Method for specific detection of asparaginase activity by immuno-magnetic separation combined with substrate peptide by lc-ms / ms

Abstract

The application relates to the field of medical detection, in particular to a method for specifically detecting asparaginase activity by LC-MS / MS in combination with immune magnetic separation and substrate peptides, which is based on the preparation of immune magnetic beads by coupling asparaginase antibodies mediated by amide reaction, the exclusive separation and enrichment of asparaginase in samples, the addition of asparagine solution after reaction based on the asparaginase specific cleavage of substrate peptide asparagine, and the realization of the direct high-sensitivity detection of asparaginase activity by LC-MS / MS. The application rapidly and exclusively separates and enriches the to-be-detected asparaginase in samples by means of magnetic separation, combines the characteristics that asparaginase specifically cleaves substrate peptide asparagine, efficiently and exclusively cleaves asparagine to generate a large amount of aspartic acid, adopts mass spectrometry to detect asparaginase activity, significantly improves the sensitivity and specificity of the detection of asparaginase activity, is simple to operate, and has important practical significance for promoting the monitoring of the in-vivo activity level of asparaginase.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of medical detection, in particular, a method for detecting asparaginase activity in a sample based on specific substrate peptides by LC-MS / MS. BACKGROUND

[0002] With the rapid development of biological medicine, protease drugs are widely developed and applied in the diagnosis and treatment of diseases. L-asparaginase, as an asparagine-specific enzyme, specifically catalyzes the hydrolysis of asparagine to form L-aspartic acid and ammonia in the blood, so that tumor cells cannot uptake enough L-asparagine to inhibit cell proliferation and achieve the purpose of anti-tumor. It is an important anti-tumor drug in clinical use for acute lymphoblastic leukemia and other diseases. However, it can easily cause serious allergic reactions such as thrombosis. Monitoring its biological activity in vivo is an important research content for evaluating drug efficacy and ensuring medication safety. The present application aims to explore a new type of high-sensitivity and exclusive analysis method for monitoring the activity of asparaginase in vivo.

[0003] Monitoring the activity level of asparaginase in vivo is of great guiding value for the monitoring and prevention of allergic reactions and the adjustment of treatment programs. However, as of now, only the adjustment of treatment programs after asparaginase allergic reactions has been treated, or the indirect evaluation of the in vivo efficacy of asparaginase drugs by monitoring the amino acid asparagine in vivo has been used. The direct monitoring of its activity level in vivo has not been widely used.

[0004] Asparaginase has high biological activity, and the clinical dosage is small, with low concentration in vivo, which brings great challenges to its accurate monitoring. At present, the asparaginase activity detection methods included in the pharmacopoeias of various countries are mainly suitable for raw materials, drug preparations and other sample types. However, there are a large number of protein and polypeptide endogenous substances with similar structures in biological samples, which interfere with the accurate detection of asparaginase activity, and the low sensitivity limits its application in the detection of asparaginase activity in vivo. The existing enzyme-linked immunosorbent assay can be applied to the detection of enzyme activity in biological samples, but it is expensive and time-consuming to operate, which is difficult to meet the actual application needs of asparaginase activity detection in large quantities of biological samples. The traditional recognized analysis methods for the biological activity of protease in vivo mainly include enzyme-linked immunosorbent assay and radioimmunoassay, but they have the disadvantages of narrow quantitative range, low selectivity and high price. It is of great practical significance to establish a specific and sensitive analysis method for the biological activity of asparaginase.

[0005] Magnetic nanoparticles have superparamagnetism, easy modification, rapid separation and other advantages, and show significant advantages for the separation and analysis of biological macromolecules susceptible to biological matrix interference. The antibody specifically recognizing asparaginase is modified on the surface of the magnetic nanoparticles, so that the asparaginase in the biological sample can be separated and enriched, and the interference of the biological sample matrix can be effectively avoided to further improve the detection sensitivity.

