Method for detecting beta-amyloid in dried blood spots and use thereof

Abstract

The application relates to the technical field of biomarker detection and analysis, and particularly discloses a detection method for beta-amyloid in a dry blood spot and application thereof. In view of the problems that the prior art must rely on an antibody enrichment and / or enzymolysis step, and complete A beta 40 and A beta 42 cannot be synchronously and high-faithfully extracted from a dry blood spot sample, thus leading to inaccurate ratio, a pretreatment method adopting mixed mode cation exchange (MCX) magnetic beads for one-step magnetic solid phase extraction (MSPE) is provided. The method does not need an antibody and does not need enzymolysis, captures complete A beta peptides under an acidic condition by using the MCX magnetic beads, and realizes synchronous elution of A beta 40 and A beta 42 with high recovery rate by using an eluent with a specific composition, so that the authenticity of the A beta 42 / A beta 40 ratio is ensured. The technology is combined with an automatic magnetic solid phase extraction instrument, and provides a feasible scheme for large-scale blood screening of Alzheimer's disease.

Description

Technical Field

[0001] This application relates to the field of biomarker detection and analysis technology, and more specifically, it relates to a method for detecting β-amyloid protein in dried blood spots and its application. Background Technology

[0002] Early diagnosis of Alzheimer's disease (AD) is of great significance for delaying disease progression. Currently, the main detection methods include PET imaging, which directly shows Aβ deposition in the brain; analytical techniques that detect the Aβ42 / Aβ40 ratio and tau protein in cerebrospinal fluid; and plasma testing, which has the advantage of being non-invasive. Among these, due to the invasiveness of cerebrospinal fluid samples, the Aβ42 / Aβ40 ratio of β-amyloid protein in plasma testing is considered a highly promising biomarker for early screening of AD.

[0003] However, the difference in the Aβ42 / Aβ40 ratio in plasma tests between AD patients and healthy individuals is only about 10%-15%, thus requiring extremely high precision and specificity from the testing methods. The plasma testing methods for Alzheimer's disease (AD) are as follows:

[0004] 1) CN117471007A discloses an LC-MS / MS method for detecting plasma β-amyloid protein. This method uses immunoprecipitation (IP) enrichment and trypsin digestion to obtain characteristic peptides as quantitative targets. However, this method has significant shortcomings:

[0005] First, relying on specific antibodies for immunoprecipitation results in large batch-to-batch variability in antibodies, high costs, and the possibility of cross-reaction with non-target subtypes.

[0006] Second, it requires trypsin digestion, and the efficiency of digestion is affected by a variety of factors, which increases the complexity of the operation and the variability of the results.

[0007] Third, the total processing time is over 14 hours, which is insufficient to meet the needs of high-throughput rapid screening in clinical settings;

[0008] 2) CN115427815A discloses a sample pretreatment method for mass spectrometry detection of β-amyloid peptide in plasma. It adopts a two-step solid phase extraction (SPE) strategy. The operation process of this method is extremely cumbersome. It requires the use of two different types of SPE columns / plates, two sample loading, multiple washing, two elutions and intermediate drying steps. The total time consumption is long. Multiple sample transfers can easily lead to the loss of target substances and it is difficult to achieve high-throughput automated processing.

[0009] In addition, some studies have attempted to test dried blood spot samples. Compared with plasma samples, dried blood spot samples are considered an ideal sample type for large-scale population screening because they are easy to collect (finger prick blood), have low transportation costs, and good stability. However, the Aβ42 / Aβ40 ratio deviates significantly from the true value during the preparation of dried blood spot samples due to the inherent characteristics of the peptides themselves. Based on this, this application provides a method for detecting β-amyloid protein in dried blood spot samples and its application. Summary of the Invention

[0010] To overcome the technical barriers when using dried blood spots as samples for detection, this application provides a method and application for detecting β-amyloid protein in dried blood spots. The method mainly utilizes MCX magnetic beads to capture intact Aβ peptides under acidic conditions and achieves simultaneous elution of Aβ40 and Aβ42 with a high recovery rate using an elution buffer with a specific composition, thus ensuring the authenticity of the Aβ42 / Aβ40 ratio.

