Method for valuing alpha fetoprotein in serum based on magnetic solid phase extraction-isotope dilution mass spectrometry

Abstract

The invention discloses a method for valuing alpha fetoprotein (AFP) in serum based on a magnetic solid phase extraction-isotope dilution mass spectrometry. The method comprises the following steps: (1) enriching the alpha fetoprotein (AFP) in the serum; (2) trypsin digestion; and (3) AFP quantitative analysis. According to the value determination method, monoclonal antibodies of two kinds of AFP are combined to magnetic beads to prepare a mixed magnetic bead solution, AFP in serum is enriched in a magnetic solid phase extraction (MSPE) mode to achieve efficient enrichment of low-abundance AFP in the serum, an organic phase is added into an elution solution to enhance elution of protein, the high recovery rate is achieved, after trypsin digestion, the content of the AFP in the serum is reduced, and the content of the AFP in the serum is reduced. The isotope labeling characteristic peptide fragment is added, and quantitative analysis is carried out on the isotope labeling characteristic peptide fragment by using isotope diluent liquid chromatography-tandem mass spectrometry (ID-LC-MS / MS), so that the quantitative result can be traced to the SI unit.

Description

Technical Field

[0001] The invention relates to the field of protein content determination, and in particular to a method for determining the value of alpha-fetoprotein in serum based on magnetic solid phase extraction-isotope dilution mass spectrometry. Background Art

[0002] Alpha-fetoprotein (AFP) is a biomarker with important clinical significance in hepatocellular carcinoma, pregnancy screening and other malignancies. Common methods for detecting AFP include enzyme-linked immunosorbent assay (ELISA), chemiluminescent immunoassay (CLIA) and radioimmunoassay (RIA). However, these methods have certain limitations, especially in high-sensitivity and high-precision quantitative analysis, which are often affected by factors such as sample processing, antibody cross-reaction, and nonspecific interference, which may lead to unstable results or insufficient precision.

[0003] Isotope dilution mass spectrometry (IDMS) adds a substance labeled with a specific isotope to the sample to be tested, and then measures the isotope ratio of the target substance in the sample by a mass spectrometer. By comparing the ratio of the natural isotope of the target substance in the sample to the labeled isotope, the concentration of the substance to be tested can be accurately calculated. This method is a highly accurate quantitative analysis technology that can overcome many limitations of traditional methods, especially in the quantification of complex samples.

[0004] However, it is still challenging to accurately quantify low-abundance proteins with large molecular weight such as AFP in matrices such as serum using IDMS. Serum is a very complex biological fluid that contains thousands of different proteins, including high concentrations of major proteins (such as albumin and globulin), whose high concentrations may cause saturation of the detection instrument and interfere with the quantification of other proteins. In contrast, AFP is a trace protein with extremely low concentrations. It is easily affected by noise, matrix effects or insufficient analytical sensitivity during the quantitative process, resulting in poor quantitative accuracy. Proteins in serum usually need to undergo certain pretreatments before quantitative analysis, including removing high-abundance proteins to prevent masking of the detection signal of low-abundance target proteins, increasing their concentrations by ultrafiltration, precipitation or other concentration techniques to improve detection sensitivity, and removing salts contained in serum or introduced during pretreatment by desalting methods. Therefore, the quantitative analysis of serum proteins is a complex process, which is affected by many factors such as sample matrix, protein diversity, detection sensitivity, method selection and experimental operation.

[0005] Therefore, for the determination of alpha-fetoprotein in serum, there is an urgent need in the art to develop a method for determining the value of AFP that can avoid the interference of serum matrix on the accurate quantification of AFP and whose results can be traced back to SI units. Summary of the invention

[0006] The purpose of the present invention is to provide a method for determining the value of alpha-fetoprotein in serum based on magnetic solid phase extraction-isotope dilution mass spectrometry. By combining magnetic solid phase extraction (MSPE) technology with isotope dilution mass spectrometry technology, a high-accuracy method for determining the value of low-abundance AFP in serum is established. The interference of serum matrix on the accurate quantification of AFP is avoided by MSPE technology, and isotope dilution mass spectrometry is used to accurately quantify AFP through three characteristic peptide segments, so that the results can be traced back to SI units.

