ELISA measurement of specific non-antigen-bound antibodies to Aβ1-42 monomer and soluble oligomers in sera from Alzheimer's disease, mild cognitively impaired, and noncognitively impaired subjects

Serum samples were obtained from the Rush Alzheimer's Disease Center (Chicago, IL) from individuals whose diagnosis on the basis of post-mortem clinical review was AD, MCI, or NCI. MCI subjects had only one impaired cognitive domain and no other apparent cause of cognitive impairment. AD patients had no other apparent cause of cognitive impairment. These individuals were participants in the Rush Memory and Aging Project, a community-based, longitudinal clinical-pathologic study of aging and AD. Details of this project were published previously [29]. The study was approved by the Institutional Review Board of Rush University Medical Center and was given exempt status by Beaumont's Human Investigation Committee. Subject summary statistics are shown in Table 2.

Aβ monomer was prepared as described previously [20, 21, 30]. Aβ1-42 (0.5 mg; AnaSpec, San Jose, CA) was disaggregated by resuspending in 0.25 ml trifluoroacetic acid (TFA, Sigma-Aldrich, Inc., St. Louis, MO) followed by hexafluoro-2-propanol (HFIP, Sigma-Aldrich). It was aliquoted into eppitubes (20 μl/tube), dried overnight (16-20 hr) at room temperature in a fume hood, and stored at -20°C. The Aβ was resuspended in HPLC-grade water adjusted to pH 3.0 with TFA (1 μl TFA per 10 ml HPLC H₂O). 0.6 ml TFA water was added to an Aβ-containing eppitube, and after thorough vortexing, this was put on ice in a separate tube. The procedure was repeated twice more on the same eppitube, yielding 1.8 ml of Aβ in TFA water. Tris base (21.8 mg) was added to bring the Tris concentration to 100 mM, and 3.8 μl of 12.1 N HCl was added to adjust the pH to 8.8. The preparation was centrifuged (11,752 × g, 5 min), passed through a 0.2 μm filter, and used immediately. The protein concentration of the filtered preparation was 6 μg/ml with the Bio-Rad Protein Assay (Bio-Rad Laboratories, Hercules, CA).

Aβ oligomers were also produced as described previously [20, 30]. 4.8 μl of 1% NH₄OH (AnaSpec) was added to an eppitube of disaggregated Aβ, and after brief vortexing, the tube sat for one min. The contents of the tube were then transferred sequentially to two more Aβ eppitubes, following this same procedure each time. The preparation was water bath sonicated for 4 min, then incubated for one hr at room temperature. After dilution in phosphate buffered saline (PBS; 0.01 M, pH 7.4, with 0.02% azide) to a final concentration of 58 μg/ml, it was used immediately or stored at 4°C for up to one week.

Western blots of Aβ monomer and soluble oligomer preparations were performed under both reducing/denaturing and native conditions as described previously [20, 30] using 4-20% Tris-HCl Ready Gels (Bio-Rad Laboratories, Hercules, CA). The molecular weight standards for the native gels were from Sigma-Aldrich's Non-Denaturing Molecular Weight Kit (cat. # MWND500). After electrophoresis, the proteins were transferred to Westran S PVDF membranes (Whatman International Ltd., Maidstone, UK). The membranes were then blocked with 10% non-fat dry milk in 0.01 M PBS, pH 7.4 for one hr at room temperature. Membranes were incubated overnight at 4°C with agitation in mouse monoclonal anti-Aβ(1-16) 6E10 (Covance Research Laboratories, Berkeley, CA; 1:5,000 dilution). After incubation in horseradish peroxidase (HRP)- conjugated anti-mouse IgG (Vector Laboratories, Inc., Burlingame, CA; 1:10,000 dilution) for 1 hr at room temperature, membranes were developed in SuperSignal West Pico chemiluminescent substrate (Thermo Scientific, Rockford, IL). Bands were detected on CL-XPosure film (Thermo Scientific).

TEM was performed as previously described [31]. Each sample was spread on a Formvar coated grid (Electron Microscopy Sciences, Fort Washington, PA) and incubated for two hr at room temperature, then rinsed with double distilled water. Samples were then fixed with 1% glutaraldehyde in 100 mM phosphate buffer, pH 7.4 for 10 min, rinsed again with water, and stained with 1% uranyl acetate for 10 min followed by alkaline lead citrate for five min. Images were taken with a Morgagni 268 transmission electron microscope (FEI Company, Hillsboro, OR) equipped with a Hamamatsu digital camera.

