβ dimers (). Levels as low as 1.56 pg/ml were detectible above background (
inset). Using strict criteria for quantitative reliability, the lower limit of quantitation was determined to be 6.25 pg/ml (), or 0.72 pM (calculated molecular weight of the synthetic dimers 8690.8 Da). The assay did not detect monomeric Aβ at concentrations as high as 10,000 pg/ml (). Aβ was maintained in monomeric form using the 0.1% HFIP to disrupt hydrogen bonding (
left lane), as otherwise many species of Aβ will aggregate spontaneously in physiological solutions. HFIP at this concentration did not interfere with the sensitivity of the assay (). Likewise, the assay was similarly sensitive to another synthetic Aβ heterogeneous oligomer preparation, produced by incubating monomeric Aβ under conditions favoring aggregation, and then cross linking the aggregates with glutaraldehyde ().
Figure 1 Sensitivity and specificity characteristics of the Aβ oligomer assay. A. Standard curve demonstrating a monotonic relationship between detected events and the concentration of synthetic Aβ1-40Ser26Cys dimer. Inset: concentrations of Aβdimer (more ...)
Amyloid-β Oligomer Assay Characteristics
The HJ3.4 antibody used in this assay did not recognize amyloid precursor protein 43
. To verify this specificity, an immunodepletion assay was performed on brain homogenate from a 9 month old 3xTg-AD mouse 38
containing high levels of human amyloid precursor protein. Incubation with 6E10 44
, a monoclonal antibody known to recognize both Aβ and amyloid precursor protein 45, 46
, depleted the lysates of immunoreactivity at the expected molecular weight of amyloid precursor protein (), whereas incubation with 82E1, a monoclonal antibody with known specificity for Aβ over amyloid precursor protein 47
did not. Incubation with HJ3.4, like incubation with 82E1, did not deplete the lysates of amyloid precursor protein immunoreactivity (). This indicated that HJ3.4 had very low binding to amyloid precursor protein in solution.
The assay was specific for oligomeric Aβ and did not recognize oligomeric forms of other peptides, unlike assays based on conformation specific antibodies 8
. Oligomeric forms of A-Dan, implicated in familial Danish dementia 48
and A-Bri, implicated in familial British dementia 49
, were not detected at concentrations as high as 200 pg/ml (). Likewise, oligomeric forms of N-terminally truncated and pyro-glutamate modified Aβ 50
were not detected by the assay ().
In principle, because the HJ3.4 antibody used to coat the plate binds to both monomeric and oligomeric Aβ, high concentrations of monomers could saturate the available binding sites on the plate and reduce the sensitivity of the assay. We found no evidence of such saturation effects at monomeric Aβ concentrations up to 2000 pg/ml (). Furthermore, when synthetic Aβ oligomers were added to frontal cortex homogenates, the levels detected were greater than 90% of the expected levels. Specifically, recovery was 96–100% when 12.5 pg/ml synthetic Aβ oligomers were added. 91–93% when 25 pg/ml were added, and 95–96% when 50 pg/ml were added. Each recovery assay was performed in 5 separate brain homogenates. Thus, the immunoassay was demonstrated to be both highly sensitive to and specific for oligomeric Aβ.
To demonstrate the utility of the assay, we assessed frontal cortex samples obtained at autopsy from normal elderly controls without significant AD pathology, non-demented (CDR 0) elderly patients with known Aβ plaque pathology, and patients with mild dementia of the Alzheimer’s type (CDR 1) and Aβ plaque pathology. The 3 groups of patients did not differ in age (p=0.83, Kruskal-Wallis Test) or post-mortem interval (p=0.46). Most were female (). As expected, Aβ immunohistochemistry revealed no plaque pathology in the normal elderly controls (). There were variable degrees of plaque coverage in both the CDR 0 non-demented elderly patients with known Aβ plaque pathology () and the patients with CDR1 mild dementia of the Alzheimer’s type (). The plaque coverage expressed as % of gray matter area was quantified in a blinded fashion on 8 slices per brain sample. The extent of Aβ plaque coverage did not differ between non-demented elderly patients with plaque pathology and patients with mild dementia of the Alzheimer’s type in this study (p=0.11, ). This is consistent with previous reports on “preclinical” AD 1, 3, 51
Characteristics of Human Brain Frontal Cortex Samples
Figure 2 Oligomerization of Aβ is tightly linked to plaque density in dementia of the Alzheimer type but not in high pathology elderly controls. A–C. Aβ immunohistochemistry using a polyclonal rabbit anti-pan-Aβ on frontal cortex (more ...)
Adjacent frontal cortex samples from the same patients were homogenized and found to contain variable levels of Aβ oligomers (). Similar results in demented patients have been reported previously using an immunoprecipitation and Western blotting-based approach 13
. Aβ oligomer levels for the normal controls were essentially at or only slightly above the lower limit of quantitation. Aβ oligomer in both the CDR 0 with plaque pathology group and the CDR 1 group were significantly elevated (p=0.0003, Mann Whitney U Tests). The Aβ oligomer levels in the CDR 0 plus pathology group were statistically lower than those in the CDR 1 group (p=0.0023). However, there was considerable overlap between groups ().