[0011] In a first aspect, this application provides a method for detecting β-amyloid protein in dried blood spots, comprising the following steps:

[0012] A) Provide dried blood spot samples;

[0013] B) Add acidic denaturing reagent and internal standard working solution to the dried blood spot sample obtained in A), and perform extraction to obtain sample extract;

[0014] C) Perform one-step magnetic solid-phase extraction (MSPE) on the sample extract obtained in B) using mixed-mode cation exchange MCX magnetic beads, wherein the extraction includes:

[0015] Under loading conditions, the MCX magnetic beads capture intact β-amyloid protein in the sample extract. Under washing conditions, interfering substances are removed. Under elution conditions, the intact β-amyloid protein is eluted from the MCX magnetic beads to obtain an eluent.

[0016] D) The eluent obtained in step C) is analyzed by liquid chromatography-tandem mass spectrometry (LC-MS / MS) to determine the content of β-amyloid protein in the dried blood spot sample.

[0017] Preferably, in step A), the dried blood spot sample is prepared by spotting whole blood onto cellulose filter paper, pure cotton lint, or a separation membrane and then drying it.

[0018] Preferably, in step B), the acidic denaturing reagent is an acetonitrile solution containing phosphoric acid.

[0019] Preferably, the volume percentage concentration of phosphoric acid in the acetonitrile solution containing phosphoric acid is 2-6%.

[0020] Preferably, in step C), the operating conditions for each step are as follows:

[0021] The sample loading conditions were acidic.

[0022] The washing conditions include washing with an acidic aqueous solution and a low proportion of organic solvent;

[0023] The elution conditions are as follows: elution is performed using an organic solvent containing ammonia, and the eluent is a mixed solution of 75% acetonitrile, 15% water and 10% ammonia.

[0024] Preferably, the β-amyloid protein includes at least one of Aβ38, Aβ40, and Aβ42.

[0025] Preferably, in step D), the LC-MS / MS detection adopts multiple reaction monitoring (MRM) mode, and the detected ion pairs include the parent ion and daughter ion of intact Aβ40 and intact Aβ42.

[0026] Secondly, this application provides a kit for detecting β-amyloid protein in dried blood spots, comprising:

[0027] Hybrid-mode cation exchange (MCX) magnetic beads;

[0028] Reagents used to prepare acidic denaturing agents;

[0029] Reagents used to prepare eluents;

[0030] And, optionally, an isotopically labeled β-amyloid internal standard.

[0031] Thirdly, this application provides an analytical system for early screening of Alzheimer's disease, comprising:

[0032] The sample preprocessing module performs steps B) and C) above;

[0033] The chromatography-mass spectrometry detection module performs step D) above;

[0034] A data analysis module that receives data from the chromatography-mass spectrometry detection module, calculates and outputs the Aβ42 / Aβ40 ratio.

[0035] Fourthly, this application provides the application of the above method in the preparation of a diagnostic kit for early screening of Alzheimer's disease, wherein the linear detection range of the method for Aβ38, Aβ40 and Aβ42 is 10-2500 pg / mL, and the spiked recovery rate is 85%-115%.

[0036] In summary, this application has the following beneficial effects:

[0037] 1. Overcame the core technical barrier of dried blood spot detection: This application achieves a high degree of synchronization of Aβ40 and Aβ42 recovery rates through the combination of MCX magnetic beads and specific elution buffer, ensuring the authenticity of the Aβ42 / Aβ40 ratio for AD diagnosis;

[0038] 2. Eliminates dependence on antibodies and enzyme digestion: This application uses chemical MCX magnetic beads to replace biological antibodies for enrichment, fundamentally avoiding problems such as batch-to-batch differences in antibodies, storage condition limitations, and cross-reaction with non-target subtypes;

[0039] Meanwhile, this method can directly detect intact Aβ peptides without the need for enzymatic digestion by trypsin or other proteases, thus completely eliminating operational variations and "enzyme digestion bias" introduced by factors such as temperature, time, pH, and enzyme-substrate ratio in the enzymatic digestion step, resulting in more accurate detection results.