[0007] To achieve the above purpose, the technical solution adopted by the present invention is as follows: A method for determining the value of alpha-fetoprotein in serum based on magnetic solid phase extraction-isotope dilution mass spectrometry comprises the following steps: (1) Purification and enrichment of AFP in serum: Activate the magnetic beads, add antibody 1 and antibody 2 in proportion, prepare a magnetic bead antibody complex (i.e., a mixed magnetic bead solution); add the magnetic bead antibody complex to the serum sample, incubate, and then wash to remove the serum; elute the AFP bound to the magnetic beads with a trifluoroacetic acid solution containing a certain proportion of organic solvent, and store at -80°C after drying for use; (2) Trypsin digestion: serum samples were affinity purified, magnetic beads were extracted and the samples were centrifuged and concentrated; a certain amount of 0.5% sodium deoxycholate solution was added; tris(2-carboxyethyl)phosphine-chloroacetamide (TCEP-CAA) was added, and denaturation, disulfide bond reduction and alkylation were carried out at room temperature; a certain amount of trypsin solution was added in proportion, and an equal amount of labeled peptide solution was added, and the digestion reaction was carried out at 37°C; formic acid was added to terminate the reaction and sodium deoxycholate was removed by precipitation; (3) Quantitative analysis of AFP: The digested samples were injected into the mass spectrometer, and the enzymatic peptides and the added isotope-labeled peptides were analyzed using multiple reaction monitoring (MRM). The peptides accurately quantified by the amino acid hydrolysis method and the labeled peptide solutions were used to prepare a low-standard solution with a mass ratio of 0.9 and a high-standard solution with a mass ratio of 1.1. The concentration of alpha-fetoprotein was calculated using the two-point method. Three characteristic peptides were selected to calculate the concentration of alpha-fetoprotein, and the average value of the three was used for quantification. The characteristic peptides were TK, FR and NR, and the isotope-labeled characteristic peptides were SIL-TK, SIL-FR and SIL-NR. The AFP concentration was calculated based on the peptide concentration using the following formula:

[0008] Where: p pep -Purity of characteristic peptide standard substances; m 标 -The mass of the isotope internal standard added to the enzymatic sample (mg); R样 -The peak area ratio of the characteristic peptides in the enzymatic hydrolysis sample and the isotope-labeled characteristic peptides; I 1-The mass ratio of the characteristic peptide in the low-standard solution and the isotope-labeled characteristic peptide; I 2-The mass ratio of the characteristic peptides in the high-standard solution and the isotope-labeled characteristic peptides; R 1-peak area ratio of characteristic peptides in low-standard solution and isotope-labeled characteristic peptides; R 2-peak area ratio of characteristic peptides in high-standard solution and isotope-labeled characteristic peptides; m - Weigh the mass of AFP solution (g); c AFP - AFP concentration (mg / g); M AFP - Molecular weight of AFP; n -The number of this characteristic peptide segment in the AFP sequence; M pep -Molecular weight of characteristic peptides.

[0009] The step (1) specifically comprises: activating the magnetic beads, adding monoclonal antibody 1 and monoclonal antibody 2 in a ratio of antibody: magnetic beads = 40 μg: 1 mg to prepare a magnetic bead antibody complex with a concentration of 1 mg / mL; taking 1 mL of AFP serum sample in a 1.5 mL centrifuge tube; adding a certain amount of magnetic bead antibody complex, incubating with rotation at 37°C for 2 h to allow AFP in the serum to bind to the magnetic bead antibody, and removing the serum on a magnetic rack; washing the magnetic beads with 1 mL of PBST solution and PBS solution for 3 times respectively; adding an acetonitrile aqueous solution containing 0.1% trifluoroacetic acid, wherein the acetonitrile: water ratio is 3:7; shaking at 1000 rpm for 15 min to elute the AFP bound to the magnetic beads; collecting the eluted solution on a magnetic rack and concentrating and drying by centrifugation.