Aβ oligomerization did not appear to be an artifact of the homogenization and assay procedures. The addition of 1000 pg/ml of Aβ1-42 added into the homogenization buffer along with brain tissue from normal control subjects before homogenization did not result in any detectible oligomer signal (). The presence of brain homogenate did not obscure or block oligomer detection, as addition of Aβdimer to homogenates resulted in the expected elevation in signal.
Interestingly, the quantitative correlation between Aβ oligomer levels and Aβ plaque coverage was very strong in the CDR 1 group (r2 =0.88), but less tight in the CDR 0 plus plaque pathology group (r2 =0.33, ). The slopes of the regression lines were statistically significantly different (F(1,19)= 52.8, p=<0.0001). Unlike plaque area or oligomer measurements in isolation, the ratio of Aβ oligomer levels to Aβ plaque coverage completely distinguished the two groups, with no overlap in these populations ().
We next repeated these analyses in parietal cortex samples and obtained qualitatively similar results. As in frontal cortex, the extent of parietal cortex Aβ plaque coverage did not differ significantly between CDR 0 plus plaque pathology subjects and CDR 1 subjects (p=0.16, ). Likewise, The Aβ oligomer levels in the CDR 0 plus pathology group were statistically lower than those in the CDR 1 group (p=0.014) but there was considerable overlap between groups (). Most importantly, the ratio of Aβ oligomer levels to Aβ plaque coverage again completely distinguished the two groups, with no overlap in these populations ().
Figure 3 Similar relationship between Aβ oligomerization and plaque pathology in parietal cortex. A. Parietal gray matter coverage by Aβ plaque pathology was not different in the non-demented elderly subjects with plaques (CDR 0 + plaques) vs. (more ...)
Several other measures did not fully distinguish CDR 1 from CDR 0 plus plaque pathology samples. Specifically, we assessed measurements of PBS-soluble Aβ1-40, PBS-soluble Aβ1-42, guanidine-soluble Aβ1-40, guanidine-soluble Aβ1-42, X-34 labeled fibrillar plaque areas, and ratios of these parameters to Aβ plaque area (–). Expected differences from normal controls and correlations between parameters were observed, but there was substantial overlap in every measure between the CDR 1 and CDR 0 plus plaque pathology groups.
Figure 4 Assessments based on overall Aβ levels did not distinguish tissue from patients with mild dementia of the Alzheimer type (CDR 1) vs. non-demented elderly patients with plaque pathology (CDR 0 + plaque). A. No difference between groups in overall (more ...)
Figure 5 Assessments based on X-34 staining of fibrillar plaque pathology did not distinguish between tissue from patients with mild dementia of the Alzheimer type (CDR 1) vs. non-demented elderly patients with plaque pathology (CDR 0 + plaque). A–I: Exemplar (more ...)
Despite the high sensitivity of the assay, oligomeric Aβ was not detected in cerebrospinal fluid samples from separate patients with mild dementia of the Alzheimer’s type (). Many of these samples had low Aβ1-42
, high tau and high phospho-tau, consistent with previously reported cerebrospinal fluid biomarkers of Alzheimer’s disease pathology 42
Characteristics of Human Cerebrospinal Fluid Samples
To determine whether the size forms of oligomeric Aβ also differed between CDR 1 and CDR 0 plus plaque pathology samples, we used two methods: immunoprecipitation-Western blotting and size exclusion chromatography. On immunoprecipitation-Western blotting, we observed primarily ~4 kDa and ~8 kDa bands consistent with monomers and dimers. These were present in both CDR 1 and CDR 0 plus plaque pathology samples. However, the dimer band could not be reliably quantified because its intensity was highly variable in our hands when aliquots of the same homogenates were assessed repeatedly.
On size exclusion chromatography, we detected oligomeric Aβ using our plate-based fluorescence immunoassay primarily in fractions 7–10 from both CDR 1 and CDR 0 plus plaque pathology samples (, representative of samples from 3 patients in each group). These fractions correspond to high molecular weight species, as a thyroglobulin protein standard (670 kDa) eluted off the column in fractions 8–10 (). The exact size of the oligomeric Aβ cannot be determined precisely, however, because aggregated Aβ likely does not behave like a globular protein. Importantly, though, the location and shape of the peak did not differ between CDR 1 and CDR 0 plus plaque pathology samples. Furthermore, there was no oligomer signal in fractions 15–19 (), where synthetic monomeric and dimeric Aβ eluted (). The same fractions were assessed for total Aβ using an indirect ELISA that does not distinguish monomeric from oligomeric species. Fractions 15–19 from both CDR 1 and CDR 0 plus plaque pathology samples contained substantial amounts of Aβ (). Our plate-based fluorescence immunoassay is capable of detecting Aβ dimers if present (), which indicates that most likely the Aβ in fractions 15–19 in monomeric. Taken together, these results indicate that and the majority of Aβ oligomeric species in these lysates are high molecular weight, and that the size of the oligomers does not distinguish between demented and non-demented subjects with Aβ plaque pathology.
Figure 6 Size exclusion chromatography indicated that Aβ oligomers were primarily high molecular weight both in non-demented patients with Aβ plaque pathology (CDR 0 + Plaques) and patients with mild dementia of the Alzheimer type (CDR 1). A. Plate-based (more ...)