[0040] 3. High-throughput automation is achieved, significantly improving detection efficiency: This application simplifies the cumbersome immunoprecipitation or multi-step SPE process in the prior art into a one-step MSPE. Utilizing the characteristic of MCX magnetic beads to achieve solid-liquid separation quickly through magnetic field, and in conjunction with an automated magnetic solid phase extraction instrument, high-throughput parallel processing of 96-well plates can be achieved, greatly reducing human operation errors and labor costs.

[0041] 4. Convenient sample collection, high patient acceptance, and suitable for large-scale screening: This application uses dried blood spots as the test sample. Only 20 μL of blood from the subject's fingertip needs to be collected and spotted onto filter paper. After drying at room temperature, it can be stored and transported for a long time. Compared with cerebrospinal fluid or plasma samples, it is more convenient and non-invasive to collect, providing a basis for large-scale screening and routine physical examinations of Alzheimer's disease. Attached Figure Description

[0042] Figure 1 Using the peak area of ​​Aβ38 as the ordinate and concentration as the abscissa, a linear regression plot was generated using the weighted least squares method (weight factor 1 / x²).

[0043] Figure 2 Using the peak area of ​​Aβ40 as the ordinate and concentration as the abscissa, a linear regression plot was generated using the weighted least squares method (weight factor 1 / x²).

[0044] Figure 3 Using the peak area of ​​Aβ42 as the ordinate and concentration as the abscissa, a linear regression plot was generated using the weighted least squares method (weight factor 1 / x²).

[0045] Figure 4 Here is the LC-MS / MS chromatogram of Aβ40 in dried blood spots;

[0046] Figure 5 Here is the LC-MS / MS chromatogram of Aβ40 in plasma;

[0047] Figure 6 Here is the LC-MS / MS chromatogram of Aβ42 in dried blood spots;

[0048] Figure 7 Here is the LC-MS / MS chromatogram of Aβ42 in plasma;

[0049] Figure 8 Here is the LC-MS / MS chromatogram of Aβ38 in dried blood spots;

[0050] Figure 9 The image shows the LC-MS / MS chromatogram of Aβ38 in plasma. Detailed Implementation

[0051] The following is in conjunction with the appendix Figure 1-9 The preparation examples and embodiments further illustrate this application in detail, and the raw materials and / or equipment used in each preparation example, embodiment and test in this application, unless otherwise specified below, can be obtained through conventional commercial means.

[0052] Instruments: LC-MS / MS (SCIEX), high-speed refrigerated centrifuge (Eppendorf), 1 / 100,000 electronic analytical balance (Sartorius), vortex mixer (Qilinbell), automated magnetic solid phase extraction system (Jiangxi Shuozhuo).

[0053] Reagents:

[0054] Ultrapure water (Milli-Q);

[0055] Methanol, acetonitrile, phosphoric acid, ammonia, and formic acid were all HPLC grade.

[0056] The β-amyloid protein standard Aβ38 was purchased from Aladdin, and Aβ40 and Aβ42 were purchased from Beijing Manhag Biotechnology Co., Ltd.

[0057] Stable isotope internal standards 15 N-Aβ38 was purchased from the biochemical company - rpeptide. 15 N-Aβ40 and 15 The brand name of N-Aβ42 is bepure, purchased from Beijing Manhag Biotechnology Co., Ltd.

[0058] HLB magnetic beads, purchased from Ajeboy, model and dosage: BNMA730-SY, 50mg / ml;

[0059] The magnetic beads MCX were purchased from Adima, and their model and dosage were: MMCX03-100, 50mg / ml, and their particle size was 3μm.

