Quantification of Amyloid- b in Plasma by Simple and Highly Sensitive Immunoaffinity Enrichment and LC-MS/MS Assay

Background: Numerous immunoassays have been developed to quantify amyloid b 1-40 (A b 40 ) and amyloid b 1-42 (A b 42 ). Nevertheless, given the low concentration of A b and the high levels of interfering factors in plasma, quantiﬁcation of plasma A b is still challenging. To overcome the problems related to the speciﬁcity of A b immunoassays, this study aimed to develop an immunoafﬁnity enrichment and LC-MS/MS (IA-MS) assay. Methods: We developed an IA-MS assay using antibody-labeled magnetic beads for puriﬁcation and LC-MS/MS for A b quantiﬁcation. To avoid the loss of A b due to aggregation in acidic buffer, we used alkaline elution buffer for immunoafﬁnity enrichment. The concentrations of the A b s in plasma samples were measured, and the correlation between the plasma and cerebrospinal ﬂuid (CSF) A b 42 /A b 40 ratio was also evaluated. Results: The intensities of the A b mass peaks were signiﬁcantly higher with the alkaline elution buffer than with the acidic elution buffer (A b 40 : 3.6-fold, A b 42: 5.4-fold). This assay exhibited high reproducibility (intra-assay and in-ter-assay precision, %CV < 15), and the working ranges of A b 40 and A b 42 were determined to be 21.7 to 692.8 pg/ mL and 5.6 to 180.6pg/mL, respectively. The concentrations of A b 40 and A b 42 in plasma were measured by IA-MS, and the plasma A b 42 /A b 40 ratio was correlated with the CSF A b 42 /A b 40 ratio ( r s ¼ 0.439, P < 0.01). Conclusions: The IA-MS assay has sufﬁcient analytic performance for measuring endogenous A b 40 and A b 42 in plasma. This assay can lead to new lines of clinical discovery related to amyloid pathology.


INTRODUCTION
Alzheimer's disease (AD) is the most common type of dementia and a major public health problem worldwide, with significant socioeconomic implications. The efficacy of potential AD treatments likely depends on how early the treatment is started. Consequently, simple screening tests that can accurately detect the early stage of AD are urgently needed. Well-known pathological features of AD are brain parenchymal plaques composed of amyloid b (Ab) (1), which is thought to initiate and propagate the formation of neurofibrillary tangles, the loss of neurons, and the synaptic degeneration that begins decades before symptoms appear (2,3). Recent studies have shown that the ratio of cerebrospinal fluid (CSF) Ab 1-42 (Ab 42 ) to Ab 1-40 (Ab 40 ) is negatively correlated with the amyloid burden in the brain visualized in in vivo amyloid positron emission tomography imaging (4,5). However, because the collection of CSF is invasive and potentially painful for the patient, plasma-based biomarkers would be highly advantageous.
Numerous immunoassays have been developed to analyze Ab 40 and Ab 42 in plasma, and the association between Ab 40 and Ab 42 levels in plasma and AD has been investigated (6,7). However, because the levels of Ab 40 and Ab 42 in plasma are much lower than those in CSF, and because plasma contains high levels of assay interferants (8), the development of immunoassays for Ab 40 and Ab 42 in plasma is still challenging. Considering that the analytic specificity of immunoassays depends mainly on the antibody and can be influenced by matrix effects or nonspecific antibody cross-reactivity (9), highly specific methods are necessary to quantify plasma Ab 40 and Ab 42 accurately and precisely.
Quantitative LC-MS/MS can be used to directly measure target peptides with analytic specificity based on each peptide's retention time, m/z, and fragmentation profile (10)(11)(12). To overcome the technical limitations of immunoassays, LC-MS/MS has been used increasingly for such applications (13)(14)(15)(16)(17). Immunoaffinity enrichment is a highly efficient sample-pretreatment method to concentrate analytes while removing the majority of the matrix (14,18). Recently, immunoaffinity enrichment and LC-MS/MS (IA-MS) assays were developed for measuring plasma Ab 40 and Ab 42 (19,20). Because Abs are prone to aggregate in acidic conditions, it is better to perform liquid chromatography separation and mass spectrometry analysis of Abs under alkaline conditions (16). Nevertheless, acidic buffers, which can generally disrupt antibody and antigen interactions more efficiently than alkaline buffers (21), were used for the elution of Abs from antibodies during immunoaffinity enrichment in previous reports. Therefore, Ab 40 and Ab 42 purified from plasma by immunoaffinity enrichment were then dried in vacuo and dissolved in an alkaline reagent before LC-MS/MS analysis (19) or were digested into much smaller peptides (20). These additional steps increase the complexity of the measurement and lead to the loss of Abs, which may decrease the sensitivity of IA-MS.
The elution of an antigen from an antibody with an alkaline buffer is a technique sometimes used in the purification of antigens that are unstable under acidic conditions. A previous study that assessed the effect of elution buffer on the recovery of Ab monomers and Ab aggregates from