[0006] LC-MS / MS is the main method for in vivo analysis of small molecule compounds, which has the advantages of ultra-high selectivity, high sensitivity, simultaneous quantitative analysis of multiple components, and other advantages. However, there are still many challenges in monitoring the activity of protease in biological samples by LC-MS / MS, including the difficulty of directly detecting enzyme activity by mass spectrometry as a mass detector, the need to use enzyme analysis or enzyme analysis to analyze the change in the amount of enzyme substrate or product to realize enzyme activity monitoring, and the low ionization efficiency of proteinase as a biological macromolecule and the weak mass spectrometry detection signal. Proteinase is mainly used for proteolysis of protein polypeptide biological macromolecules to select characteristic peptides for analysis and detection, but the detection sensitivity is limited, and the operation requirement is high, which is easy to introduce large errors. Therefore, how to combine high-sensitivity specific LC-MS / MS analysis method to detect asparaginase activity in biological samples has important practical significance. SUMMARY

[0007] The purpose of the present application is to provide a method for specific detection of asparaginase activity in samples by immuno-magnetic separation combined with substrate peptides and LC-MS. The method of the present application can be used to monitor the activity level of asparaginase in vivo, thereby effectively monitoring and preventing asparaginase allergy, and providing guidance for adjusting the drug treatment plan.

[0008] In order to achieve the above purpose, the technical scheme adopted by the present application is as follows:

[0009] The method comprises the following steps: A) coupling aspartase antibody to the surface of magnetic nanoparticles by amide reaction mediation to prepare immunomagnetic beads; the immunomagnetic beads are antibody immunofunctionalized magnetic beads prepared by coupling magnetic beads and aspartase antibody; B) adding different amounts of aspartase standard into blank biological samples to prepare aspartase-containing samples with different concentrations, and then performing specific capture and enrichment by using immunomagnetic beads to obtain corresponding immunomagnetic bead / aspartase complexes; adding aspartase substrate peptide into the immunomagnetic bead / aspartase complexes, and performing specific cleavage of the substrate peptide by aspartate to form aspartic acid and ammonia; and performing LC-MS / MS detection on the supernatant of the sample, taking the concentration of aspartase as the horizontal coordinate and the aspartic acid signal intensity detected by mass spectrometry as the vertical coordinate to establish an aspartase-mass spectrometry response signal standard curve.

[0010] The method comprises the following steps: A) coupling aspartase antibody to the surface of magnetic nanoparticles by amide reaction mediation to prepare immunomagnetic beads; the immunomagnetic beads are antibody immunofunctionalized magnetic beads prepared by coupling magnetic beads and aspartase antibody; B) adding different amounts of aspartase standard into blank biological samples to prepare aspartase-containing samples with different concentrations, and then performing specific capture and enrichment by using immunomagnetic beads to obtain corresponding immunomagnetic bead / aspartase complexes; adding aspartase substrate peptide into the immunomagnetic bead / aspartase complexes, and performing specific cleavage of the substrate peptide by aspartate to form aspartic acid and ammonia; and performing LC-MS / MS detection on the supernatant of the sample, taking the concentration of aspartase as the horizontal coordinate and the aspartic acid signal intensity detected by mass spectrometry as the vertical coordinate to establish an aspartase-mass spectrometry response signal standard curve.

[0011] Based on the above technical solution, the first aspect of the present application provides a method for specifically detecting aspartase activity by LC-MS / MS through immunomagnetic separation and substrate peptide, which comprises the following steps:

[0012] A) coupling aspartase antibody to the surface of magnetic nanoparticles by amide reaction mediation to prepare immunomagnetic beads; the immunomagnetic beads are antibody immunofunctionalized magnetic beads prepared by coupling magnetic beads and aspartase antibody; B) adding different amounts of aspartase standard into blank biological samples to prepare aspartase-containing samples with different concentrations, and then performing specific capture and enrichment by using immunomagnetic beads to obtain corresponding immunomagnetic bead / aspartase complexes; adding aspartase substrate peptide into the immunomagnetic bead / aspartase complexes, and performing specific cleavage of the substrate peptide by aspartate to form aspartic acid and ammonia; and performing LC-MS / MS detection on the supernatant of the sample, taking the concentration of aspartase as the horizontal coordinate and the aspartic acid signal intensity detected by mass spectrometry as the vertical coordinate to establish an aspartase-mass spectrometry response signal standard curve.