[0060] MAX magnetic beads, purchased from Dongna, with the following model and dosage: SI-SAX, 50mg / ml;

[0061] The magnetic beads WCX were purchased from Nanomicro, and their model and dosage were: MEWCX-1000, 50mg / ml.

[0062] The magnetic beads SCX were purchased from Dongna, and their model and dosage were: MB1057, 10mg / ml.

[0063] Preparation Example 1

[0064] The dried blood spot quality control sample was prepared through the following steps:

[0065] First, accurately weigh the standards of β-amyloid protein (Aβ38, Aβ40, Aβ42), dissolve and mix them in a 50% acetonitrile / 1% ammonia aqueous solution to prepare the standard working solution and the quality control working solution as stock solutions. Then, mix them with blank whole blood matrix in proportion to prepare the quality control samples.

[0066] After the sample is mixed evenly, 20 μL of whole blood is dropped onto 8 mm filter paper, air-dried at room temperature for 4 hours, and then placed in an aluminum foil sealed bag containing desiccant for frozen storage in the dark for later use.

[0067] A standard curve was constructed based on the obtained quality control samples. The concentration range of Aβ38, Aβ40 and Aβ42 in the standard curve was 10-2510 pg / mL.

[0068] The sampling points for Aβ38 were: 10, 40, 160, 400, 1000, and 2500 pg / mL.

[0069] The sampling points for Aβ40 are: 10, 40, 160, 448, 1254, and 2509 pg / mL;

[0070] The sampling points for Aβ42 were: 10, 40, 160, 448, 1254, and 2509 pg / mL.

[0071] The quality control samples include:

[0072] Quality control sample group 1 includes corresponding quality control samples of Aβ38, Aβ40 and Aβ42, each with a concentration of 100 pg / mL.

[0073] Quality control sample group 2 includes corresponding quality control samples of Aβ38, Aβ40 and Aβ42, each with a concentration of 2000 pg / mL.

[0074] Preparation Example 2

[0075] An internal standard working solution is dissolved in a 50% acetonitrile / 1% ammonia aqueous solution.15 N-Aβ38, 15 N-Aβ40, 15 N-Aβ42 standard, the concentration of each internal standard is 150 pg / mL.

[0076] Preparation Example 3

[0077] An automated SPE (Sequencing Precipitation Extraction) plate reagent method involves adding the following reagents in sequence to the corresponding wells of a 96-well plate:

[0078] A1-H1 wells: 200 μL methanol + 20 μL MCX magnetic beads;

[0079] A2-H2 well: 200 μL 4% phosphoric acid solution;

[0080] Wells A3-H3: To be added with sample extraction solution;

[0081] A4-H4 wells: 500 μL acidic aqueous solution (4% phosphoric acid);

[0082] A5-H5 wells: 500 μL of low-proportion organic solvent (10% acetonitrile);

[0083] Wells A6-H6: 200 μL eluent (75% acetonitrile + 15% water + 10% ammonia).

[0084] Preparation Example 4

[0085] A pre-treatment reconstitution solution is prepared, comprising: 5% acetonitrile, 0.05% Triton X-100, 0.5% formic acid, and 94.85% purified water.

[0086] Example 1

[0087] The detection method for β-amyloid protein in dried blood spots, and the detection steps are as follows:

[0088] A) Provide dried blood spot samples

[0089] Following the method in Preparation Example 1, take a dried blood spot filter paper containing a whole blood standard curve or quality control sample. For the actual sample, take 20 μL of the subject's fingertip blood and spot it on an 8 mm filter paper. After drying at room temperature for 4 hours, the dried blood spot sample is obtained and can be used normally.

[0090] B) Add acidic denaturing reagent and internal standard working solution, and perform extraction.

