A sample pretreatment kit for simultaneously detecting abeta40, abeta42 and ranibizumab and application thereof

Abstract

This invention provides a sample pretreatment kit and its application for the simultaneous detection of Aβ40, Aβ42, and lencanezumab, belonging to the field of biodetection technology. The kit includes: mixed magnetic beads, internal standard solution, trypsin digestion solution, enzyme digestion enhancer, and ammonia; the mixed magnetic beads are composed of protein A magnetic beads and streptavidin magnetic beads; the internal standard solution is composed of… 15 N-labeled Aβ40, 15 This kit consists of N-labeled Aβ42 and lencanezumab internal standard peptides. The pretreatment kit eliminates the need for anti-IgG1 antibodies, requiring only Protein A for detection, thus shortening sample processing time, greatly simplifying the sample handling process, and increasing throughput. The kit can simultaneously detect Aβ40, Aβ42, and lencanezumab, providing more reliable data support for clinical diagnosis and treatment efficacy evaluation, and significantly promoting the development of companion detection technologies for Alzheimer's disease biomarkers and therapeutic drugs.

Description

Technical Field

[0001] This invention belongs to the field of biological detection technology, and in particular relates to a sample pretreatment kit and its application for the simultaneous detection of Aβ40, Aβ42 and lencanezumab. Background Technology

[0002] In the field of Alzheimer's disease diagnosis, amyloid Aβ40, as an important blood biomarker, is closely related to the onset and progression of Alzheimer's disease due to changes in its blood levels. When Alzheimer's-related lesions occur, the metabolism of Aβ40 becomes abnormal, leading to its abnormal accumulation in the blood. Therefore, detecting Aβ40 levels is crucial for the early diagnosis and monitoring of Alzheimer's disease. Meanwhile, lencanezumab, as a drug for treating Alzheimer's disease, specifically binds to and clears amyloid plaques in the brain, thereby slowing disease progression and improving patients' cognitive function.

[0003] Currently, clinical detection of Alzheimer's disease biomarkers often uses cerebrospinal fluid (CSF) as a sample, detecting levels of amyloid proteins such as Aβ40 and Aβ42, as well as Tau protein, to aid in diagnosis. However, CSF collection requires lumbar puncture, which is an invasive procedure. While blood samples are readily available, the concentration of biomarkers in plasma is only about 1% of that in CSF, and its complex composition with numerous interfering substances makes it difficult for existing detection technologies to accurately detect low concentrations of biomarkers such as Aβ40 and Aβ42 in blood. Furthermore, the results show poor correlation with CSF biomarker levels, failing to accurately reflect the disease state. Regarding drug concentration detection, traditional methods often employ single immunoassays, such as enzyme-linked immunosorbent assay (ELISA). This method has limitations in sensitivity and specificity, making it difficult to simultaneously meet the demand for efficient and accurate detection of multiple biomarkers and drugs.

[0004] In the field of Alzheimer's disease (AD) diagnosis, existing technologies face limitations in two key dimensions: test samples and testing techniques. Regarding test samples, while cerebrospinal fluid (CSF) testing has high diagnostic value, its invasive nature leads to low patient acceptance, hindering large-scale screening applications. Blood tests, while convenient for sampling, face multiple challenges: numerous interfering factors, extremely low biomarker concentrations (Aβ40 only 350–400 pg / mL, Aβ42 only 60–90 pg / mL), and poor correlation with CSF test results, all of which severely affect the accuracy of the results.

[0005] In terms of detection technology, traditional immunoassay methods, represented by ELISA, suffer from significant limitations in sensitivity and specificity, failing to meet the clinical need for simultaneous and accurate detection of Alzheimer's disease biomarkers and the therapeutic drug lencanizumab. In contrast, liquid chromatography-tandem mass spectrometry (LC-MS / MS) technology, due to its superior specificity and sensitivity, can significantly reduce interference factors and achieve efficient detection of low-concentration target substances. Particularly noteworthy is that LC-MS / MS detection of biomarkers in blood shows better correlation with cerebrospinal fluid biomarker levels, more accurately reflecting the patient's true disease status. However, LC-MS / MS technology still faces significant technical challenges in detecting Aβ40 and Aβ42 in blood, primarily due to the extremely low concentration of Aβ protein in human blood, far lower than its levels in cerebrospinal fluid (ng level). Of particular concern is the severe lack of effective methods in the current technological system capable of simultaneously detecting Alzheimer's disease biomarkers and therapeutic drugs. Summary of the Invention

[0006] Therefore, the purpose of this invention is to provide a sample pretreatment kit and its application for the simultaneous detection of Aβ40, Aβ42 and lencanezumab.

[0007] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0008] This invention provides a sample pretreatment kit for the simultaneous detection of Aβ40, Aβ42 and lencanezumab. The sample pretreatment kit includes: mixed magnetic beads, internal standard solution, trypsin digestion solution, enzyme digestion enhancer and ammonia.

[0009] The mixed magnetic beads consist of protein A magnetic beads and streptavidin magnetic beads;

[0010] The internal standard solution is composed of 15 N-labeled Aβ40, 15 It consists of N-labeled Aβ42 and lencanetanumab internal standard peptide, wherein the lencanetanumab internal standard peptide is a universal monoclonal antibody labeled with SILu MAB stable isotope.

[0011] In this invention, the mass ratio of the protein A magnetic beads to the streptavidin magnetic beads is preferably 0.5–1.5:1, more preferably 1:1; 15 The final concentration of N-labeled Aβ40 is preferably 20–30 ng / mL, more preferably 22–28 ng / mL, and even more preferably 24, 25, or 26 ng / mL; 15The final concentration of N-labeled Aβ42 is preferably 20–30 ng / mL, more preferably 22–28 ng / mL, and even more preferably 24, 25, or 26 ng / mL; the final concentration of the lencanetumab internal standard peptide is 5–20 μg / mL; the concentration of the trypsin digest is preferably 0.5–1.5 μg / μL, more preferably 0.7–1.2 μg / μL, and even more preferably 1 μg / μL; the concentration of the enzymatic digestion enhancer is preferably 0.5–1.5 μg / μL, more preferably 0.7–1.2 μg / μL, and even more preferably 1 μg / μL. In this invention, since lencanetumab itself is an antibody with an IgG1 structure, this invention has found that pritein A has a high affinity for human IgG1, eliminating the need for enrichment with anti-IgG antibodies and greatly simplifying the sample processing procedure.

[0012] In this invention, the preferred volume ratio of the mixed magnetic beads, internal standard solution, trypsin hydrolysate, enzymatic hydrolysis enhancer, and ammonia is (10-50):(15-25):(1-3):(0.5-1.5):(4-6), more preferably (35-45):(17-23):(1.5-2.5):(0.7-1.2):(4.5-5.5), and even more preferably 40:20:2:1:5.

[0013] In this invention, the sample pretreatment kit further includes 1×PBS buffer, glycine-hydrochloric acid buffer, formic acid, 20%–30% Tween 20, guanidine hydrochloride, and anti-Aβ1-16 antibody. The concentration of the anti-Aβ1-16 antibody is preferably 2–3 μg / μL, more preferably 2.2–2.8 μg / μL, and even more preferably 2.5 μg / μL; the volume ratio of the mixed magnetic beads, 1×PBS buffer, glycine-hydrochloric acid buffer, formic acid, 20%–30% Tween 20, guanidine hydrochloride, and anti-Aβ1-16 antibody is preferably (10–50):(16) 00~3000):(50~150):(0.5~1.5):(4~6):(40~50):(1~10), further preferably (35~45):(1610~2000):(75~120):(0.7~1.3):(4.5~5.5):(42~48):(1.5~7), more preferably 40:1620:100:1:5:45:2.

[0014] This invention provides an application of the above-described sample pretreatment kit in the preparation of the following products a and / or b:

[0015] a. Detect the levels of Aβ40, Aβ42, and lencanezumab;

[0016] b. Diagnose Alzheimer's disease.

[0017] This invention provides a method for simultaneously detecting the concentrations of Aβ40, Aβ42, and lencanemab, comprising the following steps:

[0018] Plasma samples were pretreated to obtain characteristic peptides, which were then quantified using an internal standard method combined with liquid chromatography-tandem mass spectrometry to detect the concentrations of Aβ40, Aβ42, and lencanezumab.

[0019] The pretreatment involves using the aforementioned sample pretreatment kit to capture, denature, and enzymatically digest Aβ40, Aβ42, and lencanezumab in plasma samples using magnetic beads.

[0020] In this invention, the method is used for non-diagnostic purposes.