[0010] The monoclonal antibody 1 is mAb 4A3, and the monoclonal antibody 2 is mAb 5H7; the antibodies are required to react with human alpha-fetoprotein but have no cross-reaction with human globulin.

[0011] The step (2) specifically comprises: affinity purification of the serum sample, and the experiment is carried out by the two-point method; after magnetic bead extraction, the sample is concentrated and dried by centrifugation; 0.5% sodium deoxycholate solution is added for re-dissolution; tris(2-carboxyethyl)phosphine-chloroacetamide is added, and the mixture is incubated at room temperature for 1 h; trypsin solution is added in proportion, and an equal amount of labeled peptide solution is added, and the enzyme cleavage reaction is carried out at 37°C; 10% formic acid is added to a final concentration of 1%, the reaction is terminated, and sodium deoxycholate is precipitated; the supernatant is taken after centrifugation, and dried; and the mobile phase is used for re-dissolution to obtain the enzyme-cleaved peptides TK, FR and NR.

[0012] The sequences of the characteristic peptides TK, FR and NR are TFQAITVTK, FIYEIAR and NIFLASFVHEYSR respectively; the sequences of the isotope-labeled characteristic peptides SIL-TK, SIL-FR and SIL-NR are TFQAITVTK* 、 FIYEIAR* and NIFLASFVHEYSR*.

[0013] Compared with the prior art, the outstanding effects of the present invention are: (1) The quantitative method of the present invention binds two monoclonal antibodies of AFP to magnetic beads to prepare a mixed magnetic bead solution, enriches AFP in serum by magnetic solid phase extraction (MSPE) to achieve efficient enrichment of low-abundance AFP in serum, and enhances protein elution by adding an organic phase to the elution solution to achieve a high recovery rate. After trypsin digestion, isotope-labeled characteristic peptides are added and quantitatively analyzed by isotope dilution liquid chromatography tandem mass spectrometry (ID-LC-MS / MS), so that the quantitative results can be traced back to SI units.

[0014] (2) The determination method of the present invention is an efficient, simple and reliable detection method, which can provide a reference for the determination of proteins in other serum samples.

[0015] The method for determining the value of alpha-fetoprotein in serum based on magnetic solid phase extraction-isotope dilution mass spectrometry according to the present invention will be further described below in conjunction with the accompanying drawings and specific examples. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 This is the experimental flow chart of the method for determining the AFP value in serum based on magnetic solid phase extraction-isotope dilution mass spectrometry.

[0017] Figure 2 Optimize results for hybrid magnetic bead enrichment efficiency.

[0018] Figure 3 Optimization results for magnetic bead elution solution composition.

[0019] Figure 4This is the trypsin digestion time curve.

[0020] Figure 5 LC-MS / MS mass spectra. Among them, a is the total ion current; b and c are the extracted ion chromatograms of SIL-TK and TK, respectively; d and e are the extracted ion chromatograms of SIL-FR and FR, respectively; f and g are the extracted ion chromatograms of SIL-NR and NR, respectively. DETAILED DESCRIPTION

[0021] Reagents: Acetonitrile was purchased from Thermo Fisher Scientific, USA; Ultrapure water was purified by Millipore water purification system; Trifluoroacetic acid (TFA) was purchased from Sigma-Aldrich, USA; Formic acid was purchased from Thermo Fisher Scientific, USA; Sodium deoxycholate (SDC) was purchased from Shanghai Macklin Company; Tri(2-carboxyethyl)phosphine (TCEP) was purchased from Thermo Fisher Scientific; Chloroacetamide (CAA) was purchased from Sigma-Aldrich; Trypsin was purchased from Promega, USA; Sodium hydroxide and ammonium bicarbonate were purchased from Shanghai Macklin Company; Magnetic beads were purchased from Invitrogen; AFP antibody was purchased from Hytest, Finland; The peptides TFQAITVTK (TK), FIYEIAR (FR), NIFLASFVHEYSR (NR), TFQAITVTK* (SIL-TK), FIYEIAR* (SIL-FR), and NIFLASFVHEYSR* (SIL-NR) were synthesized by Anhui Guoping Pharmaceutical Co., Ltd. The sequences of TK, FR, and NR are shown in SEQ ID NOs: 1-3.