[0091] Take the dried blood spot obtained in A), add 100 μL of water to moisten it, then add 50 μL of the internal standard working solution of Preparation Example 2 and 300 μL of acid denaturing reagent (4% acetonitrile phosphate solution), vortex to mix for 5 min, centrifuge at 16000 rpm for 10 min at 4℃, and take 300 μL of supernatant into a new tube, which is the sample extract;

[0092] Step C) Perform one-step MSPE using MCX magnetic beads

[0093] Take out the 96-well plate prepared in Preparation Example 3 and add the sample extract obtained in step B) to wells A3-H3.

[0094] Place the 96-well plate on the automated magnetic solid phase extraction instrument (Jiangxi Shuozhuo) and run the preset program. The program will automatically perform the following operations:

[0095] Sample loading conditions: Under acidic conditions (the sample extract in wells A3-H3 is acidic), MCX magnetic beads capture intact β-amyloid protein in the sample extract.

[0096] Washing conditions: Wash sequentially with 500 μL of 4% phosphoric acid solution in wells A4-H4 and 500 μL of 10% acetonitrile in wells A5-H5 to remove interfering substances.

[0097] Elution conditions: Use 200 μL of elution buffer (75% acetonitrile + 15% water + 10% ammonia) from wells A6-H6 to elute intact β-amyloid protein from the MCX magnetic beads to obtain the eluent.

[0098] Step D) LC-MS / MS detection

[0099] Take 200 μL of the eluent (wells A6-H6) obtained in step C), dry it with nitrogen, and reconstitute the residue with 20 μL of the reconstituted solution obtained in Example 4. After mixing and centrifuging, take the supernatant for sample testing.

[0100] Chromatographic conditions: Column: ACQUITY UPLC BEH C18 (2.1×100mm, 1.7μm); Column temperature: 40℃; Flow rate: 0.4 mL / min; Injection volume: 10 μL; Autosampler temperature: 4℃; Mobile phase A: water (containing 0.1% formic acid), Mobile phase B: acetonitrile (containing 0.1% formic acid).

[0101] The gradient elution procedure is shown in Table 1:

[0102] Table 1 Gradient elution program for liquid chromatography

[0103]

[0104] Mass spectrometry conditions: Ion source: ESI source; Detection mode: Multiple reaction monitoring (MRM); Scan mode: Positive ion mode; Spray voltage: 5500V; Curtain gas (CUR): 35psi; Collision gas (CAD): 8psi; Nebulizer gas (GS1): 60psi; Injection voltage (EP): 13.5V; Collision chamber exit voltage (CXP): 10V. MRM ion pair parameters are shown in Table 2.

[0105] Table 2 Ion pair information and parameters

[0106]

[0107] Analysis of the ion pair parameters in Table 2 shows that this method enables direct mass spectrometry detection of intact Aβ38, Aβ40, and Aβ42 in dried blood spots without enzymatic digestion or antibodies. Furthermore, each target analyte undergoes dual calibration using both ion pairs and an isotope internal standard, as detailed below:

[0108] 1) Q1 / Q3 values: Q1 of Aβ38-1 is 827.3 (+5 charge), Q1 of Aβ40-1 is 866.8 (+5), and Q1 of Aβ42-1 is 903.9 (+5), corresponding to the parent ion of the complete peptide;

[0109] Furthermore, the Q1 values ​​of the second ion pair (-2 channel) of each analyte are 1033.5, 1083.2, and 1129.3 (+4 charge), respectively, indicating that this application detects intact Aβ protein without the need for enzymatic digestion.

[0110] 2) Dual ion pair monitoring: Two ion pairs are set for each Aβ subtype to meet the qualitative requirements of 4 recognition points and effectively eliminate false positives.

[0111] 3) Isotope internal standard: 15 The N-labeled internal standard is highly consistent with the parameters of declustering voltage DP / collision energy CE / collision chamber ejection voltage CXP of the natural peptide, which can effectively ensure the accuracy of quantitative correction.

[0112] In summary, the data in Table 2 demonstrate from the perspective of mass spectrometry detection that this application has for the first time established an LC-MS / MS method that can directly extract and accurately quantify intact Aβ38 / Aβ40 / Aβ42 from dried blood spots without antibody enrichment or protease digestion.