[0021] Preferably, the method for capturing and denaturing magnetic beads includes the following steps: after washing the mixed magnetic beads, anti-Aβ1-16 antibody is added for conjugation and incubation. Plasma sample, internal standard solution, 20%-30% Tween 20, 1×PBS and guanidine hydrochloride are added to the magnetic beads with conjugated antibody for capture incubation to obtain the captured solution.

[0022] The enzymatic hydrolysis method includes the following steps: the captured solution is washed with 1×PBS, the supernatant is discarded, and the washed captured product is obtained. The washed captured product is eluted with glycine-hydrochloric acid buffer to obtain the eluted captured product. Trypsin hydrolysis solution, enzymatic hydrolysis enhancer and ammonia are added, and enzymatic hydrolysis is incubated to obtain the enzymatic hydrolysate. The supernatant is collected, centrifuged, and the supernatant after centrifugation is collected.

[0023] In this invention, the coupling incubation time is 0.5–3 hours, more preferably 1–2 hours, and even more preferably 1 hour or 2 hours; the coupling incubation temperature is 35–40°C, more preferably 36–38°C, such as 36°C, 37°C, or 38°C; the capture incubation time is 0.5–3 hours, more preferably 1–2 hours, and even more preferably 1 hour or 2 hours; the capture incubation temperature is 35–40°C, more preferably 36–38°C, such as 36°C, 37°C, or 38°C. This invention, by using magnetic beads to capture Aβ40, Aβ42, and lencanemab in plasma samples, can successfully obtain plasma samples containing one or more of Aβ40, Aβ42, and lencanemab.

[0024] In this invention, the enzymatic incubation time is preferably 2-3 hours, more preferably 2.5 hours, and the enzymatic incubation temperature is 35-40°C, more preferably 36-38°C, such as 36°C, 37°C, or 38°C. This invention obtains characteristic peptides through enzymatic hydrolysis. The amino acid sequence of the characteristic peptide of lencanezine monoclonal antibody is shown in SEQ ID NO. 9; the amino acid sequence of the internal standard peptide of SILu MAB stable isotope-labeled universal monoclonal antibody is shown in SEQ ID NO. 12; the amino acid sequence of the characteristic peptide of Aβ40 is shown in SEQ ID NO. 3; and the amino acid sequence of the characteristic peptide of Aβ42 is shown in SEQ ID NO. 4.

[0025] Preferred liquid chromatography conditions:

[0026] Chromatographic column: Waters HSS T3 column, 2.1 × 50 mm, 1.8 μm;

[0027] Mobile phase A was 0.1% formic acid aqueous solution; mobile phase B was 0.1% formic acid acetonitrile solution; gradient elution was used at a flow rate of 0.3 mL / min.

[0028] The device employs a positive ion electrospray ionization source, with the spray voltage set to 5500V and the ion source temperature maintained at 500℃; the atomizing gas pressure is 50psi, the auxiliary heating gas pressure is 50psi, and the curtain gas pressure is 30psi; the scanning mode is MRM.

[0029] The parameters for the compound ion pairs in the mass spectrometer are as follows:

[0030]

[0031] The gradient elution method is as follows:

[0032] time min Mobile phase A% Mobile phase B% 0 90 10 0.5 90 10 3.5 10 90 4.5 10 90 4.6 90 10 5.5 90 10 .

[0033] In this invention, each amino acid of the characteristic peptides of Aβ40 and Aβ42 in the compound ion pair parameters of mass spectrometry is represented by... 15 N-marker.

[0034] In this invention, the above-mentioned pretreatment kit is used to simultaneously detect Aβ40, Aβ42 and lencanemab, and the characteristic peptides are quantified by liquid chromatography-tandem mass spectrometry. The limit of quantitation for Aβ40 is 20 pg / mL, the limit of quantitation for Aβ42 is 10 pg / mL, and the limit of quantitation for lencanemab is 1 μg / mL. The recoveries of Aβ40, Aβ42 and lencanemab are all in the range of 85% to 115%, the detection accuracy is high, the relative standard deviation (RSD) is <15%, and the detection precision is high.

[0035] Compared with the prior art, the present invention has the following beneficial effects:

[0036] This invention provides a sample pretreatment kit and its application for the simultaneous detection of Aβ40, Aβ42, and lencanezumab. This pretreatment kit eliminates the need for anti-IgG1 antibodies; detection can be completed using only Protein A, shortening sample processing time, greatly simplifying the sample processing workflow, and increasing throughput. The kit can simultaneously detect Aβ40, Aβ42, and lencanezumab, providing more reliable data support for clinical diagnosis and treatment efficacy evaluation, and strongly promoting the development of companion detection technologies for Alzheimer's disease biomarkers and therapeutic drugs. Attached Figure Description

[0037] Figure 1 The ion chromatograms for Aβ40 extraction are shown below, where a is the Aβ40 extraction ion chromatogram for quantitative ion channels (ion pair: 1085.6>968.5), b is the Aβ40 extraction ion chromatogram for qualitative ion channels (ion pair: 1085.6>755.5), and c is the Aβ40 extraction ion chromatogram for internal standard ion channels (ion pair: 1097.6>979.5).

[0038] Figure 2 The ion chromatograms for Aβ42 extraction are shown below, where a is the Aβ42 extraction ion chromatogram for quantitative ion channels (ion pair: 635.4>1067.6), b is the Aβ42 extraction ion chromatogram for qualitative ion channels (ion pair: 635.4>968.5), and c is the Aβ42 extraction ion chromatogram for internal standard ion channels (ion pair: 642.4>1079.6).

[0039] Figure 3 The responses of Aβ40 and Aβ42 (25 pg / mL) to different chromatographic columns were analyzed. a1 and a2 are the chromatograms of Aβ40 and Aβ42 analyzed on a C18 (2.1 × 50 mm, 2.6 μm) column, respectively; b1 and b2 are the chromatograms of Aβ40 and Aβ42 analyzed on a T3 (2.1 × 50 mm, 1.8 μm) column, respectively.

[0040] Figure 4The image shows the TIC spectra of lencanide monoclonal antibody peptides. In the image, EEQ… represents EEQYNSTYR, EGG… represents EGGYYYGR, AED… represents AEDTAAVYYCAR (SEQ ID NO. 13), ALP… represents ALPAPIEK (SEQ ID NO. 14), EPQ… represents EPQVYTLPPSR (SEQ ID NO. 15), STS… represents STSGGTAALGCLVK (SEQ ID NO. 16), NQV… represents NQVSLTCLVK (SEQ ID NO. 17), WQQ… represents WQQGNVFSCSVMHEALHNHYTQK (SEQ ID NO. 18), FNW… represents FNWYVDGVEVHNAK (SEQ ID NO. 19), GPS… represents GPSVFPLAPSSK (SEQ ID NO. 20), and TPE… represents TPEVTCVVVDVSHEDPEVK (SEQ ID NO. 19). NO.21), SYY... represents SYYTMDYWGQGTTVTVSSASTK (SEQ ID NO.8), NSL... represents NSLFLQMSSLR (SEQ ID NO.22), GFY... represents GFYPSDIAVEWESNGQPENNYK (SEQ ID NO.23), TTP... represents TTPPVLDSDGSFFLYSK (SEQ ID NO.24), VVS... represents VVSVLTVLHQDWLNGK (SEQ ID NO.25), GLE... represents GLEWVAYISSGSSTIYYGDTVK (SEQ ID NO.7);

[0041] Figure 5 The TIC image shows the peptide profile of lencanide after filtering from a human protein database. In the image, EGG… represents EGGYYYGR, SYY… represents SYYTMDYWGQGTTVTVSSASTK (SEQ ID NO.8), and GLE… represents GLEWVAYISSGSSTIYYGDTVK (SEQ ID NO.7).

[0042] Figure 6 The images show the primary and secondary mass spectra of EGGYYYGR (from a high-resolution mass spectrometer). From left to right, they are the primary mass spectrum of EGGYYYGR, the secondary mass spectrum of EGGYYYGR, and the fragment ion coverage map.

[0043] Figure 7 Extraction ion chromatograms of the lencanide characteristic peptide (EGGYYYGR) and the internal standard peptide (DTLMISR);

[0044] Figure 8The sample processing procedure for Aβ40 and Aβ42 in the control group;

[0045] Figure 9 This is the sample processing flow for Aβ40 and Aβ42 of the present invention;

[0046] Figure 10 The extracted ion chromatograms are for Aβ40 and Aβ42 samples (both at a concentration of 100 pg / mL) processed by the control group method and the present invention's human plasma sample processing method. Here, a1 is the Aβ40 chromatographic peak detected using the control group method, b1 is the Aβ40 chromatographic peak detected using the present invention's human plasma sample processing method (a1 response: 1.59e5, b1 response: 2.5e5), a2 is the Aβ42 chromatographic peak detected using the control group method, and b2 is the Aβ42 chromatographic peak detected using the present invention's human plasma sample processing method (a2 response: 4.13e4, b2 response: 6.96e4).