[0013] B) adding different amounts of aspartase standard into blank biological samples to prepare aspartase-containing samples with different concentrations, and then performing specific capture and enrichment by using immunomagnetic beads to obtain corresponding immunomagnetic bead / aspartase complexes; adding aspartase substrate peptide into the immunomagnetic bead / aspartase complexes, and performing specific cleavage of the substrate peptide by aspartate to form aspartic acid and ammonia; and performing LC-MS / MS detection on the supernatant of the sample, taking the concentration of aspartase as the horizontal coordinate and the aspartic acid signal intensity detected by mass spectrometry as the vertical coordinate to establish an aspartase-mass spectrometry response signal standard curve.

[0014] C) After the sample to be tested is mixed with the immunomagnetic beads and incubated, a substrate peptide asparagine solution is added for LC-MS / MS detection, and the aspartic acid signal intensity obtained is substituted into a standard curve, so that the activity of asparaginase in the sample to be tested can be calculated.

[0015] Further, the sample to be tested is a blood sample.

[0016] Further, the method for specifically detecting asparaginase activity in vivo by combining immunomagnetic separation with a substrate peptide by LC-MS / MS comprises the following steps:

[0017] (a) Fe3O4 magnetic nanoparticles are prepared by a hydrothermal method, and then an agarose molecular layer is introduced in situ on the surface of the magnetic nanoparticles, and N-hydroxysuccinimide (NHS) activated ester groups are further modified to prepare NHS magnetic beads;

[0018] (b) The NHS activated magnetic nanoparticle dispersion solution is removed, the supernatant is removed by magnetic separation, and pre-cooled hydrochloric acid solution is quickly added and mixed to wash, and the supernatant is removed by magnetic separation;

[0019] (c) Asparaginase monoclonal antibody is added, and the mixture is mixed uniformly at 4°C in the dark for 12 hours, and the supernatant is removed by magnetic separation;

[0020] (d) Under the condition of an external magnetic field, the magnetic beads are washed with 100mM Tris-HCl (containing NaCl 150mM, pH8.0) buffer solution for 4 times, and then dispersed in 100mM Tris-HCl (containing NaCl 150mM, pH8.0) again, and sealed at room temperature for 2 hours in the dark, and the supernatant is removed by magnetic separation;

[0021] (e) The magnetic beads are washed 4 times by blowing and mixing with 5wt% BSA in 10mM PBS (containing NaCl 137mM, pH7.4), and then dispersed in 5wt% BSA in 10mM PBS (containing NaCl 137mM, pH7.4), and sealed at 4°C for 12 hours in the dark;

[0022] (f) The supernatant is removed by magnetic separation, and the magnetic beads are dispersed after being washed 4 times with PBS containing 0.1wt% BSA, to obtain antibody immunofunctional magnetic beads;

[0023] (g) The antibody immunofunctional magnetic bead suspension prepared is separated by an external magnetic field to remove the supernatant, and diluted with 5 times the volume of 2wt% BSA in PBS;

[0024] (h) The sample to be tested containing asparaginase or the standard sample is added, and mixed at room temperature for 2 hours;

[0025] (i) The supernatant is removed by an external magnetic field, and washed 3 times with a washing buffer, and the supernatant is removed by an external magnetic field.

[0026] (j) adding aspartame phosphate buffer, shaking at 37℃ for 15 min; adding 4 times volume of internal standard containing acetonitrile, vortexing for 1 min, centrifuging, taking supernatant, diluting 600 times with 85% acetonitrile-water mixed solution, vortexing, and transferring to a sample vial;

[0027] (k) analyzing and detecting by LC-MS / MS method.

[0028] Further, in step c, 1 times volume of 0.5 mg / mL aspartame monoclonal antibody is added.

[0029] Further, in step j, 5.0 mg / mL aspartame-containing phosphate buffer (pH 8.0, 100 mM) is added.

[0030] Further, in step j, the volume ratio of acetonitrile to water in the acetonitrile-water mixed solution is 85:15.