[0113] Furthermore, through dual ion pair double confirmation, isotope internal standard parallel correction, and DP / CE parameters optimized for different charge states, this method achieves pg / mL-level detection sensitivity and specificity in the complex matrix of dried blood spots, providing a truly non-invasive, high-throughput, and standardized technical solution for early screening of Alzheimer's disease.

[0114] Furthermore, to further verify the feasibility and accuracy of the detection method described in Example 1, this application conducted linear range evaluation and spike recovery rate investigation based on this method, as detailed below:

[0115] 1) Linear range evaluation

[0116] First, take the dried blood spot standard curve sample prepared according to the method of Preparation Example 1, with a concentration range of 10-2500 pg / mL, and measure it according to the steps of Example 1;

[0117] Linear regression was performed using weighted least squares (weight factor 1 / x²) with the peak area ratio of each analyte to its corresponding internal standard as the ordinate and concentration as the abscissa. The results are as follows: Figure 1-3 And Table 3:

[0118] Table 3. Linearity and Linear Correlation Coefficient Table

[0119]

[0120] The results in the table above show that the correlation coefficients r of Aβ38, Aβ40, and Aβ42 are all greater than 0.995, proving that the method has good linearity in the concentration range of 10-2500 pg / mL and meets the requirements for quantitative analysis.

[0121] 2) Spike recovery verification

[0122] Two quality control samples with low and high concentrations (quality control sample group 1: 100 pg / mL; quality control sample group 2: 2000 pg / mL) were taken, and each concentration was measured three times. The recovery rate was calculated and recorded in Table 4.

[0123] Table 4. Comparison of Spiked Recovery Rates

[0124]

[0125] The results in the table above show that the spiked recoveries of each analyte are between 85% and 115%, which meets the acceptance criteria of the guidelines for biological sample analysis methods. This indicates that the target analyte loss is small and matrix interference is controllable during the pretreatment process, and the quantitative results are accurate and reliable.

[0126] Comparative Example 1

[0127] A sample pretreatment method for mass spectrometry detection of β-amyloid peptide in plasma differs from Example 1 in that it employs the two-step solid-phase extraction (SPE) method disclosed in patent CN115427815A to pretreat the same dried blood spot sample obtained in Example 1. The steps are as follows:

[0128] First, the dried blood spot extract is contacted with an acidic or basic denaturing agent, and then the first SPE (reverse phase or ion exchange SPE) and the second SPE (cation or anion exchange SPE) are performed sequentially. Each SPE step includes multiple washing and elution steps. Finally, the eluent is dried and reconstituted before being injected for analysis.

[0129] The samples processed by the above method were then analyzed under the same LC-MS / MS conditions as in Example 1, and the results are shown in Table 5:

[0130] Table 5 Performance Test Comparison Table of Example 1 and Comparative Example 1

[0131]

[0132] As can be seen from the table above, the one-step MCX magnetic bead method in Example 1 of this application is superior to the two-step SPE method in Comparative Example 1 in terms of the recovery rates of Aβ40 and Aβ42.

[0133] In addition, the two-step SPE method requires the use of two different types of SPE columns / plates, and involves two sample loading, multiple washing, two elutions and intermediate drying steps, with a total time of about 1 hour and 35 minutes.

[0134] In summary, the one-step MSPE method in this application only takes about 30 minutes (excluding nitrogen blowing concentration). Compared with the prior art, this application not only has a higher recovery rate, but is also simpler to operate and takes less time, making it more suitable for high-throughput automated clinical testing.