[0047] Figure 11 Extraction ion chromatograms of Aβ40 and Aβ42 samples (concentration 100 pg / mL) after enzymatic hydrolysis for 2.5 h and 16 h are shown. In the figure, a1 is the Aβ40 chromatographic peak detected in the 2.5 h enzymatic hydrolysis group, b1 is the Aβ42 chromatographic peak detected in the 2.5 h enzymatic hydrolysis group (a1 response: 7.71e4, b1 response: 9.95e3), a2 is the Aβ40 chromatographic peak detected in the 16 h enzymatic hydrolysis group, and b2 is the Aβ42 chromatographic peak detected in the 16 h enzymatic hydrolysis group (a2 response: 9.49e4, b2 response: 1.26e4).

[0048] Figure 12 Linearity plot of Aβ40 (linear range: 20 pg / mL to 1000 pg / mL);

[0049] Figure 13 Linearity plot of Aβ42 (linear range: 10 pg / mL to 500 pg / mL);

[0050] Figure 14 Linearity graph of lencanizate (linear range: 1 μg / mL to 50 μg / mL);

[0051] Figure 15 The results from left to right show the detection limits of Aβ40 (20 pg / mL), Aβ42 (10 pg / mL), and lencanemab (1 μg / mL). Detailed Implementation

[0052] In this invention, unless otherwise specified, all raw material components are commercially available products well known to those skilled in the art.

[0053] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0054] In the following embodiments, 15 N 40 -Aβ40 15 N 40 This means that Aβ40 has 40 amino acids, and each amino acid is labeled with a unique identifier. 15 N, a total of 40 were labeled. 15 N 42 -Aβ42 15 N 42 This means that Aβ42 has 42 amino acids, and each amino acid is labeled with a unique identifier. 15 N, a total of 42 were marked. GAIIGLMVGGVV- 15 N 12 middle 15 N 12 This means that the peptide segment has a total of 12 amino acids, and each amino acid is labeled. 15 N, a total of 12 were marked. GAIIGLMVGGVVIA- 15 N 14 middle 15 N 14 This means that the peptide segment has a total of 14 amino acids, and each amino acid is labeled. 15 N, a total of 14 were labeled.

[0055] Preparation of calibrators, quality control samples, and internal standards according to the present invention:

[0056] (1) Preparation of stock solution

[0057] Using DMSO as the solvent, stock solutions of Aβ40 and Aβ42 at concentrations of 1 mg / mL were prepared, respectively. Similarly, using DMSO as the solvent, stock solutions of Aβ40 and Aβ42 at concentrations of 0.5 mg / mL were prepared. 15 N 40 -Aβ40 stock solution and 0.5 mg / mL 15 N 42 -Aβ42 stock solution. Dissolve SILu MAB standard (purchased from Sigma, catalog number: MSQC3) in 0.1% formic acid acetonitrile solution to prepare a stock solution with a concentration of 0.1 mg / mL. Lencanetumab injection (100 mg / mL, purchased from Beijing Xingshitang Pharmaceutical, National Drug Approval Number SJ20240001) can be used directly as a stock solution.

[0058] (2) Preparation of secondary stock solution

[0059] Take 6 μL of Aβ40 stock solution and 3 μL of Aβ42 stock solution, place them in the same reagent bottle, and dilute to 6 mL with 1% ammonia-acetonitrile solution to prepare secondary stock solutions with a concentration of 1 μg / mL Aβ40 and 0.5 μg / mL Aβ42. The 1% ammonia-acetonitrile solution is obtained by mixing 1 mL of ammonia solution with 99 mL of acetonitrile.

[0060] (3) Internal standard secondary stock solution

[0061] Take 5 μL 15 N 40 -Aβ40 stock solution and 5 μL 15 N 42 -Aβ42 stock solution was placed in the same reagent bottle and diluted to 5 mL with 1% ammonia and acetonitrile to prepare a concentration of 0.5 μg / mL. 15 N 40 -Aβ40 internal standard secondary stock solution and concentration of 0.5 μg / mL 15 N 42 -Aβ42 internal standard secondary stock solution. The 1% ammonia-acetonitrile solution is obtained by mixing 1 mL of ammonia solution with 99 mL of acetonitrile.

[0062] (4) Preparation of blank matrix

[0063] Weigh 12g of bovine serum albumin, add 360mL of water and 40mL of 10×PBS, then add 40μL of surfactant, 40μL of preservative, and 40μL of antioxidant, and mix. The surfactant is Tween 20, the preservative is Proclin 950, and the antioxidant is 1MDTT.

[0064] (5) Preparation of working solution for analyte

[0065] Take 125 μL of 1 μg / mL Aβ40 secondary stock solution, 125 μL of 0.5 μg / mL Aβ42 secondary stock solution, and 62.5 μL of lencanide injection, and dilute with blank matrix to 125 mL to prepare the analyte working solution (Aβ40: 1 ng / mL; Aβ42: 0.5 ng / mL; lencanide: 50 μg / mL).

[0066] (6) Preparation of internal standard working solution

[0067] Take 500 μL of 0.5 μg / mL solution respectively. 15 N 40 -Aβ40 internal standard secondary stock solution or 500 μL of 0.5 μg / mL solution. 15 N 42 The internal standard secondary stock solution of -Aβ42 was diluted to 10 mL with a blank matrix to prepare 25 ng / mL solutions.15 N 40 -Aβ40 internal standard working solution and concentration of 25 ng / mL 15 N 42 The internal standard working solution of -Aβ42 was prepared, and then dispensed into 2.5 mL vials at a rate of 0.2 mL each, followed by freeze-drying.

[0068] (7) Preparation of calibrators and quality control samples (see Table 1)

[0069] Table 1. Preparation process of calibrators and quality control materials for Alzheimer's disease comorbidity testing kits.

[0070]

[0071] After preparation, both calibrators and quality control samples were dispensed in 0.5 mL portions into 2.5 mL vials and then freeze-dried.

[0072] In this embodiment, the streptavidin magnetic beads used are directly prepared by mixing commercially available streptavidin magnetic beads and protein A magnetic beads in a 1:1 volume ratio. The particle size of each magnetic bead is 1 μm. The mixed magnetic beads are dispensed into brown vials in 5 mL portions and stored at room temperature.

[0073] Preparation of trypsin hydrolysate: 100 μg of lyophilized trypsin (purchased from Promega, catalog number: V5117) was dissolved in deionized water to a concentration of 1 μg / μL and then dispensed as trypsin hydrolysate.

[0074] Preparation of enzymatic hydrolysis enhancers: Trypsin enhancer (full name ProteaseMAX) TM Surfactant (Trypsin Enhancer, purchased from Promega, catalog number: V2071) 1 mg was dissolved in deionized water to a concentration of 1 μg / μL and then dispensed to obtain the enzyme hydrolysis enhancer.

[0075] 25% Tween 20: Dilute Tween (purchased from McLean, product number: T6335) with water by 4 times its volume.

[0076] Glycine-hydrochloric acid buffer: purchased from Guangzhou Yitao Biotechnology Co., Ltd., product number: ED-8290-500mL.

[0077] Preparation of denaturant:

[0078] Weigh 57.3g of guanidine hydrochloride, dissolve it in 100mL of deionized water, and dispense 10mL into brown vials. Store at room temperature. In actual production, the preparation volume can be adjusted according to the production volume.

[0079] Preparation of buffer solution:

[0080] Dilute the commercially available 10×PBS buffer 10 times with deionized water to obtain a 1×PBS solution, which can be stored at room temperature. In actual production, the preparation volume can be adjusted according to the production volume.

[0081] In the following examples, the anti-Aβ1-16 antibody was purchased from Abcam, catalog number: ab126649.

[0082] Example 1

[0083] Optimization of Aβ protein and internal standard liquid chromatography-mass spectrometry conditions

[0084] Aβ40 is a small protein of 40 amino acids, with the amino acid sequence: DAEFRHDSGYEVHHQKLVFFAEDVGSNKGA IIGLMVGGVV (SEQ ID NO.1). Aβ42 is a small protein composed of 42 amino acids, with the amino acid sequence: DAEFRH DSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVVIA (SEQ ID NO.2). The internal standard for Aβ40... 15 N 40 -Aβ40 and Aβ42 internal standards 15 N 42 -Aβ42 all belong to 15 Fully marked N, of which 15 N 40 -Aβ40 is labeled with 40 amino acids. 15 N 42 -Aβ42 is labeled with 42 amino acids, and the labeling site is located at the N atom at the N end of each amino acid.