[0031] Further, the LC-MS / MS method for analysis and detection comprises the following steps:

[0032] (a) Chromatographic conditions: Shimadzu GLInertsil HILIC column (2.1x75 mm, 3 μm), mobile phase: 0.05% formic acid water (A)-0.05% formic acid acetonitrile (B) (15:85, v / v), isocratic elution, flow rate 0.3 mL / min, column temperature 35℃, injection volume 1 μL, analysis time 2 min;

[0033] (b) Mass spectrometric conditions: Shimadzu 8045 triple quadrupole mass spectrometer, ion source is ESI, scanning mode is multi-reaction monitoring (MRM), detection in positive ion mode, m / z of aspartic acid parent ion and daughter ion is 134.0, 74.0 respectively, collision energy is -30 eV, m / z of aspartame parent ion and daughter ion is 133.0, 74.0 respectively, CE is -35 eV.

[0034] The present application has the advantages of:

[0035] 1. The immunomagnetic beads prepared by the NHS magnetic nanoparticles have the advantages of high efficiency, rapidness, and easy operation.

[0036] 2. The immunofunctionalized magnetic beads are used to specifically separate and enrich the aspartame enzyme to be detected, so that the sample matrix interference is avoided.

[0037] 3. The aspartase activity is detected by using asparaginase to specifically cut asparagine to generate aspartic acid, and the decrease of asparagine or the increase of aspartic acid is detected by mass spectrometry.

[0038] 4. The LC-MS / MS analysis method is established to quantitatively analyze the change of aspartic acid and asparagine, and the detection sensitivity is further improved.

[0039] In summary, the detection method has high sensitivity, good specificity and simple operation. The asparaginase to be detected in the sample is rapidly and specifically separated and enriched by magnetic separation, the asparaginase is specifically cut asparagine as a substrate peptide to generate a large amount of aspartic acid, the asparaginase activity is detected by mass spectrometry, and the sensitivity of the asparaginase activity detection is significantly improved. The method has important practical significance for promoting the in-vivo activity level monitoring of asparaginase. BRIEF DESCRIPTION OF DRAWINGS

[0040] Figure 1 It is a TEM characterization diagram of the magnetic nanoparticles prepared in Example 1 of the present application.

[0041] Figure 2 It is a TEM characterization diagram of the NHS magnetic nanoparticles prepared in Example 1 of the present application.

[0042] Figure 3 It is a diagram of the concentration optimization results of the asparagine solution added in Example 3 of the present application.

[0043] Figure 4 It is an ion scanning diagram of aspartic acid in Example 3 of the present application.

[0044] Figure 5 It is a mass spectrometry detection diagram of the asparaginase-containing sample in Example 3 of the present application. DETAILED DESCRIPTION

[0045] The specific embodiments provided by the present application are described in detail below in combination with examples.

[0046] Example 1: Preparation of immunofunctional magnetic beads

[0047] Take 0.5 g FeCl3·6H2O dissolved in 20 mL ethylene glycol, after ultrasonic to form a clear solution, add 1.5 g of NaOAc dissolved uniformly, then transfer the mixed solution to a stainless steel autoclave lined with polytetrafluoroethylene, heat at 200℃ for 12 h. After cooling, the black product was washed with absolute ethanol and ultrapure water three times, respectively, to obtain monodisperse Fe3O4 magnetic nanoparticles. Take 1.0 g of the prepared monodisperse Fe3O4 magnetic nanoparticles and ultrasonically disperse them in 2 mL of phosphate buffer solution (10 mM, pH 7.0). After ultrasonic dispersion for 20 min, add 1.5 mL of epichlorohydrin and mechanically stir at room temperature for 2 h. Then, mechanically stir at 50℃ for 40 min, add 2 mg / mL chitosan solution and react overnight. Under the action of a magnetic field, Fe3O4-chitosan magnetic nanoparticles are obtained. Then, activate the nanoparticles with N-hydroxysuccinimide and ultrasonically disperse them in N,N-dimethylacetamide to obtain a 10 mg / mL N-hydroxysuccinimide-activated magnetic nanoparticle dispersion. Remove the supernatant by magnetic separation, quickly add 2 volumes of 4℃ pre-cooled hydrochloric acid solution, vortex thoroughly for 30 s, and then remove the supernatant by magnetic separation. Add 1 volume of asparaginase monoclonal antibody (0.5 mg / mL) and rotate to mix at 4℃ in the dark for 12 h. Remove the supernatant by magnetic separation, and then wash the magnetic beads 4 times with 1 volume of 100 mM Tris-HCl (containing NaCl 150 mM, pH 8.0) buffer. Redisperse the magnetic beads in an equal volume of 100 mM Tris-HCl (containing NaCl 150 mM, pH 8.0), block at room temperature in the dark for 2 h, remove the supernatant by magnetic separation, and then wash the magnetic beads 4 times with an equal volume of 10 mM PBS (containing NaCl 137 mM, pH 7.4) containing 5% BSA. Redisperse the magnetic beads in an equal volume of 10 mM PBS (containing NaCl 137 mM, pH 7.4) containing 5% BSA, and further block at 4℃ in the dark for 12 h. After blocking, remove the supernatant by magnetic separation, redisperse the magnetic beads by washing with PBS containing 0.1% BSA 4 times, and then obtain antibody-immunized magnetic beads.