[0135] Comparative Examples 2-4

[0136] A method for detecting β-amyloid protein in dried blood spots differs from Example 1 in that the magnetic beads used in step C) are different, as shown in the table below:

[0137] Table 6 Comparative Table of Magnetite Bead Selection in Examples 2-4

[0138]

[0139] Then, the samples treated by the methods in Comparative Examples 2-4 were analyzed under the same LC-MS / MS conditions as in Example 1, and the results are shown in Table 7:

[0140] Table 7 Performance test results of Comparative Examples 2-4

[0141]

[0142] As shown in the table above, the MCX magnetic beads selected in Example 1 of this application significantly improve performance compared to Comparative Examples 2-4. This indicates that not all magnetic beads are suitable. The specific analysis is as follows:

[0143] 1) HLB magnetic beads (reverse mechanism): hydrophobic interfering substances compete for adsorption, resulting in poor selectivity. Therefore, different Aβ isoforms cannot be recovered simultaneously, and the performance is significantly lower than that of Example 1.

[0144] 2) SCX-1 magnetic beads (strong cation exchange): Although it is also a solid-phase extraction material based on the principle of cation exchange, its effect is extremely poor, with a recovery rate of only 14% for Aβ42;

[0145] This indicates that the MCX magnetic beads selected in this application are not simple cation exchangers. Under acidic conditions, they can efficiently capture intact Aβ peptides through cation exchange mechanisms and enhance retention and effectively exclude interfering substances through a reverse phase mechanism.

[0146] 3) WCX magnetic beads (weak cation exchange): They have poor compatibility with acidic loading conditions and low release efficiency of target peptides in the elution process, resulting in almost no effective capture and recovery of target peptides. Their recovery rate of Aβ42 is only 7%, and their performance is significantly lower than that of Example 1.

[0147] In summary, only MCX magnetic beads, with their optimized mixed-mode cation exchange mechanism, can specifically capture positively charged intact Aβ peptides under acidic loading conditions, while enhancing retention through hydrophobic interactions and achieving efficient elution with alkaline organic solvents. This results in high recovery rates (nearly 100%) for Aβ38, Aβ40, and Aβ42, with good synchronicity between subtypes.

[0148] Comparative Examples 5-9

[0149] A method for detecting β-amyloid protein in dried blood spots differs from Example 1 in that the processing conditions in steps B) and / or C) are different, as shown in the table below:

[0150] Table 8 Comparison of Operating Conditions for Examples 5-9

[0151]

[0152] Then, the samples treated by the method in Comparative Examples 5-9 were analyzed under the same LC-MS / MS conditions as in Example 1, and the results are shown in Table 9:

[0153] Table 9 Performance test results of Comparative Examples 5-9

[0154]

[0155] As shown in the table above, the process conditions selected in Example 1 of this application significantly improve performance compared to Comparative Examples 5-9. This indicates that not all conditions are applicable, as detailed below:

[0156] 1) Protein precipitation is necessary and requires specific conditions: 4% acetonitrile phosphate can both precipitate proteins and provide an acidic environment. Omitting or replacing it (such as guanidine hydrochloride) will lead to a significant decrease in recovery rate (especially Aβ42).

[0157] 2) The polarity of the washing solution needs to be precisely controlled: a low proportion of acetonitrile (10%) can effectively remove interference without eluting the target peptide; a high proportion of organic solvent (80% methanol) will elute the target substance prematurely and reduce the recovery rate;

[0158] 3) One elution is sufficient: Two elutions not only waste time, but also fail to achieve 100% recovery, and may even affect sensitivity due to the dilution effect;

[0159] 4) Key to the formulation of the reconstitution solution: The reconstitution solution containing Triton X-100 and formic acid can prevent adsorption and ensure the synchronization of subtypes; using TFA will lead to abnormal fluctuations in recovery rate and distortion of ratio.

[0160] In summary, the combination of conditions used in Embodiment 1 of this application constitutes a synergistically optimized overall technical solution. Any deviation from any single step will make it difficult to achieve the same excellent extraction effect, especially for Aβ42, a key marker with extremely low abundance and easy loss.