[0085] When using LC-MS / MS technology for quantification, it is possible to quantify Aβ40 and Aβ42 proteins in their complete sequences, as well as characteristic peptides after trypsin digestion. The purpose of this invention is to achieve the combined detection of Aβ protein and lencanezumab. Quantitative detection of lencanezumab requires an enzymatic digestion process, which inevitably digests the complete Aβ protein. Therefore, this invention utilizes the characteristic peptides after enzymatic digestion to achieve the quantitative detection of Aβ40 and Aβ42.

[0086] (1) After enzymatic hydrolysis, Aβ40, Aβ42, 15 N 40 -Aβ40 and 15 N 42 Optimization of Aβ42 mass spectrometry conditions

[0087] Dissolve Aβ40 and Aβ42 standard powders in the first solvent to prepare Aβ40 stock solutions and Aβ42 stock solutions with a concentration of 0.5 mg / mL, respectively. Similarly, ... 15 N40 -Aβ40 and 15 N 42 -Aβ42 standard powder was prepared into a solution of 0.5 mg / mL using the first solvent. 15 N 40 -Aβ40 stock solution and 15 N 42 -Aβ42 stock solution.

[0088] The Aβ40 and Aβ42 stock solutions were diluted with a second solvent to prepare a 1 μg / mL Aβ40 analyte working solution or an Aβ42 analyte working solution. Similarly, 15 N 40 -Aβ40 stock solution and 15 N 42 The Aβ42 stock solution was diluted with a second solvent to a concentration of 1 μg / mL. 15 N 40 -Aβ40 internal standard mixed working solution or 15 N 42 -Aβ42 internal standard mixed working solution.

[0089] The first solvent is dimethyl sulfoxide (DMSO), and the second solvent is a solution containing 20% ​​acetonitrile. The 20% acetonitrile solution is prepared by mixing 20 mL of acetonitrile and 80 mL of water, then adding 0.1 mL of ammonia water and mixing.

[0090] Take 10 μL of 1 μg / mL analyte working solution and 10 μL of 1 μg / mL internal standard working solution, respectively, and place them in 1.5 mL centrifuge tubes. Add 2 μL of trypsin (0.5 μg / μL) and 80 μL of deionized water, respectively. Incubate at 37 °C for 4 h. Add 1 μL of formic acid, mix well, and dilute to 0.5 mL with 20% acetonitrile. Place the tubes in an OrbitrapExploris 240 high-resolution mass spectrometer and use DDAMS (Digital-Distributed Atomic Mass Spectrometry) to obtain Aβ40, Aβ42, and... 15 N 40 -Aβ40 and 15 N 42 -Aβ42 and its peptide profiling data. The working solution of the above analytes is the Aβ40 analyte working solution, and the corresponding internal standard working solution is... 15 N 40 -Aβ40 internal standard working solution, or the above analyte working solution is an Aβ42 analyte working solution, and the corresponding internal standard working solution is: 15 N 42 -Aβ42 internal standard mixed working solution.

[0091] Since Aβ40 and Aβ42 contain 40 identical amino acids, the terminal peptides of the sequence after enzymatic digestion are the characteristic peptides: the characteristic peptide of Aβ40 is GAIIGLMVGGVV (SEQ ID NO.3); the characteristic peptide of Aβ42 is GAIIGLMVGGVVIA (SEQ ID NO.4).

[0092] The parent ion m / z of GAIIGLMVGGVV is 1085.6 ([M+H]). + (single charge), fragment ions m / z are 755.5 and 968.5.

[0093] The m / z of the single-charge precursor ion GAIIGLMVGGVVIA is 1269.8 ([M+H]). + The fragment ions (single-charged) have m / z values ​​of 1180.6 and 1067.6. The doubly charged parent ion has an m / z value of 635.4 ([M+2H]). 2+ (Double charge), fragment ions have m / z values ​​of 968.5 and 1067.6.

[0094] GAIIGLMVGGVV isotope markers ( 15 N 12 The parent ion m / z of -GAIIGLMVGGVV) is 1097.6 ([M+H]). + (single charge), the fragment ions have m / z values ​​of 763.5 and 979.5.

[0095] GAIIGLMVGGVVIA isotope markers ( 15 N 14 The precursor ion m / z of -GAIIGLMVGGVVIA is 642.4 ([M+2H]). 2+ (Double charge), fragment ions m / z are 979.6 and 1079.6.

[0096] The enzymatically digested samples were placed in a Waters Acquity UPLC I Class liquid chromatograph-tandem TQS mass spectrometer. Ion source temperature, cone voltage, and collision voltage were optimized. Chromatograms of Aβ40, Aβ42, and the internal standard are shown below. Figure 1 and Figure 2 .

[0097] Table 2 Figure 1 and Figure 2 The results showed that GAIIGLMVGGVV and 15 N 12 -GAIIGLMVGGVV showed the strongest responses in the 1085.6 / 968.5 and 1097.6 / 979.5 ion channels, while GAIIGLMVGGVVIA and 15 N 14-GAIIGLMVGGVVIA showed the strongest responses in the 635.4 / 1067.6 and 642.4 / 1079.6 ion channels. Ultimately, the monocharged precursor ion of GAIIGLMVGGVV (1085.6 / 968.5) and the dicharged precursor ion of GAIIGLMVGGVVIA (635.4 / 1067.6) were selected as the quantitative ion.

[0098] The specific ion pair parameters are shown in Table 2.

[0099] Table 2 Ion pair parameters of GAIIGLMVGGVV and GAIIGLMVGGVVIA and their internal standards

[0100]

[0101]

[0102] Furthermore, this invention optimizes mass spectrometry and liquid chromatography conditions on different models of mass spectrometers. The selected liquid chromatography-tandem mass spectrometers include AB SCIEX 5500Plus, AB SCIEX 6500Plus, AB SCIEX 7500, and Waters TQ-S. The liquid chromatography system connected in series with the AB SCIEX mass spectrometer is Jasper HPLC or ExionAD, and the liquid chromatography system connected in series with the TQ-S is UPLC Class I.

[0103] The liquid chromatography and mass spectrometry conditions for AB SCIEX 5500Plus, AB SCIEX 6500Plus, and AB SCIEX 7500 are the same, as shown in Table 3.

[0104] Table 3 shows the mass spectrometry and liquid chromatography conditions for AB SCIEX (Aβ40 and Aβ42).

[0105]

[0106] The liquid chromatography and mass spectrometry conditions for Waters TQ-S are shown in Table 4.

[0107] Table 4. Mass spectrometry and liquid chromatography conditions (Aβ40 and Aβ42) for Waters TQ-S.

[0108]

[0109]

[0110] The preparation method for mobile phase A as described in Tables 3 and 4 is as follows: Add 1 mL of formic acid to 1 L of deionized water and mix well to obtain a 0.1% formic acid aqueous solution. The preparation method for mobile phase B is as follows: Add 1 mL of formic acid to 1 L of acetonitrile and mix well to obtain a 0.1% formic acid-acetonitrile solution.

[0111] This invention focuses on the screening of chromatographic columns. Using a Waters UPLC I class tandem Xevo TQ-S mass spectrometer, and employing the ion pair parameters, mass spectrometry conditions, and liquid chromatography conditions listed in Table 4, different chromatographic columns were used to detect Aβ40 (50 pg / mL) and Aβ42 (25 pg / mL). The selected chromatographic columns were C18 and T3 columns, with column lengths of 50 mm and 100 mm, column inner diameters of 2.1 mm and 3 mm, and particle sizes of 1.7 μm, 1.8 μm, 2.5 μm, and 2.6 μm.

[0112] Figure 3 The results showed that C18 (2.1×50 mm, 2.6 μm) and T3 (2.1×50 mm, 1.8 μm) were selected as the analytical columns for Aβ40 and Aβ42, respectively. The response value of Aβ40 was 2.6e4, and the response value of Aβ42 was 5e3. T3 (2.1×50 mm, 1.8 μm) was selected as the analytical column for Aβ40 and Aβ42, respectively. The response value of Aβ40 was 3.4e4, and the response value of Aβ42 was 1.4e4.

[0113] Example 2: Optimization of liquid chromatography-mass spectrometry (LC-MS) conditions for lencanizate and internal standard.