[0048] Take an appropriate amount of the synthesized NHS magnetic nanoparticles and perform transmission electron microscopy (TEM) characterization to evaluate the morphology and distribution of the NHS magnetic nanoparticles used in this method.

[0049] Figure 1 TEM characterization of the magnetic nanoparticles prepared in this example. Figure 2 TEM characterization of the NHS magnetic nanoparticles prepared in this example.

[0050] AsFigure 1 and Figure 2 As shown in the figure, the prepared magnetic nanoparticles have uniform particle size distribution, and the surface of the NHS magnetic nanoparticles is successfully coated with chitosan.

[0051] An appropriate amount of the prepared magnetic nanoparticles was taken for Dynamic Light Scattering (DLS) characterization to evaluate the particle size distribution of the NHS magnetic beads prepared by the method.

[0052] The DLS characterization results showed that the prepared NHS magnetic beads have uniform particle size distribution, about 396 ± 31 nm.

[0053] Example 2: Immune functionalized magnetic beads specifically enrich asparaginase in samples

[0054] The prepared antibody immune functionalized magnetic bead suspension was removed, and the supernatant was removed by an external magnetic field. The suspension was diluted with 5 volumes of PBS containing 0.1% BSA, and the supernatant was removed by an external magnetic field. The sample to be tested containing asparaginase or the standard sample was added, and the reaction was uniformly rotated at room temperature for 2 hours. Then, the supernatant was removed by an external magnetic field, and the suspension was washed 3 times with 10 mM PBS (containing NaCl 137 mM, 0.05% Tween-20 (w / v), pH 7.4) washing buffer. The supernatant was removed by an external magnetic field.

[0055] Example 3: Specifically sensitive analysis of asparaginase activity by LC-MS / MS

[0056] The optimal concentration of asparagine phosphate solution (100 mM, pH 8.0) was added to the enriched sample, and the reaction was incubated at 37°C for 15 min. Then, 4 volumes of internal standard acetonitrile were added, and the mixture was vortexed for 1 min. The supernatant was quantitatively removed by centrifugation, diluted 600 times with acetonitrile-water mixed solution (85:15, v / v), vortexed, transferred to an injection vial, and analyzed by the established LC-MS / MS method.

[0057] The LC-MS / MS method was as follows: Shimadzu GLInertsil HILIC column (2.1×75mm, 3μm), mobile phase: 0.05% formic acid water (A)-0.05% formic acid acetonitrile (B) (15:85, v / v), isocratic elution, flow rate: 0.3mL / min, column temperature: 35℃, injection volume: 1μL, analysis time: 2min. A Shimadzu 8045 triple quadrupole mass spectrometer was used, with ESI as the ion source and multiple reaction monitoring (MRM) as the scanning mode. In positive ion mode, the m / z of the aspartic acid precursor ion and daughter ion were 134.0 and 74.0, respectively, with a collision energy of -30eV. The m / z of the asparagine precursor ion and daughter ion were 133.0 and 74.0, respectively, with a CE of -35eV.