[0161] Comparative Example 10

[0162] To further verify the accuracy of dried blood spots as samples, this application also simultaneously conducted plasma testing for comparison. The difference from Example 1 is that only the test sample was changed to plasma. The test results are shown below. Figure 4-9 And through comparison Figure 4 and Figure 5 , Figure 6 and Figure 7 , Figure 8 and Figure 9 It can be known that:

[0163] After processing with this method, the chromatographic peak shape, signal-to-noise ratio, and retention time of each Aβ subtype in dried blood spot samples were highly consistent with those of plasma samples, indicating that this method successfully overcame the matrix interference of dried blood spots and achieved analytical performance comparable to that of plasma samples. Given the significant advantages of dried blood spots in sampling, transportation, and storage, dried blood spots are an ideal sample type to replace plasma for large-scale AD screening.

[0164] This specific preparation example is merely an explanation of this application and is not intended to limit it. After reading this specification, those skilled in the art can make modifications to this preparation example without contributing any inventive step, but such modifications are protected by patent law as long as they fall within the scope of the claims of this application.

Claims

1. A method for detecting β-amyloid protein in dried blood spots, characterized in that, Includes the following steps: A) Provide dried blood spot samples; B) Add acidic denaturing reagent and internal standard working solution to the dried blood spot sample obtained in A), and perform extraction to obtain sample extract; C) One-step magnetic solid-phase extraction of the sample extract obtained in B) using mixed-mode cation exchange MCX magnetic beads, wherein the extraction includes: Under loading conditions, the MCX magnetic beads capture intact β-amyloid protein in the sample extract. Under washing conditions, interfering substances are removed. Under elution conditions, the intact β-amyloid protein is eluted from the MCX magnetic beads to obtain an eluent. D) The eluent obtained in step C) is analyzed by liquid chromatography-tandem mass spectrometry (LC-MS / MS) to determine the content of β-amyloid protein in the dried blood spot sample.

2. The method for detecting β-amyloid protein in dried blood spots according to claim 1, characterized in that, In step A), the dried blood spot sample is prepared by spotting whole blood onto cellulose filter paper, pure cotton lint, or a separation membrane and then drying it.

3. The method for detecting β-amyloid protein in dried blood spots according to claim 1, characterized in that, In step B), the acidic denaturing reagent is an acetonitrile solution containing phosphoric acid.

4. The method for detecting β-amyloid protein in dried blood spots according to claim 3, characterized in that, The volume percentage concentration of phosphoric acid in the acetonitrile solution containing phosphoric acid is 2-6%.

5. The method for detecting β-amyloid protein in dried blood spots according to claim 1, characterized in that, In step C), the operational conditions for each step are as follows: The sample loading conditions were acidic. The washing conditions include washing with an acidic aqueous solution and a low proportion of organic solvent; The elution conditions are as follows: elution is performed using an organic solvent containing ammonia, and the eluent is a mixed solution of 75% acetonitrile, 15% water and 10% ammonia.

6. The method for detecting β-amyloid protein in dried blood spots according to claim 1, characterized in that, The β-amyloid protein includes at least one of Aβ38, Aβ40, and Aβ42.

7. The method for detecting β-amyloid protein in dried blood spots according to claim 1, characterized in that, In step D), the LC-MS / MS detection adopts multiple reaction monitoring mode, and the detected ion pairs include the parent ion and daughter ion of intact Aβ40 and intact Aβ42.

8. An analytical system for early screening of Alzheimer's disease, characterized in that, include: The sample preprocessing module that performs steps B) and C) of the method according to any one of claims 1-7; The chromatography-mass spectrometry detection module that performs step D) of the method according to any one of claims 1-7; A data analysis module that receives data from the chromatography-mass spectrometry detection module, calculates and outputs the Aβ42 / Aβ40 ratio.

9. The use of the method according to any one of claims 1-7 in the preparation of a diagnostic kit for early screening of Alzheimer's disease, characterized in that, The method exhibits a linear detection range of 10–2500 pg / mL for Aβ38, Aβ40, and Aβ42, with spiked recoveries ranging from 85% to 115%.

Images