[0114] (1) Screening of characteristic peptides

[0115] Lencanemab is a monoclonal antibody with an IgG1 structure and a molecular weight of approximately 140 kDa. The strategy for mass spectrometry quantification of monoclonal antibodies is as follows: the monoclonal antibody is digested with trypsin, and the peptide spectra are acquired using high-resolution mass spectrometry. The data is then imported into Skyline software to identify and screen subsequent peptides for quantification.

[0116] Amino acid sequence of the light chain of lencanamide:

[0117] DVVMTQSPLSLPVTPGAPASISCRSSQSIVHSNGNTYLEWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLRISRVEAEDVGIYYCFQGSHVPPTFGPGTKLEI KRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ ID NO.5).

[0118] Amino acid sequence of the heavy chain of lencanamide:

[0119] EVQLVESGGGLVQPGGSLRLSCSASGFTFSSFGMHWVRQAPGKGLEWVAYISSGSSTIYYGDTVKGRFTISRDNAKNSLFLQMSSLRAEDTAVYYCAREGGYYYGRSYYTMDYW GQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDK THTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTI SKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO.6).

[0120] In this invention, 100 mg / mL lencanemab injection solution was used as a standard. Peptide spectra after enzymatic digestion were acquired using an OrbitrapExploris 240 high-resolution mass spectrometer (Thermo Scientific). The data were then imported into Skyline software to obtain information on several candidate peptides on the light and heavy chains. Figure 4 In Skyline software, the peptide digestion method was set to trypsin, the missed cleavage site was set to 0, and human plasma protein data was used as background filter. The final candidate peptide information for the three characteristic peptides is shown below. Figure 5 All three peptides are located in the heavy chain of lencanetan antibody.

[0121] from Figure 5 As shown in the (TIC plot), the peptides GLEWVAYISSGSSTIYYGDTVK (SEQ ID NO.7) and SYYTMDYWGQGTTVTVSSASTK (SEQ ID NO.8) have too long retention times and low mass spectrometry responses. Therefore, the peptide EGGYYYGR (SEQ ID NO.9) was selected as the quantitative peptide for lencanizate.

[0122] (2) Selection of lencanezomib internal standard

[0123] Since lencanizate sample processing requires immunocapture and enzymatic digestion, using isotopically labeled monoclonal antibodies as internal standards for lencanizate quantitative analysis can calibrate the entire process, including immunocapture, enzymatic digestion, and mass spectrometry. This invention selects a commercially available SILu MAB stable isotopically labeled universal monoclonal antibody standard as the internal standard (purchased from Sigma, catalog number: MSQC3). The amino acid sequence of this internal standard is as follows:

[0124] The amino acid sequence of the SILuMab heavy chain is as follows:

[0125] EVQLVESGGGLVQPGGSLRLSCVASGFTLNNYDMHWVRQGIGKGLEWVSKIGTAGDRYYAGSVKGRF

[0126] TISRENAKDSLYLQMNSLRVGDAAVYYCARGAGRWAPLGAFDIWGQGTMVTVSSASTKGPSVFPLAP

[0127] SSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYIC

[0128] NVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPK DTLMISR TPEVTCVVVDVSH

[0129] EDPEVK FNWYVDGVEVHNAK TKPREEQYNSTYRV VSVLTVLHQDWLNGK EYKCKVSNKALPAPIE

[0130] KTISKAKGQPREPQVYTLPPSRDELTK NQVSLTCLVKGFYPSDIAVEWESNGQPENNYK TTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO. 10).

[0131] The amino acid sequence of the SILuMab light chain is as follows:

[0132] QSALTQPRSVSGSPGQSVTISCTGTSSDIGGYNFVSWYQQHPGKAPKLMIYDATKRPSGVPDRFSGSKS

[0133] GNTASLTISGLQAEDEADYYCCSYAGDYTPGVVFGGGTKLTVLGQPKAAPSVTLFPPSSEELQANKATL

[0134] VCLISDFYPGAVTVAWKADSSPVK AGVETTTPSK QSNNK YAASSYLSLTPEQWK SHRSYSCQVTHEGSTVEKTVAPTECS (SEQ ID NO. 11).

[0135] The bolded underlined portions of the amino acid sequences of the SILuMab heavy and light chains represent... 15 N isotope labeled sequence.

[0136] In the SILuMab-labeled peptides, DTLMISR and the lencanide-quantitative peptide EGGYYYGR are both located on the heavy chain of the monoclonal antibody and have similar peptide lengths. Therefore, this invention selects DTLMISR (SEQ ID NO.12) on the heavy chain of SILuMab as the internal standard peptide.

[0137] (3) Optimization of LC-MS / MS conditions for lencanezumab and internal standard

[0138] The peptide profiling data of lencanizate were processed using BiopharamFinder 5.1 software, and the fragment ion information of EGGYYYGR was analyzed. The analysis results are shown in [the table below]. Figure 6 The precursor ion of EGGYYYGR, 482.7111 (double charge), and the fragment ion, 558.2667 (y4 ion), showed the highest response. Therefore, the quantitative ion pair was set to 482.8 / 558.2, and the qualitative ion pair was set to 482.8 / 395.2 (since the tandem quadrupole used for quantitative analysis is a low-resolution mass spectrometer, the quantitative precursor ion is 482.8 Da, the quantitative fragment ion is 558.2 Da, the qualitative precursor ion is 482.8 Da*, and the quantitative fragment ion is 395.2 Da).

[0139] It should be noted that the deviation of compound ions under high-resolution mass spectrometry is generally within 5 ppm, while the mass deviation of tandem quadrupole mass spectrometers (such as the 6500, TQ-S, etc.) is generally ±0.5 Da. Figure 6 The permissible deviation for a mass-to-charge ratio of 482.7 in a quadrupole mass spectrometer is 482.7 ± 0.5 Da.

[0140] The ion pair information for the internal standard peptide DTLMISR can be obtained from the COA file of the standard. The ion pair information is 423.2 / 629.4 (parent ion: 423.2 Da, fragment ion: 629.4 Da).

[0141] The enzymatically digested sample was placed in the autosampler of a Shimadzu LC-30 tandem SCIEX 6500+ liquid chromatograph, and the ion source temperature, declustering voltage, and collision energy were optimized. The final LC-MS / MS parameters are shown in Table 5, and the chromatograms are shown below. Figure 7 .

[0142] Table 5. LC-MS / MS conditions for the characteristic peptide (EGGYYYGR) and internal standard peptide (DTLMISR) of lenkanemab.

[0143]

[0144]

[0145] The preparation method for mobile phase A described in Table 5 is as follows: Add 1 mL of formic acid to 1 L of deionized water and mix well to obtain a 0.1% formic acid aqueous solution. The preparation method for mobile phase B is as follows: Add 1 mL of formic acid to 1 L of acetonitrile and mix well to obtain a 0.1% formic acid-acetonitrile solution.

[0146] Example 3: Pretreatment Optimization of Aβ40 and Aβ42

[0147] The concentrations of Aβ40 and Aβ42 in human plasma are extremely low, generally in the pg range (1×10⁻⁶). -12 g) Conventional protein precipitation, liquid-liquid extraction or solid-phase extraction cannot achieve a satisfactory enrichment level.

[0148] Control group: Immunocapture (IP) was used as the enrichment method for Aβ40 and Aβ42 in plasma samples.

[0149] (1) 0.5 mg of epoxy magnetic beads (Dynabeads M-270) and 1.67 μg of antibody (anti-Aβ1-16) were coupled according to the Thermo epoxy magnetic beads instructions (approximately 16-24 h) to obtain the antibody-coupled magnetic beads.

[0150] (2) Add an internal standard (25 ng / mL) to 500 μL of calibrator, quality control sample or plasma sample to be tested. 15 N 40 -Aβ40 internal standard working solution or 15 N 42 -Aβ42 internal standard working solution), vortex mix for 30 min. The standard is standard 1 to standard 6 in Table 1, and the quality control is quality control 1 to quality control 3.

[0151] (3) Add the antibody-conjugated magnetic beads to the plasma sample to be tested and incubate at 26°C for 3 hours.

[0152] (4) Wash the magnetic beads three times with PBS buffer.

[0153] (5) Elution: Elute with 0.1 mL of 50% acetonitrile solution containing 2% ammonia, and collect all the eluent. The 50% acetonitrile solution containing 2% ammonia is prepared by mixing 50 mL of acetonitrile with 48 mL of water, and then adding 2 mL of ammonia solution.

[0154] (6) The eluent is dried under vacuum and dissolved in a 30% ethanol solution containing 1% ammonia. The 30% ethanol solution containing 1% ammonia is obtained by mixing 30 mL of anhydrous ethanol with 69 mL of water and then adding 1 mL of ammonia.