IMPACT STATEMENT
With the developed method, amyloid b 1-40 (Ab 40 ) and amyloid b 1-42 (Ab 42 ) levels in patient plasma samples can be measured with high sensitivity and reliability. The assay includes an immunoaffinity enrichment method that uses alkaline elution buffer for the elution of amyloid bs from antibodies without the need for a buffer change or enzymatic digestion before quantification by LC-MS/MS. The assay requires only 250 mL of plasma for measurement of Ab 40 and Ab 42 , benefiting patients by reducing physical burden during blood collection. This assay will advance the development of tests for the diagnosis of Alzheimer's disease. human AD brains by immunoaffinity enrichment showed that elution under acidic conditions (pH 2.7) resulted in almost no recovery of Ab. In contrast, elution under alkaline conditions (pH 10.5) resulted in nearly complete recovery of Ab (22). We hypothesized that adopting alkaline elution buffer for IA-MS would improve the sensitivity of Ab detection.
In this study, we developed a simple and highly sensitive IA-MS assay for the measurement of Ab 40 and Ab 42 in routinely available volumes of plasma. This assay uses alkaline elution buffer for the elution of Abs from the antibody in the immunoaffinity enrichment process, followed by injection into an LC-MS/MS system without changing the buffer. The assay was tested and validated in terms of the calibration curves, recovery, dilution linearity, intra-assay precision, inter-assay precision, and limit of quantification (LOQ). The ratio of plasma Ab 42 and Ab 40 was also investigated and compared with CSF measurements.

Materials
Ab 40 (sequence: DAEFRHDSGYEVHHQKLVF FAEDVGSNKGAIIGLMVGGVV) and Ab 42 (sequence: DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVG GVVIA) were purchased from AnaSpec. As the internal standard of Ab 40 and Ab 42 , uniformly 15 N-labeled Ab 42 ( 15 N-Ab 42 ), which is 55 Da heavier than Ab 42 , was purchased from rPeptide. The lyophilized peptide stocks of Ab 40 , Ab 42 , and 15 N-Ab 42 were dissolved in 10 mM NaOH, dispensed into tubes, and stored at À80 C. Mouse monoclonal anti-Ab antibodies (6E10 IgG1) were purchased from BioLegend. M-270 Epoxy-activated Dynabeads were purchased from Thermo Fisher Scientific. For immunoaffinity enrichment, anti-Ab antibodies were covalently immobilized on beads following the manufacturer's recommended protocol and incubated in 0.2% BSA-PBS, pH 7.4, for 1 h at room temperature to block the surface of the beads. Anti-Ab monoclonal antibody (mAb)coated magnetic beads were stored at 4 C. BSA was purchased from Proliant Health and Biologicals. Ammonium acetate and ultrapure water were purchased from FUJIFILM Wako Pure Chemical Industries. Acetonitrile was purchased from Kanto Chemical, and 28% NH 4 OH was purchased from Nacalai Tesque.

Immunoaffinity Enrichment
The workflow for the IA-MS assay of Ab 40 and Ab 42 in plasma is shown in Fig. 1. In total, 250 mL of plasma samples, calibrators, and control samples were added to a 1.5-mL tube (Eppendorf). In addition 250 mL of 3% BSA-PBS containing 500 pg/mL of 15 N-Ab 42 was added to every sample and gently mixed by pipetting. After incubation for 30 min at room temperature, 40 mL of a suspension (4 mg of mAb on 0.4 mg of beads) of anti-Ab mAb-coated magnetic beads were added, gently mixed by pipetting, and incubated with end-over-end rotation for 1 h at room temperature. After incubation, the magnetic beads were pulled to the side of each tube using a magnetic stand and washed 3 times with 1 mL of 3% BSA-PBS, 2 times with 1 mL of 50 mM ammonium acetate, and 2 times with 1 mL of ultrapure water. The Abs were eluted from the beads in 25 mL of 30% acetonitrile with 1.68% NH 4 OH in water and transferred to polypropylene microvolume vial inserts (Tomsic) set in glass 12 Â 32 mm screw-neck vials (Waters). The eluted samples were placed in the LC-MS autosampler.