Antibody concentrations to the Aβ1-42 monomer and soluble oligomer preparations were measured by ELISA in AD, MCI, and NCI serum samples. A separate ELISA plate was required for each serum sample. The plate arrangement is shown in Figure 1. Samples were randomized as to the order in which they were evaluated. A volume of 100 μl was placed in each well for each step of the procedure. The Aβ monomer and soluble oligomer preparations were incubated at 0.9 μg/ml in Tris buffer (0.1 M, pH 8.8) overnight at 4°C on a 96-well Nunc Maxisorp plate (Nalge Nunc International, Rochester, NY). As a "specificity control" the same concentration of bovine serum albumin (BSA, Sigma-Aldrich) in Tris buffer was filtered and placed in adjacent wells. After incubation overnight at 4°C, wells were washed three times with PBS with 0.1% Tween-20 (Sigma-Aldrich) (hereafter, PBS-T; this wash step was repeated after all subsequent incubations). The plate was then treated with SuperBlock (SuperBlock Blocking Buffer in PBS, Thermo Scientific) as per the manufacturer's instructions, followed by addition of antibody-antigen complex dissociated and undissociated serum samples. These samples were diluted 1:100 in PBS (pH 7.2) with 0.1% Tween-20 and 1% BSA (hereafter, PBS-T-BSA) and assayed in quadruplicate. Positive controls were dissociated and undissociated preparations of an IvIg product, Gamunex Immune Globulin Intravenous (Human), 10% (Talecris Biotherapeutics, Inc., Research Triangle Park, NC), diluted 1:1,000. A normal control serum sample from an individual not participating in the Rush Memory and Aging Project was included on all plates to allow data to be normalized between plates. Dissociation of serum antibody-antigen complexes with pH 3.5 dissociation buffer was performed as previously described [20] using the procedure described by Li et al. [25] with slight modifications. To produce the standard curve, four-fold dilutions of mouse monoclonal 6E10 anti-Aβ antibody (1:4,000 [250 ng/ml], 1:16,000 [62.5 ng/ml], 1:64,000 [15.6 ng/ml], and 1:256,000 [3.9 ng/ml]) in PBS-T-BSA were placed in wells previously coated with Aβ monomer, Aβ oligomers, or BSA. Blank wells received PBS-T-BSA at this step. Secondary antisera were biotinylated goat anti-mouse IgG (Vector Laboratories, Inc., Burlingame, CA; 1:1,000 dilution) for the wells previously receiving mouse 6E10 antibody and biotinylated goat anti-human IgG (H + L) (Jackson ImmunoResearch Laboratories, West Grove, PA; 1:1,000 dilution) for wells previously incubated with serum samples. After incubation with streptavidin-alkaline phosphatase (Zymed Laboratories, Invitrogen, Carlsbad, CA; 1:1,000 in PBS-T), para-nitrophenol phosphate (Sigma-Aldrich) was added (5 mg in 40 ml of 1 M diethanolamine buffer, pH 9.8). The plate was read at 405 nm with a Vmax kinetic microplate reader (Molecular Devices Corp., Sunnyvale, CA) until the standard curve OD reached 1.0. Softmax Pro software version 3.0 (Molecular Devices) was used to generate the best-fit plot of the standard curve, using the log-logit option.

To calculate specific anti-Aβ antibody concentrations, the mean antibody concentration measured when each serum sample was incubated on BSA-coated wells was subtracted from the antibody concentrations measured on wells coated with the soluble Aβ conformations. Densitometric analysis of western blots indicated that approximately 30% of the total band intensity in the Aβ oligomer preparation was due to the Aβ monomer band [20]. Therefore, after calculating the mean anti-monomer antibody concentration of each sample, 30% of this was subtracted from its antibodies to the oligomer preparation to determine its anti-oligomer antibody concentration. The antibody levels measured in each experiment were normalized for interassay variation by multiplying them by the overall mean concentration (from all 30 experiments) of anti-Aβ oligomer antibodies in antibody-antigen-dissociated serum from the normal control sample, then dividing by the observed concentration of the anti-Aβ oligomer antibody in this control sample in the experiment. This normalization procedure was based on anti- Aβ oligomer levels in dissociated sera, rather than the other anti-Aβ measurements, because the most consistent findings across experiments were detected for dissociated anti-Aβ oligomer antibody measurements.

Spearman's correlation coefficient was used to assess the association between antibody concentrations to Aβ monomer and oligomeric Aβ using pooled data from all groups and also within each group. Differences in mean antibody levels between groups and between sample preparation methods (either dissociated or undissociated) were assessed with repeated measures ANOVA using restricted maximum likelihood estimation with an appropriate variance structure. Main effects models were used when there was no evidence of interaction. Tukey-Kramer p-values and confidence intervals were used for multiple comparisons as appropriate. The significance of differences between groups was evaluated using one-way ANOVA (for subject age), the Kruskal-Wallis test (for post-mortem intervals [PMI]), and exact versions of Pearson's chi-square tests (for gender, apolipoprotein E [apoE] status, and use of anti-inflammatory medications). P-values ≤ 0.05 were considered statistically significant. All p-values were two-tailed. Statistical analyses were performed using The SAS System for Windows version 9.2.