[0022] Human serum samples were prepared by Guangdong Provincial Hospital of Traditional Chinese Medicine.

[0023] instrument: Liquid chromatography-mass spectrometry: Xevo TQ-XS triple quadrupole mass spectrometer, Waters, USA; Pipette: Eppendorf Research, Germany; Balance: ME235S, minimum division value 0.01 mg, Satorius, Germany; Balance: XP26, minimum graduation value 0.001 mg, Mettler Toledo, Sweden; Chemiluminescent immunoassay analyzer: UniCel DxI 800 Access, Beckman Coulter, USA.

[0024] Combination Figure 1 As shown, a method for determining the value of alpha-fetoprotein in serum based on isotope dilution mass spectrometry comprises the following steps: (1) Enrichment of serum alpha-fetoprotein (AFP) AFP in serum is extracted by magnetic bead affinity purification. The principle is that the magnetic beads covalently bind to the monoclonal antibody of AFP to form an antibody-magnetic bead complex, and then the AFP in serum is extracted by the specific binding of antigen and antibody.

[0025] Specifically, the magnetic beads were activated, and monoclonal antibody 1 (mAb 4A3 from Hi-Peptide Biotech) and monoclonal antibody 2 (mAb 5H7 from Hi-Peptide Biotech) were added at a ratio of 40 μg:1 mg to prepare a magnetic bead-antibody complex at a concentration of 1 mg / mL; 1 mL of serum sample was placed in a 1.5 mL centrifuge tube; 50 μL of the magnetic bead-antibody complex was added, and the mixture was rotated and incubated at room temperature for 2 h to allow the AFP in the serum to be fully bound, and the supernatant was removed (for subsequent calculation of the enrichment efficiency of AFP); the magnetic beads were washed 3 times with PBST; and then washed 3 times with PBS; a final concentration of 0.1% trifluoroacetic acid in acetonitrile (acetonitrile: water = 3:7) was added, and the AFP bound to the magnetic beads was eluted by shaking at 1000 rpm for 15 min; the magnetic beads were adsorbed with a magnetic rack, the eluted solution was collected, and centrifuged and concentrated to dryness.

[0026] (2) Investigation of magnetic bead enrichment efficiency In step (1), the AFP in the serum is extracted using the magnetic bead antibody complex, the discarded serum is collected, and its AFP concentration is detected using a chemiluminescent immunoassay to obtain the magnetic bead extraction efficiency.

[0027] Magnetic bead enrichment efficiency: The enrichment efficiency of magnetic beads is a potential source of measurement bias and must be properly evaluated. Limited by the non-specific adsorption of magnetic beads and the sensitivity of antibodies, the quantification of AFP enriched by magnetic beads will not reflect the true content of AFP in serum. Therefore, the enrichment efficiency needs to be tested, and the results are shown in Table 1: Table 1 Chemiluminescence immunoassay results

[0028] The 6 AFP serum parallel samples were tested using a chemiluminescent immunoassay. The results are shown in Table 1. The average concentration of serum AFP before enrichment was 43.07 ng / mL, and the average concentration of AFP in serum after magnetic bead enrichment was 0.03 ng / mL. The average enrichment efficiency was greater than 99.9%, and it can be considered that AFP in serum was completely enriched. Figure 2 shown.

[0029] (3) Enzyme-cleaved peptides TK, FR and NR Serum samples were affinity purified. After magnetic bead enrichment, centrifugation and concentration were performed; a certain amount of 0.5% sodium deoxycholate solution (SDC) was added for reconstitution; tris(2-carboxyethyl)phosphine-chloroacetamide (TCEP-CAA) was added and incubated at room temperature for 1 h; an equal amount of labeled peptide solution was added, 2 μg of trypsin solution was added, and the samples were incubated at 37°C for 30 h; 10% formic acid was added to a final concentration of 1% to terminate the reaction and precipitate to remove SDC; after centrifugation and concentration, the samples were reconstituted in a 10% ACN (containing 0.1% FA) aqueous solution to obtain the enzyme-cleaved peptides TK, FR and NR.