LC-MS/MS
An ACQUITY UPLC H-Class Bio System (Waters) was used for chromatographic separation. Overall, 10 mL of the solution of Abs eluted from the anti-Ab mAb-coated magnetic beads were injected into the system and separated on an ACQUITY UPLC Peptide BEH C18 column (300 Å, 1.7 mm, 2.1 mm x 150 mm, Waters) maintained at 50 C. Mobile phase A was composed of 0.1% NH 4 OH in water (pH 10.74), and mobile phase B was composed of 90% acetonitrile with 0.01% NH 4 OH in water. Gradient elution of the Abs bound to the column was performed at 200 ml/min with the following linear gradient: 10% mobile phase B from 0 to 1.0 min, 10 to 55% mobile phase B from 1.0 to 5.5 min, 55% mobile phase B from 5.5 to 6.7 min, 55 to 10% mobile phase B from 6.7 to 7.0 min, and 10% mobile phase B from 7.0 to 8.5 min. Ab 40 was eluted at 6.28 min, and Ab 42 and 15 N-Ab 42 were eluted at 6.37 min.
Ab 40 and Ab 42 were quantified by a Xevo TQ-XS triple-quadrupole mass spectrometer (Waters) in positive mode with an electrospray ionization source. The mass spectrometric parameters were optimized via the direct infusion of Ab 40 , Ab 42 , and 15 N-Ab 42 solutions into the mass spectrometer.
The instrument was operated in multiple reaction monitoring mode. The optimal instrumental parameters, including the multiple reaction monitoring transitions, cone voltages, and collision energies, of Ab 40 , Ab 42 , and 15 N-Ab 42 are listed in Supplemental Data Table 1. The data-processing method was designed with MassLynx V4.1 software (Waters). The peak areas of Ab 40 , Ab 42 , and 15 N-Ab 42 were calculated with QuanLynx software (Waters).

Analytic Validation
Calibration curve analysis was performed by measuring calibration samples (n ¼ 6). The standard curves of both Ab 40 and Ab 42 were generated using a linear 1/Â regression of a calibrator. The precision was determined as the %CV of the analysis of each calibration sample. The accuracy was determined with the calibration standards as the back-calculated Ab 40 and Ab 42 concentrations. Recovery was determined by spiking pooled plasma with Ab 40 (90.6, 179.0, and 369.1 pg/mL) and Ab 42 (22.0, 44.5, and 95.0 pg/mL) before immunoaffinity enrichment. Dilution linearity was assessed by diluting pooled plasma spiked with Ab 40 (4503.1 pg/mL) and Ab 42 (1038.4 pg/mL) up to 1:32 in 3% BSA-PBS before analysis and analyzed according to a previous study (23). Intraassay precision (%CV) was determined by measuring the concentrations in 6 replicates of the control samples in a single run. Inter-assay precision (%CV) was determined by analyzing the control samples in 7 different runs. The LOQ was determined to be the lowest concentration that could be measured with an inter-assay %CV <20. The working range was determined to be the range between the LOQ and the highest concentration of the calibration samples.

Plasma and CSF Samples
Forty-four commercially available paired plasma and CSF samples (2 from individuals with AD, 33 from individuals with mild cognitive impairment, and 9 from cognitively normal [CN] individuals) were obtained from PrecisionMed. Sample collection was approved by the institutional review board at PrecisionMed (protocols 7800 and 1009). All participants signed informed consent. The concentrations of Ab 40 and Ab 42 in plasma were determined by IA-MS assay. CSF samples collected from the same patients were measured by inhouse Ab 40 and Ab 42 immunoassays, which were developed according to previous studies (24,25).
The correlation between the plasma Ab 42 /Ab 40 ratio and the CSF Ab 42 /Ab 40 ratio was evaluated.

Statistical Analysis
Spearman's rank correlation was used to analyze correlations between the plasma Ab 42 /Ab 40 ratio and the CSF Ab 42 /Ab 40 ratio. We report Spearman's rank correlation coefficient r s values with corresponding P values. Values of P < 0.05 were considered statistically significant. These analyses were performed with StatFlex V6 (Artech).