All calculations were based on a significance level of 0.05, with 80% power to detect specified differences using the F test for the group effect from repeated measures ANOVA. The standard deviation of concentration and the mean concentration of anti-Aβ antibodies in NCI sera (averaged between dissociated and undissociated samples) were estimated from the data. The power analysis calculations specified that the mean anti-Aβ antibody concentration in AD subjects would be increased by a given percentage (20%, 25%, 30%, 40%, or 50%) from the antibody concentration in the NCI group. The calculations used NCPASS 2005 software with equal group sample sizes.

Western blots of the Aβ conformations, performed on gels run under both reducing/denaturing and native conditions, were published previously [30]. The Aβ monomer preparation produced a single band in both blots. The blot of the reducing/denaturing gel of the oligomer preparation contained bands corresponding to Aβ monomer, dimer, tetramer, pentamer, and higher-order oligomers. Western blots of the this preparation run on a native gel produced a protein smear in which individual bands were difficult to visualize.

Spherical structures were present in both the Aβ monomer and Aβ oligomer preparations. The diameter of the spherical structures in the oligomer preparation ranged from 50 to 100 nm while the diameter of the largest spherical structure in the monomer preparation was approximately 20 nm. TEM images are shown in Figure 2.

There were no significant differences for serum antibody concentrations to the Aβ monomer preparation between the three groups (p = 0.73 for combined data from undissociated and dissociated serum samples), although the mean concentrations of these antibodies tended to be increased in AD vs. NCI sera (by 20% in undissociated samples and 29% in dissociated samples). 95% Tukey confidence intervals for differences in the mean antibody levels indicated that the possibility of large differences between these groups could not be excluded: MCI - NCI: (-0.280, 0.431); AD - NCI: (-0.243, 0.468); AD - MCI: (-0.318, 0.392). Anti-Aβ monomer antibody levels were significantly increased after antibody-antigen complex dissociation (pooled data from all subjects: p = 0.0011; 95% confidence interval for dissociated - undissociated: [0.073, 0.258]), but none of the within-group differences were statistically significant after Tukey-Kramer adjustment of p-values. Data are shown in Figure 3.

Results were generally similar to those for anti-Aβ monomer antibodies. There were no significant differences between the levels of anti-Aβ oligomer antibodies beween AD, MCI, and NCI serum samples (p = 0.58 for pooled data), although the mean levels again tended to be increased in AD vs. NCI sera (30% increase in undissociated sera, 13% increase in dissociated sera), and 95% Tukey confidence intervals for the differences in mean antibody levels indicated that the possibility of large differences between the groups could not be excluded: MCI - NCI: (-0.161, 0.301); AD - NCI: (-0.137, 0.325); and AD - MCI: (-0.207, 0.255). In contrast to the anti-monomer antibodies, antibody- antigen dissociation did not increase mean anti-Aβ oligomer antibody levels (p = 0.65; 95% confidence interval for dissociated - undissociated = (-0.121, 0.072). Data are shown in Figure 4.

When the population means for serum anti-Aβ monomer antibody concentrations for NCI, MCI, and AD subjects were modeled as 0.440 μg/ml, 0.495 μg/ml, and 0.550 μg/ml, specifying a 25% increase in anti-Aβ monomer antibody levels for AD vs. NCI subjects similar to the findings in the present study, power analysis indicated that 328 samples per group would have been required for 80% probability of statistically significant results at the 0.05 level. For anti-Aβ oligomer antibodies, when the population means for NCI, MCI, and AD were modeled as 0.433 μg/ml, 0.487 μg/ml, and 0.541 μg/ml, resulting in a 25% increase in these antibodies between AD and NCI subjects, 150 samples per group would have been required for 80% probability of significance at the 0.05 level. Tables 3 and 4 indicate the approximate numbers of samples per group that would have been required for 80% probability to achieve significance at the 0.05 level for specified increases in AD vs. NCI antibodies to Aβ monomer and oligomers, respectively, between 20% and 50%.

Antibody levels to Aβ monomer and soluble Aβ oligomers were strongly associated. For pooled data from all subjects, Spearman rank correlations were 0.798 for undissociated serum preparations and 0.564 for dissociated preparations. When evaluated for each group, these associations remained positive (data not shown).

There were no significant differences between groups for subject age, apoE status, PMI, or use of anti-inflammatory medications. The gender differences between the groups were statistically significant (p = 0.02) because the majority of the AD group was male (8 males and 2 females) while the other two groups were predominantly females (NCI, 2 males and 8 females; MCI, 3 males and 7 females).