[0030] By optimizing the composition of the magnetic bead elution solution and improving the sample recovery rate, the accuracy of the quantitative results can be improved. Using 0.1% TFA elution solution without adding organic solvent as the control group, the effects of adding 30% acetonitrile, methanol, n-hexane, acetone, isopropanol and ethanol to 0.1% TFA on the elution effect of the magnetic beads were investigated. The elution effect was investigated by the peak area of ​​endogenous peptides and added labeled peptides. The results are shown in Figure 2. Figure 3 When the eluent contains 30% acetonitrile, the elution of AFP can be effectively enhanced.

[0031] The trypsin digestion time was optimized. The enzymatic digestion efficiency of the samples after 6 h, 12 h, 18 h, 24 h, 30 h, 36 h, 42 h and 48 h of digestion was investigated by the peak area ratio of endogenous peptides and added labeled peptides. Figure 4 As shown in the figure, FR peptide and TK peptide reached the maximum concentration after 30 h of enzymatic digestion, and NR peptide reached the maximum concentration after 24 h of enzymatic digestion, so the optimal enzymatic digestion time was 30 h.

[0032] The mass spectrometry conditions of the characteristic peptides and the labeled peptides are shown in Table 2, and the liquid phase gradient conditions are shown in Table 3. The chromatographic column is an ACQUITY UPLC BEH C18 column (2.1mm×100mm, 1.7 µm); the mobile phase A is water containing 0.1% formic acid, and the mobile phase B is acetonitrile containing 0.1% formic acid; the flow rate is 0.4 mL / min, the column temperature is 40°C, and the injection volume is 10 μL. The above conditions were used to test the enzymatically hydrolyzed samples, and the total ion current of the characteristic peptides and the labeled peptides was obtained ( Figure 5 ).

[0033] Table 2 Main mass spectrometry parameters

[0034] Table 3 Mobile phase gradient

[0035] (4) Quantitative analysis of AFP: The samples after enzyme digestion were analyzed by mass spectrometry, and the concentration of AFP was calculated by the concentration of peptide segments, which was obtained from the peak area of ​​the MRM spectrum. Three peptide segments were selected to calculate the AFP concentration, and the average value of the three was used for quantification. The AFP concentration was calculated based on the peptide concentration using the following formula:

[0036] Where: p pep -Purity of characteristic peptide standard substances; m 标 -The mass of the isotope internal standard added to the enzymatic sample (mg); R 样 -The peak area ratio of the characteristic peptides in the enzymatic hydrolysis sample and the isotope-labeled characteristic peptides; I 1-The mass ratio of the characteristic peptide in the low-standard solution and the isotope-labeled characteristic peptide; I 2-The mass ratio of the characteristic peptides in the high-standard solution and the isotope-labeled characteristic peptides; R 1-peak area ratio of characteristic peptides in low-standard solution and isotope-labeled characteristic peptides; R 2-peak area ratio of characteristic peptides in high-standard solution and isotope-labeled characteristic peptides; m - Weigh the mass of AFP solution (g); c AFP - AFP concentration (mg / g); M AFP - Molecular weight of AFP; n -The number of this characteristic peptide segment in the AFP sequence; M pep -Molecular weight of characteristic peptides.

[0037] AFP quantitative results: AFP serum samples (about 40 ng / mL) were affinity purified and quantified by enzyme digestion. Six parallel samples were set up, with 3 technical repeats in each group, and the bracket method was used for calculation. Detailed data are shown in Table 4. The AFP content in serum, as well as the SD value and CV value were calculated according to the above formula, in units of (ng / mL).

[0038] Table 4 Results of MSPE-ID-LC-MS / MS standard value

[0039] The data in Table 4 show that the concentration of AFP in pregnant women's serum measured by LC-MS / MS is 41.42 ng / mL. This is close to the result of 43.07 ng / mL measured by chemiluminescent immunoassay. In addition, by adding the purity standard substance of alpha-fetoprotein solution (GBW09227) to the alternative matrix (5% BSA), the measured spike recovery rate was 101%, which verified the correctness of the method. Therefore, the method of the present invention is an efficient, simple and reliable detection method.