Impact of Alkaline Elution Buffer on IA-MS Sensitivity
To assess the impact of alkaline elution buffer on IA-MS sensitivity, 190 pg/mL Ab 40 and 103 pg/ mL Ab 42 spiked in 3% BSA-PBS were purified with anti-Ab mAb-coated magnetic beads, eluted with 30% acetonitrile with 1.68% NH 4 OH in water (alkaline elution buffer) and analyzed by LC-MS/MS. The same samples were also analyzed with an IA-MS assay in which Abs were eluted with 30% acetonitrile with 0.1% trifluoroacetic acid in water (acidic elution buffer), followed by drying and then dissolution in the same volume of alkaline elution buffer. Under both conditions, Ab 40 was detected at 6.28 min, and Ab 42 was detected at 6.37 min. Compared with the acidic elution buffer, the alkaline elution buffer significantly increased the intensities of the mass peaks of both Ab 40 (3.6-fold) and Ab 42 (5.4-fold) (Fig. 2).

Recovery
To assess the interference of the plasma matrix, an aliquot of pooled plasma and 3% BSA-PBS was spiked with different concentrations of Ab 40 and Ab 42 and assayed with IA-MS. Recovery was calculated by comparing the Ab values in plasma spiked with Abs with the Ab values in 3% BSA-PBS spiked with Abs. The recoveries of Ab 40 were 96.4% (90.6pg/mL spike), 95.3% (179.0-pg/mL spike), and 92.1% (369.1-pg/mL spike), and the recoveries of Ab 42 were 104.1% (22.0-pg/mL spike), 112.9% (44.5-pg/mL spike), and 104.1% (95.0-pg/mL spike) ( Table 1).

Assay Precision, LOQ, and Working Range
Intra-and inter-assay precision and accuracy were determined at the low, medium-low, medium-high, and high control levels. The intra-assay precision for Ab 40 and Ab 42 ranged from 5.6% to 7.0% and from 3.9% to 13.0%, respectively. The inter-assay precision for Ab 40 and Ab 42 ranged from 7.5% to 9.5% and from 5.8% to 13.2%, respectively ( Table 2). The LOQs of Ab 40 and Ab 42 were determined to be 21.7 pg/mL and 5.6 pg/mL, respectively. The working ranges of Ab 40 and Ab 42 were determined to be 21.7-692.8 pg/mL and 5.6-180.6 pg/mL, respectively.

Correlation Study
The concentrations of Ab 40 and Ab 42 were measured in plasma samples by IA-MS. Plasma levels of Ab 40 and Ab 42 ranged from 94.9 to 306.3 pg/mL and from 10.7 to 34.1 pg/mL, respectively. The plasma Ab 42 /Ab 40 ratio was significantly correlated with the CSF Ab 42 /Ab 40 ratio (r s ¼ 0.439, P < 0.01) (Fig. 4).