[0155] (7) Add 0.5 μg of trypsin, incubate at 37°C with shaking for 14 h, then add 1 μL of 1% formic acid aqueous solution to terminate the reaction and obtain the enzymatic hydrolysis product. The 1% formic acid aqueous solution is obtained by mixing 99 mL of water with 1 mL of formic acid.

[0156] (8) The enzymatic hydrolysis product is dried under vacuum and dissolved in 50% acetonitrile (containing 5% ammonia) to obtain an enzymatic hydrolysis product solution.

[0157] (9) Centrifuge the enzymatic hydrolysis product solution obtained in step (8) at 10,000 rpm, collect the supernatant, transfer it to a vial for LC-MS / MS analysis.

[0158] Current literature reporting methods, such as Figure 8 (Reference 1: DOI:10.1016 / j.jpba.2024.116396), in which the pre-coupling of magnetic beads takes 16 to 24 hours according to the Thermo Fisher protocol.

[0159] The method for processing human plasma samples according to the present invention is as follows:

[0160] (1) Transfer 30 μL of streptavidin magnetic beads (10 μg / μL) into a 1.5 mL centrifuge tube, add 200 μL of 1×PBS buffer and wash twice, then discard the washing solution.

[0161] (2) Add 2 μL of 2.5 μg / μL anti-Aβ protein antibody (anti-Aβ1-16 antibody purchased from Abcam, catalog number: ab126649), and incubate the centrifuge tube at 37°C for 1 h.

[0162] (3) Add 450 μL of calibrator, quality control material or human plasma sample, and 20 μL of internal standard working solution (25 ng / mL). 15 N 40 -Aβ40 internal standard working solution or solution with a concentration of 25 ng / mL 15 N 42The solution contained 5 μL of 25% Tween 20 and 20 μL of 1×PBS (internal standard working solution of -Aβ42), and was incubated at 37°C for 1.5 h. The standards were standards 1 to 6 in Table 1, and the quality control products were quality control products 1 to 3.

[0163] (4) Place the centrifuge tube back on the magnetic rack and let it stand for about 1 minute. After the magnetic beads are adsorbed, remove the supernatant. Add 200 μL of 1×PBS each time to wash the tube. Repeat the washing operation 3 times.

[0164] (5) Elution: Add 100 μL of glycine-hydrochloric acid buffer (pH 2.5), shake for 2 min, place on a magnetic rack, and transfer the supernatant to a new 1.5 mL centrifuge tube.

[0165] (6) Add 1 μL of 1 μg / μL trypsin hydrolysate, 1 μL of 1 μg / μL enzyme enhancer and 5 μL of ammonia water, and incubate at 37°C with shaking for 2.5 h. Then add 1 μL of formic acid to terminate the reaction and obtain the enzymatic hydrolysate.

[0166] (7) Centrifuge the enzymatic hydrolysis product obtained in step (6) at 10,000 rpm for 5 min, collect the supernatant after centrifugation, transfer it to a vial for LC-MS / MS analysis.

[0167] The LC-MS / MS analysis was performed using a Waters UPLC I class tandem Xevo TQ-S mass spectrometer, with the ion pair parameters, mass spectrometry conditions, and liquid chromatography conditions listed in Table 4. Aβ40 (100 pg / mL) and Aβ42 (100 pg / mL) were detected using a Waters HSS T3 column (2.1 × 50 mm, 1.8 μm).

[0168] The beneficial effects of the human plasma sample processing method of the present invention compared with the control group are as follows:

[0169] Advantage 1: Compared with the control group, the human plasma sample processing method of the present invention requires only 1 hour to couple magnetic beads and 1.5 hours for immune capture, while the control group requires 16-24 hours for magnetic bead coupling and 3 hours for immune capture.

[0170] Advantage 2: Compared with the control group, the human plasma sample processing method of the present invention does not require drying and reconstruction, while the control group requires two nitrogen blowing reconstruction processes. The processing method of the present invention not only improves the mass spectrometry signal of the analyte, but also greatly increases the throughput of sample processing.

[0171] Advantage 3: This invention uses glycine-hydrochloric acid buffer for elution. After alkalization of the eluent, it is directly enzymatically hydrolyzed. The enzymatic hydrolysis time is only 2.5 hours, while the control group takes 14 hours.

[0172] Furthermore, by comparing the control group method (elution with 50% acetonitrile ammonia solution, followed by reconstruction) with the elution method of this invention (elution with glycine-hydrochloric acid buffer), it was found that the Aβ40 and Aβ42 responses were significantly improved in the samples treated by this invention (see [link to original text]). Figure 10 ).

[0173] Regarding the enzymatic hydrolysis time, this invention compares enzymatic hydrolysis of 2.5h and overnight enzymatic hydrolysis (16h). The method for overnight enzymatic hydrolysis is to change "after shaking and incubating at 37℃ for 2.5h" in step (6) above to "after shaking and incubating at 37℃ for 16h", while the other steps remain unchanged.

[0174] Figure 11 The results showed that, through comparison, overnight enzymatic hydrolysis ( Figure 11 After adding a2 and b2), the analyte response improved slightly, but not significantly. Therefore, an enzymatic digestion time of 2.5 h was chosen. Figure 11 (a1 and b1 in the text).

[0175] Example 4: Optimization of lencanemab pretreatment

[0176] Since lencanetumab itself is an antibody with an IgG1 structure, some literature reports the use of anti-human IgG antibody conjugated with magnetic beads for immunocapture, followed by overnight enzyme digestion. The control group (application number 202410827868.2, publication number CN118777453A) was captured using Protein G magnetic beads, and lencanetumab was eluted by high-speed vortexing with 0.25% formic acid aqueous solution for 10 min.

[0177] This invention targets the structural characteristics of lencanizumab. Protein A has a high affinity for human IgG1, eliminating the need for enrichment with anti-IgG antibodies. After enrichment, lencanizumab requires denaturation, reduction, alkylation, and enzymatic digestion. Since the characteristic peptide EGGYYYGR of lencanizumab and the characteristic peptide DTLMISR of the internal standard SILuMab are both located in the heavy chain region, denaturation, reduction, alkylation, and enzymatic digestion are all performed on magnetic beads. After alkylation, the magnetic beads are washed to remove denaturing and reducing agents before enzymatic digestion. This invention uses Protein A magnetic beads for enrichment, and the specific method is as follows:

[0178] (1) Magnetic bead washing: Transfer 15 μL of protein A magnetic beads to a 1.5 mL centrifuge tube, add 200 μL of 1×PBS buffer, wash twice, and discard the washing solution.

[0179] (2) Sample incubation: Transfer 30 μL of human plasma sample and 20 μL of internal standard (SILu MAB stable isotope labeled universal monoclonal antibody) to a centrifuge tube containing magnetic beads, add 200 μL of 1×PBS buffer, and incubate in a constant temperature shaker at 1000 rpm and 25°C for 30 min.

[0180] (3) Denaturation and reduction treatment: Add 50 μL of 8M urine solution and 50 μL of 25mM TCEP solution, and incubate at 40℃ for 15 min.

[0181] (4) Washing: Add 200 μL of 50 mM ammonium bicarbonate solution to wash the magnetic beads, remove the supernatant by magnetic adsorption, remove residual reagents, and ensure the purity of the system.

[0182] (5) Enzymatic hydrolysis reaction: Add 80 μL of 50 mM ammonium bicarbonate solution, 1 μL of enzyme enhancer and 1 μL of rpsin hydrolysate to a centrifuge tube, and incubate with shaking at 37°C and 1000 rpm for 2.5 h.

[0183] (6) Reaction termination: After incubation, add 3 μL of 10% formic acid aqueous solution and mix well to terminate the reaction and stabilize the enzymatic hydrolysis product.

[0184] (7) Sample preparation: The enzymatic hydrolysis product obtained in step (6) is centrifuged at 10,000 rpm for 5 min, and the supernatant after centrifugation is collected and transferred to a vial for LC-MS / MS analysis.

[0185] Preparation of 50mM ammonium bicarbonate solution: Weigh 198mg of ammonium bicarbonate and dissolve it in 50mL of deionized water.

[0186] Preparation of 8M urine solution: Weigh 24g of urea and dissolve it in 50mL of deionized water.

[0187] Preparation of 25mM TCEP solution: Weigh 358mg (tris(2-carboxyethyl)phosphonic acid hydrochloride) and dissolve it in 50mL of deionized water.