[0040] In summary, the present invention uses a mixed magnetic bead immunoenrichment technique to pre-treat serum AFP. Low-abundance AFP in serum is specifically purified by magnetic bead immunoenrichment, and the interference of high-abundance proteins is removed to reduce the detection limit and improve the accuracy of detection. By performing chemiluminescent immunoassay on the serum before and after enrichment, it is shown that within the measurement concentration range, the magnetic bead extraction efficiency is greater than 99.5%. Therefore, the use of the mixed magnetic bead enrichment technique to enrich AFP in serum has good reliability.

[0041] The present invention investigates the protein elution efficiency and the elution efficiency of different eluent types on magnetic bead-enriched AFP. By investigating the serum spike recovery rate, it is found that when the eluent is an aqueous solution containing 0.1% trifluoroacetic acid and 30% acetonitrile, the AFP recovery rate is 101%. At this time, the protein elution efficiency is the highest and will not cause excessive impurity proteins to interfere with the detection of AFP.

[0042] In addition, the present invention also investigates the enzyme cleavage time. The present invention uses 0.5% sodium deoxycholate solution and tris(2-carboxyethyl)phosphine-chloroacetamide solution to simultaneously denature, reduce disulfide bonds and alkylate the sample. A certain amount of trypsin is added to perform enzyme cleavage at 37°C. Through the investigation of the enzyme cleavage time, it is found that the protein is completely cleaved in 30 h.

[0043] The optimization of magnetic bead enrichment efficiency, protein elution efficiency and enzyme cleavage efficiency is only for enhancing the response intensity of mass spectrometry signals in the present invention and does not participate in the concentration calculation.

[0044] The present invention uses isotope dilution mass spectrometry to quantify serum AFP. The experimental principle is to calculate the concentration of the characteristic peptide segment based on the peak area ratio of the labeled peptide segment to the characteristic peptide segment and then obtain the concentration of the protein. The AFP concentration is calculated by the average value of three characteristic peptide segments. The present invention eliminates the error introduced by the enzyme cleavage process by adding an equal amount of labeled peptide segments as internal standards in the sample. The efficient enrichment of AFP in serum is achieved by preparing mixed magnetic beads, and the recovery rate of AFP in serum is improved by optimizing the composition of the magnetic bead eluent. In addition, the denaturation and disulfide bond reduction of the protein are achieved simultaneously by a one-step method, simplifying the pre-treatment steps. The detection of low-abundance AFP in serum is achieved through method optimization.

[0045] The embodiments described above are merely descriptions of preferred implementation modes of the present invention and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A method for determining the value of alpha-fetoprotein in serum based on magnetic solid phase extraction-isotope dilution mass spectrometry, characterized in that: The following steps are involved: (1) Purification and enrichment of AFP in serum: Activate the magnetic beads, add antibody 1 and antibody 2 in proportion to prepare a magnetic bead antibody complex; add the magnetic bead antibody complex to the serum sample, incubate, and then wash to remove the serum; Elute the AFP bound to the magnetic beads with an elution solution; (2) Trypsin digestion: affinity purification of serum samples followed by centrifugation and concentration; addition of sodium deoxycholate solution; addition of tris(2-carboxyethyl)phosphine-chloroacetamide for denaturation, disulfide bond reduction, and alkylation; Add trypsin solution and labeled peptide solution to carry out enzyme cleavage reaction; add formic acid to terminate the reaction; (3) Quantitative analysis of AFP: The digested samples were injected into the mass spectrometer, and the enzymatic peptides and the added isotope-labeled peptides were analyzed using multiple reaction monitoring. The low-standard solution and high-standard solution were prepared using the peptides accurately quantified by amino acid hydrolysis and the labeled peptide solutions, and the concentration of AFP was calculated using the two-point method.