DISCUSSION
The strategy to integrate immunoaffinity enrichment and LC-MS/MS while using stable isotopelabeled internal standards enables robust analysis of peptides. Although immunoaffinity enrichment presents problems such as matrix effects and nonspecific antibody cross-reactivity, these disadvantages can be corrected by adding internal standards to the sample before immunoaffinity enrichment and using a mass spectrometer for the detection, which provides highly specific peptide detection. In contrast, because the recovery rate of immunoaffinity enrichment depends on the affinity of antibodies, this strategy cannot be adapted for peptides at low concentrations without acquiring high-performance antibodies. To solve the problem related to antibodies, we used anti-Ab antibody clone 6E10, which has enough affinity (dissociation constant, K D ¼ 22.3 nM) (26) and can be used to capture endogenous plasma Abs (27). The use of immunoaffinity enrichment with an acidic elution buffer is the typical strategy for the purification of peptides, including Abs, from a wide variety of biological samples. However, previous studies have demonstrated that the aggregation of Abs increases between pH 2 and 9 (28). The aggregation models for Ab 42 computed using a Merck molecular force-field method showed that when Ab 42 was in an acidic environment, aggregation was accelerated by the intermolecular ion binding of Asp23 with Lys28. In contrast, at pH 9.5, no aggregation of Ab 42 was observed because the side chain of Lys28 contained unprotonated amino groups (29). These mechanical insights indicate the benefit of alkaline conditions to the solubility of Abs.
In this study, we developed an IA-MS assay for the measurement of Ab 40 and Ab 42 with 30% acetonitrile and 1.68% NH 4 OH (pH 11.64) used for the elution of Abs from antibodies during immunoaffinity enrichment, followed by injection into an LC-MS/MS system without changing the buffer. The use of alkaline elution buffer significantly increased the sensitivity of IA-MS and enabled the measurement of endogenous Ab 40 and Ab 42 in plasma. Because this assay is applicable to the analysis of other Ab species, it can be used for more detailed studies of amyloid pathology, such as metabolism and kinetics. This assay is also applicable to the analysis of other biomarkers for neurological diseases, such as a-synuclein, which is unstable under acidic conditions (30), and may enable the comprehensive analysis of biomarkers for neurological disorders.
We used 15 N-Ab 42 as the internal standard for both Ab 40 and Ab 42 , and the area ratio of the Abs and the internal standard were used for the quantification analysis. Although stable isotope-labeled internal standards corresponding to each Ab are typically used for mass spectrometry-based methods (19,20), the use of a common internal standard for Ab 40 and Ab 42 measurements has several benefits. First, the errors caused by the preparation and addition of the internal standard can be eliminated during the Ab 42 /Ab 40 ratio calculation, which enables accurate assessment. Second, extended scanning time can be allocated to Ab 40 and Ab 42 in LC-MS/MS, compared with using internal standards corresponding to each Abs, and enables the acquisition of reliable MS peaks. Finally, using only one internal standard is simple and may be cost-and labor-effective when measuring a large number of samples. In contrast, because Ab 40 and 15 N-Ab 42 do not have an identical amino acid sequence, if the effect of matrix components and reactivity to the antibody are different between Ab 40 and 15 N-Ab 42 , accurate Ab 40 measurements become challenging. This is the risk of using a common internal standard. In this study, however, we confirmed the excellent analytic performance of both Ab 40 and Ab 42 measurements. Considering the importance of assessing the Ab 42 /Ab 40 ratio accurately and precisely, we  ................................................................................................ concluded that the use of 15 N-Ab 42 as a common internal standard was highly advantageous.
Plasma Ab biomarkers were recently developed, and their potential clinical utility in predicting the brain Ab burden at the individual level was reported (27,31,32). In this study, the plasma Ab 42 /Ab 40 ratio measured using IA-MS was correlated moderately (r s ¼ 0.439) and significantly (P < 0.01) with the CSF Ab 42 /Ab 40 ratio. This result agreed with previous studies that showed the r value of the correlation between the plasma and CSF Ab 42 /Ab 40 ratio to be 0.215-0.631 and statistically significant (6,33). In addition, as shown in Fig. 4C, the plasma Ab 42 /Ab 40 ratio with AD tended to be lower than that with normal cognition or mild cognitive impairment. This result agreed with a previous report showing that the plasma Ab 42 / Ab 40 ratio is lower in patients with AD than in controls (P < 0.0001) and with mild cognitive impairment (P ¼ 0.003) (33). Although these results are quite informative, plasma collected from more AD patients needs to be analyzed to clarify the implications of these data.
Notably, this IA-MS assay requires only 250 mL of plasma for the measurement of Ab 40 and Ab 42 . This is 8-20 times less than the plasma usage noted in previous reports (19,20). Blood collection is less invasive than CSF collection. Nevertheless, given the advanced age of patients with AD, the ability to measure small amounts of plasma may be beneficial because it reduces the physical burden on the patient during blood collection and enables a larger cohort study to elucidate the clinical utility of Ab biomarkers for AD diagnosis.
Because the number of patients with AD is increasing, simple screening tests for its diagnosis are urgently needed. During the past 15-20 years, the sensitivity of LC-MS/MS has improved and rivals or surpasses that of some immunoassays (34). Nevertheless, the adoption of LC-MS/MS in clinical laboratories has been limited given the high level of technical expertise required. To improve the usability of LC-MS/MS in the clinical setting, the first fully automated LC-MS/MS technology was recently developed and validated for 25-hydroxy vitamin D 2 and D 3 assays in a routine clinical laboratory (35). An automated immunoaffinity enrichment method for LC-MS/MS using an automated magnetic bead processor was also reported (36) and is adoptable for immunoaffinity enrichment of Abs. If these technologies are integrated with the IA-MS assay and factors such as cost and ease of maintenance are improved in the near future, this IA-MS strategy can be applied in the clinical setting and lead to new lines of biological and clinical discovery related to amyloid pathology.

SUPPLEMENTAL MATERIAL
Supplemental material is available at The Journal of Applied Laboratory Medicine online. Author Contributions: All authors confirmed they have contributed to the intellectual content of this paper and have met the following 4 requirements: (a) significant contributions to the conception and design, acquisition of data, or analysis and interpretation of data; (b) drafting or revising the article for intellectual content; (c) final approval of the published article; and (d) agreement to be accountable for all aspects of the article thus ensuring that questions related to the accuracy or integrity of any part of the article are appropriately investigated and resolved.