[0188] The LC-MS / MS analysis was performed using an Exion liquid chromatograph connected in series with an AB SCIEX 6500Plus mass spectrometer, under the LC-MS / MS conditions in Table 5, using a Waters HSS T3 column (2.1×50mm, 1.8μm) to detect lencanetumab.

[0189] Compared with existing patented technologies, this invention achieves a breakthrough in antibody analysis and processing, mainly by integrating denaturation, reduction, and enzymatic digestion into a single operation within the magnetic bead system. Taking lencanezumab with a humanized IgG1 structure as an example, its characteristic peptide EGGYYYGR is located in the Fab region of the heavy chain. This invention utilizes Protein A magnetic beads to specifically conjugate antibody Fc fragments to construct a stable "magnetic bead-antibody complex" reaction system, and innovatively achieves in-situ processing throughout the entire process.

[0190] Key technological advantages include: First, significantly shortening processing time; the denaturation and reduction steps can be completed in just 15 minutes, compared to the control group (application publication number CN118777453A), which uses a 95℃ high-temperature heating for 30 minutes followed by cooling and no reduction treatment, resulting in a processing efficiency improvement of over 50%. Second, simplifying the operation process; utilizing the separation characteristics of magnetic beads, denaturant removal can be completed simply by aspirating and discarding the supernatant, completely eliminating the traditional elution step. Third, the integrated processing mode achieves seamless connection from coupling to enzymatic digestion, eliminating the need for additional intermediate processing steps, greatly improving the consistency and stability of analysis and detection, and providing a more efficient technical path for antibody characteristic peptide analysis based on LC-MS / MS.

[0191] In addition, in the LC-MS / MS conditions of the control group, the injection volume was 20 μL and the analysis time for each sample was 12 min. In the LC-MS / MS conditions of the present invention, the minimum injection volume was 5 μL and the analysis time for each sample was 4 min (see Table 5).

[0192] Example 5: Optimization of pretreatment for combined detection of Aβ40, Aβ42, and lencanemab

[0193] Because of the significant differences in molecular weight and structure between Aβ protein and lencanizate, a mixed magnetic bead method is used for co-detection. Since streptavidin magnetic beads have extremely high specificity with biotinylated antibodies, anti-Aβ1-16 antibody is added to the mixed magnetic beads. After the coupling reaction is complete, the sample is added. Aβ40 and Aβ42 in the sample specifically bind to the anti-Aβ1-16 antibody, while lencanizate binds to protein A.

[0194] (1) Magnetic bead washing: Protein A magnetic beads and streptavidin magnetic beads were mixed at a mass ratio of 1:1 to obtain mixed magnetic beads. 40 μL of the mixed magnetic beads was transferred to a 1.5 mL centrifuge tube, and 0.5 mL of 1×PBS was added. The mixed magnetic beads were gently vortexed to disperse them, and then placed on a magnetic rack to stand for 1 min. After the solution became clear, the supernatant was aspirated with a pipette. The washing was repeated twice to effectively remove impurities from the surface of the magnetic beads and ensure the specificity of subsequent reactions.

[0195] (2) Antibody conjugation: Add 100 μL of 1×PBS to the centrifuge tube after washing, followed by 2 μL of anti-Aβ1-16 antibody (2.5 μg / μL). Place the centrifuge tube at 37°C for conjugation incubation. The conjugation incubation time is controlled within 1 h.

[0196] In this process, by setting different combinations of antibody addition amount and incubation time, the effect of these on the coupling efficiency of magnetic beads and antibodies was explored, so as to determine the optimal coupling conditions. In an optimal scheme, the amount of antibody used is 2μL, i.e. 5μg, and the coupling time is 1h.

[0197] (3) Sample incubation: Take 450 μL of plasma sample, 20 μL of internal standard solution, 5 μL of 25% Tween 20, 20 μL of 1×PBS, and 45 μL of 5M guanidine hydrochloride solution. Mix thoroughly and transfer the resulting mixture to the centrifuge tube from step (2). Incubate at 37°C for 1 hour. The internal standard solution consists of the following components: 25 ng / mL. 15 N 40 -Aβ40 internal standard working solution, 25 ng / mL 15 N 42 -Aβ42 internal standard working solution and 10 μg / mL SILu MAB stable isotope-labeled universal monoclonal antibody.

[0198] In this step, by changing parameters such as capture time, the effects on the capture effects of Aβ40, Aβ42 and lencanezumab were studied to screen out the most suitable capture conditions. In an optimal scheme, the immune capture time is 1.5 h.

[0199] (4) Washing: Add 0.5 mL of 1×PBS to the centrifuge tube, shake to disperse the magnetic beads, and then magnetically remove the supernatant. Repeat twice to further remove unbound impurities and improve enrichment purity.

[0200] (5) Elution: Add 100 μL of glycine-hydrochloric acid buffer (pH 2.5) to the centrifuge tube and elute by shaking for 30 s.

[0201] (6) Add 2 μL of trypsin hydrolysate, 1 μL of enzymatic hydrolysis enhancer and 5 μL of ammonia water, and incubate at 37°C with shaking for 2.5 h. Then add 1 μL of formic acid to terminate the reaction and obtain the enzymatic hydrolysis product.

[0202] (7) Centrifuge the enzymatic hydrolysis product obtained in step (6) at 10,000 rpm for 5 min, collect the supernatant after centrifugation, transfer it to a vial for LC-MS / MS analysis.

[0203] The LC-MS / MS analysis was performed using an AB SCIEX 6500 instrument with the ion pair parameters, mass spectrometry conditions, and liquid chromatography conditions specified in Table 6, using a Waters HSS T3 column (2.1 × 50 mm, 1.8 μm) to detect Aβ40, Aβ42, and lencanezumab.

[0204] Table 6. Ion pair parameters, mass spectrometry conditions, and liquid chromatography conditions for SCIEX 6500 (Aβ40, Aβ42, and lencanemab)

[0205]

[0206] Example 6: Preparation of components for the Alzheimer's disease comorbidity detection kit

[0207] The Alzheimer's disease comorbidity test kit involved in this invention includes components such as calibrators, quality control products, internal standards, magnetic beads, denaturants, reducing agents, buffer solutions, trypsin, and enzyme enhancers. The specific components are shown in Table 7.

[0208] Table 7. Components and Specifications of the Alzheimer's Disease Companion Testing Kit

[0209]

[0210] The calibrators and quality control samples are identical in composition except for the concentrations of Aβ40, Aβ42, and lencanezumab. There are six calibrators at various concentrations (calibrators 1-6 in Table 1), and three quality control samples at various concentrations (quality control samples 1-3 in Table 1). The mixed magnetic beads are obtained by mixing protein A magnetic beads and streptavidin magnetic beads at a mass ratio of 1:1. The 5M guanidine hydrochloride solution is prepared by weighing 57.3 g of guanidine hydrochloride and dissolving it in 100 mL of deionized water.

[0211] Preparation of the trypsin hydrolysate: 100 μg of lyophilized trypsin (purchased from Promega, catalog number: V5117) was dissolved in deionized water to a concentration of 1 μg / μL and then dispensed as the trypsin hydrolysate.

[0212] Preparation of the enzyme enhancer: trypsin enhancer (full name ProteasMAX) TM Surfactant (Trypsin Enhancer, purchased from Promega, catalog number: V2071) 1 mg was dissolved in deionized water to a concentration of 1 μg / μL and then dispensed to obtain the enzyme enhancer.

[0213] The internal standard solution consists of the following components at a concentration of 25 ng / mL. 15 N 40- Aβ40 internal standard working solution, 25 ng / mL 15 N42 -Aβ42 internal standard working solution and 10 μg / mL SILu MAB stable isotope-labeled universal monoclonal antibody.

[0214] Example 7: Reagent Kit Method Validation

[0215] (1) Linearity verification

[0216] The mixed working solution containing Aβ40, Aβ42, and lencanemab was diluted with 1% BSA solution (w / v) to prepare linear validation samples with concentrations from L1 to L66 (concentrations shown in Table 8). Aβ40, Aβ42, and lencanemab were detected using the Alzheimer's disease comorbidity detection kit of Example 6 according to the method of Example 5. The theoretical values ​​of the linear validation samples were plotted as the x-axis, and the measured values ​​as the y-axis. A linear equation was fitted, and the results are shown in Table 8. Figures 12-14 As shown in Table 8.

[0217] Table 8. Linearity Validation Results of the Alzheimer's Disease Companion Monitoring Kit

[0218]

[0219]

[0220] Figures 12-14 The results showed that the linear detection range of Aβ40 was 20 pg / mL to 1000 pg / mL, the linear detection range of Aβ42 was 10 pg / mL to 500 pg / mL, and the linear detection range of lencanizate was 1 μg / mL to 50 μg / mL.