2. The method for determining the value of alpha-fetoprotein in serum based on magnetic solid phase extraction-isotope dilution mass spectrometry according to claim 1, characterized in that: The step (1) specifically comprises: activating the magnetic beads, adding monoclonal antibody 1 and monoclonal antibody 2 in a ratio of antibody: magnetic beads = 40 μg: 1 mg, preparing a magnetic bead antibody complex with a concentration of 1 mg / mL; taking 1 mL of AFP serum sample in a 1.5 mL centrifuge tube; adding a certain amount of magnetic bead antibody complex, incubating with rotation at 37°C for 2 h to allow AFP in the serum to bind to the magnetic bead antibody, and removing the serum on a magnetic rack; washing the magnetic beads with 1 mL of PBST solution and PBS solution for 3 times respectively; adding an acetonitrile aqueous solution containing 0.1% trifluoroacetic acid, wherein the acetonitrile: water ratio is 3:7; shaking at 1000 rpm for 15 min to elute the AFP bound to the magnetic beads; collecting the eluted solution on a magnetic rack and concentrating and drying by centrifugation.

3. The method for determining the value of alpha-fetoprotein in serum based on magnetic solid phase extraction-isotope dilution mass spectrometry according to claim 2, characterized in that: The monoclonal antibody 1 is mAb 4A3, and the monoclonal antibody 2 is mAb 5H7; the antibodies are required to react with human alpha-fetoprotein but have no cross-reaction with human globulin.

4. The method for determining the value of alpha-fetoprotein in serum based on magnetic solid phase extraction-isotope dilution mass spectrometry according to claim 1, characterized in that: The step (2) specifically comprises: affinity purification of the serum sample, and the experiment is carried out by the two-point method; after magnetic bead extraction, the sample is centrifuged and concentrated to dryness; 0.5% sodium deoxycholate solution is added for re-dissolution; tris(2-carboxyethyl)phosphine-chloroacetamide is added, and the mixture is incubated at room temperature for 1 h; trypsin solution is added in proportion, and an equal amount of labeled peptide solution is added, and enzyme cleavage reaction is carried out at 37°C; 10% formic acid is added to a final concentration of 1%, the reaction is terminated, and sodium deoxycholate is precipitated; and enzyme cleavage peptides TK, FR, and NR are obtained.

5. The method for determining the value of alpha-fetoprotein in serum based on magnetic solid phase extraction-isotope dilution mass spectrometry according to claim 1, characterized in that: In the step (3), three characteristic peptide segments are selected to calculate the alpha-fetoprotein concentration, and the average value of the three is used for quantification; the characteristic peptide segments are TK, FR and NR, and the isotope-labeled characteristic peptide segments are SIL-TK, SIL-FR and SIL-NR; The AFP concentration was calculated based on the peptide concentration using the following formula: ； Where: p pep -Purity of characteristic peptide standard substances; m 标 -Mass of isotope internal standard added to the enzymatic sample, mg; R 样 -The peak area ratio of the characteristic peptides in the enzymatic hydrolysis sample and the isotope-labeled characteristic peptides; I 1-The mass ratio of the characteristic peptide in the low-standard solution and the isotope-labeled characteristic peptide; I 2-The mass ratio of the characteristic peptides in the high-standard solution and the isotope-labeled characteristic peptides; R 1-peak area ratio of characteristic peptides in low-standard solution and isotope-labeled characteristic peptides; R 2-peak area ratio of characteristic peptides in high-standard solution and isotope-labeled characteristic peptides; m - Weigh the mass of AFP solution, g; c AFP - AFP concentration, mg / g; M AFP - Molecular weight of AFP; n -The number of this characteristic peptide segment in the AFP sequence; M pep -Molecular weight of characteristic peptides.

6. The method for determining the value of alpha-fetoprotein in serum based on magnetic solid phase extraction-isotope dilution mass spectrometry according to claim 5, characterized in that: The sequences of the characteristic peptide segments TK, FR and NR are TFQAITVTK, FIYEIAR and NIFLASFVHEYSR respectively; the sequences of the isotope-labeled characteristic peptide segments SIL-TK, SIL-FR and SIL-NR are TFQAITVTK*, FIYEIAR* and NIFLASFVHEYSR* respectively.

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