[0221] The results in Table 8 show that the linear concentration deviations are all < ±15%, and the correlation coefficients (R) of the linear equations are all > 0.99, which meets the performance requirements of the kit.

[0222] (2) Limit of Quantitation Validation

[0223] The mixed working solution containing Aβ40, Aβ42 and lencanemab was diluted with 1% BSA solution (w / v) to prepare a limit of quantitation verification sample, wherein the concentrations of Aβ40, Aβ42 and lencanemab were 20 pg / mL, 10 pg / mL and 1 μg / mL, respectively. Aβ40, Aβ42 and lencanemab were detected using the Alzheimer's disease comorbidity test kit of Example 6 according to the method of Example 5. The verification results are shown in Table 9.

[0224] Table 9. Validation results of the limit of quantitation (LOQ) of the Alzheimer's disease comorbidity testing kit.

[0225] Analytes Aβ40 Aβ42 lencanide monoclonal antibody Concentration unit pg / mL pg / mL μg / mL Theoretical concentration 20 10 1 Detection concentration (repeated 1 time) 18.84 11.03 0.93 Concentration detection (repeated 2 times) 19.51 9.03 0.87 Concentration detection (repeated 3 times) 18.90 9.81 0.93 Concentration detection (repeated 4 times) 20.99 8.84 0.95 Concentration detection (repeated 5 times) 20.01 9.95 0.93 mean 19.65 9.73 0.92 deviation(%) -1.8 -2.7 -7.8 RSD (%) 4.5 8.9 3.3

[0226] Figure 14The results showed that the limit of quantification (LOQ) for Aβ40 was 20 pg / mL, for Aβ42 it was 10 pg / mL, and for lencanizate it was 1 μg / mL.

[0227] The results in Table 9 show that at the limit of quantitation concentration, the deviations between the detected values ​​and the theoretical values ​​are all < ±15%, and the relative standard deviations (RSDs) are all < 15%, which meets the performance requirements of the kit.

[0228] (3) Accuracy verification

[0229] Take 980 μL of mixed plasma sample from normal individuals, add 20 μL of working solutions of three different concentrations of analytes, and use the Alzheimer's disease comorbidity detection kit of Example 6 to detect the background concentration of plasma before spiking and the concentration of the sample after spiking according to the method of Example 5, and calculate the recovery rate.

[0230] Table 10. Accuracy verification results of the Alzheimer's disease comorbidity testing kit.

[0231]

[0232]

[0233] *The concentration of the working solution is 50 times the spiked concentration in the table.

[0234] The results in Table 10 show that the recoveries of the three analytes were all within the range of 85% to 115% at the limit of quantitation concentration, and the accuracy met the performance requirements of the kit.

[0235] (4) Precision verification

[0236] Human mixed plasma samples were divided into two equal parts. One part was retained as a low-concentration precision validation sample (PL), and the other part was added to the analyte working solution as a high-concentration precision validation sample (PH). The two samples were tested separately using an Alzheimer's disease comorbidity test kit. Each sample was tested five times, and the precision of the test results was calculated.

[0237] Table 11 Precision validation results of the Alzheimer's disease comorbidity testing kit

[0238]

[0239] The results in Table 11 show that the relative standard deviations (RSDs) of the three analytes in both the low-concentration and high-concentration precision validation samples are less than 15%, and the precision meets the performance requirements of the kit.

[0240] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A sample pretreatment kit for simultaneous detection of Aβ40, Aβ42 and lencanezumab, characterized in that, The sample pretreatment kit includes: mixed magnetic beads, internal standard solution, trypsin hydrolysate, hydrolysis enhancer, and ammonia. The mixed magnetic beads consist of protein A magnetic beads and streptavidin magnetic beads; The internal standard solution consists of 15 N-labeled Aβ40, 15 N-labeled Aβ42 and a ranibizumab internal standard peptide, which is a SILu MAB stable isotope labeled universal monoclonal antibody; The mass ratio of protein A magnetic beads to streptavidin magnetic beads is 0.5~1.5:1; 15 The final concentration of N-labeled Aβ40 was 20-30 ng / mL; 15 The final concentration of N-labeled Aβ42 is 20-30 ng / mL; the final concentration of the lencanezine monoclonal antibody internal standard peptide is 5-20 μg / mL; the concentration of the trypsin hydrolysate is 0.5-1.5 μg / μL; and the concentration of the hydrolysis enhancer is 0.5-1.5 μg / μL. The volume ratio of the mixed magnetic beads, internal standard solution, trypsin hydrolysate, enzymatic hydrolysis enhancer, and ammonia is (10~50):(15~25):(1~3):(0.5~1.5):(4~6). The sample pretreatment kit also includes 1×PBS buffer, glycine-hydrochloric acid buffer, formic acid, 20%~30% Tween 20, guanidine hydrochloride and anti-Aβ 1-16 antibody; The concentration of the anti-Aβ 1-16 antibody is 2~3 μg / μL; the volume ratio of the mixed magnetic beads, 1×PBS buffer, glycine-hydrochloric acid buffer, formic acid, Tween 20 with a volume fraction of 20%~30%, guanidine hydrochloride and anti-Aβ 1-16 antibody is (10~50):(1600~3000):(50~150):(0.5~1.5):(4~6):(40~50):(1~10); The enzymatic hydrolysis enhancer is a trypsin enhancer, full name ProteaseMAX™ Surfactant, Trypsin Enhancer, purchased from Promega, catalog number V2071.

2. The use of the sample pretreatment kit according to claim 1 in the preparation of the following products a and / or b: a. Detect the levels of Aβ40, Aβ42, and lencanezumab; b. Diagnose Alzheimer's disease.

3. A method for simultaneously detecting the concentrations of Aβ40, Aβ42, and lencanezumab, characterized in that, The method is used for non-diagnostic purposes and includes the following steps: Plasma samples were pretreated to obtain characteristic peptides, which were then quantified using an internal standard method combined with liquid chromatography-tandem mass spectrometry to detect the concentrations of Aβ40, Aβ42, and lencanezumab. The pretreatment involves using the sample pretreatment kit described in claim 1 to perform magnetic bead capture, denaturation, and enzymatic digestion of Aβ40, Aβ42, and lencanezumab in the plasma sample; The method for capturing and denaturing magnetic beads includes the following steps: after washing the mixed magnetic beads, anti-Aβ 1-16 antibody is added for conjugation and incubation. Plasma sample, internal standard solution, 20%~30% Tween 20, 1×PBS and guanidine hydrochloride are added to the magnetic beads with conjugated antibody for capture incubation to obtain the captured solution. The enzymatic hydrolysis method includes the following steps: the captured solution is washed with 1×PBS, the supernatant is discarded, the washed captured product is obtained, the washed captured product is eluted with glycine-hydrochloric acid buffer, the eluted captured product is obtained, trypsin hydrolysis solution, enzymatic hydrolysis enhancer and ammonia are added, enzymatic hydrolysis is incubated, the enzymatic hydrolysis solution is obtained, the supernatant is collected, centrifuged, and the supernatant after centrifugation is collected. The conjugation incubation time is 0.5–3 h, and the conjugation incubation temperature is 35–40 °C; the capture incubation time is 0.5–3 h, and the capture incubation temperature is 35–40 °C; the enzymatic digestion incubation time is 2–3 h, and the enzymatic digestion incubation temperature is 35–40 °C; the amino acid sequence of the characteristic peptide of lencanizate is shown in SEQ ID NO. 9; the amino acid sequence of the internal standard peptide of SILu MAB stable isotope-labeled universal monoclonal antibody is shown in SEQ ID NO. 12; the amino acid sequence of the characteristic peptide of Aβ40 is shown in SEQ ID NO. 3; the amino acid sequence of the characteristic peptide of Aβ42 is shown in SEQ ID NO. 4; Liquid chromatography conditions: Chromatographic column: Waters HSS T3 column, 2.1 × 50 mm, 1.8 μm; Mobile phase A was 0.1% formic acid aqueous solution; mobile phase B was 0.1% formic acid acetonitrile solution; gradient elution was used at a flow rate of 0.3 mL / min. A positive ionization electrospray ionization source was used, with the spray voltage set to 5500V and the ion source temperature maintained at 500℃; the atomizing gas pressure was 50psi, the auxiliary heating gas pressure was 50psi, and the curtain gas pressure was 30psi; the scanning mode was MRM. The parameters for the compound ion pairs in the mass spectrometer are as follows: The gradient elution method is as follows: 。

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