[0058] Mass spectrometry was used to scan the asparagine substrate peptide and aspartic acid.

[0059] Figure 3 This graph shows the optimized concentration of the asparagine substrate solution added in this embodiment. Figure 3 As shown, the linear correlation between the reduction in asparagine concentration and the asparaginase concentration was best when the concentration of the added substrate asparagine solution was 5.00 mg / mL.

[0060] Figure 4 This is the ion scan of aspartic acid in this embodiment. Figure 4 As shown, the m / z of the aspartic acid parent ion and daughter ion are 134.0 and 74.0, respectively, and the m / z of the asparagine parent ion and daughter ion are 133.0 and 74.0, respectively.

[0061] The obtained samples were analyzed by mass spectrometry.

[0062] Figure 5 This is a mass spectrometry image of the asparaginase-containing sample in this embodiment. Figure 5 As shown, the serum sample containing 0.05 IU / mL asparaginase showed good peak shape in mass spectrometry and no other interfering impurity peaks, indicating that the analytical method of the present invention is specific and sensitive and can meet the needs of in vivo asparaginase activity detection.

[0063] The preferred embodiments of the present invention have been described in detail above, but the present invention is not limited to the embodiments described. Those skilled in the art can make various equivalent modifications or substitutions without departing from the spirit of the present invention, and these equivalent modifications or substitutions are all included within the scope defined by the claims of this application.

Claims

1. A method for specifically detecting asparaginase activity by immunomagnetic separation combined with asparagine using LC-MS / MS, characterized in that, Includes the following steps: A) Immunomagnetic beads are prepared by coupling asparaginase antibodies to the surface of magnetic nanoparticles via an amide reaction; the immunomagnetic beads are antibody-functionalized magnetic beads prepared by coupling magnetic beads with asparaginase antibodies; the preparation method of the antibody-functionalized magnetic beads includes the following steps (a)-(f): (a) Ferric oxide magnetic nanoparticles were prepared by hydrothermal method, and then an agarose molecular layer was introduced in situ on the surface of the magnetic nanoparticles to further modify the NHS activated ester groups, thus preparing NHS magnetic beads. (b) Take the NHS-activated magnetic nanoparticle dispersion, remove the supernatant by magnetic separation, quickly add pre-cooled hydrochloric acid solution, mix and wash, and remove the supernatant by magnetic separation. (c) Add asparaginase monoclonal antibody, mix and react at 4°C in the dark by rotating for 12 h, and remove the supernatant by magnetic separation. (d) Under an external magnetic field, wash the sample four times with 100mM Tris-HCl buffer, redisperse it in 100mM Tris-HCl, and seal it at room temperature in the dark for 2 hours by rotation. Remove the supernatant by magnetic separation. (e) The magnetic beads were washed four times by blowing and mixing with 10mM PBS containing 5% BSA, and then redispersed in 10mM PBS containing 5% BSA. The mixture was then sealed at 4°C in the dark for 12 hours. (f) Remove the supernatant by magnetic separation, wash repeatedly with PBS containing 0.1% BSA 4 times and then disperse to obtain antibody-immunofunctionalized magnetic beads. B) Different concentrations of asparaginase-containing samples were prepared by adding different amounts of asparaginase standards to blank biological samples. These samples were then specifically captured and enriched using immunomagnetic beads to obtain the corresponding immunomagnetic bead / asparaginase complexes for each sample. Asparagine was added to the immunomagnetic bead / asparaginase complex, where asparaginase specifically cleaved the asparagine to form aspartic acid and ammonia. The sample supernatant was detected by LC-MS / MS. A standard curve of the asparaginase-mass spectrometry response signal was established, with the asparaginase concentration as the x-axis and the aspartic acid signal intensity detected by mass spectrometry as the y-axis. C) After mixing and incubating the sample with immunomagnetic beads, add asparagine phosphate buffer for LC-MS / MS detection. Substitute the obtained aspartic acid signal intensity into the standard curve to calculate the asparaginase activity in the sample.

2. The method for specific detection of in vivo asparaginase activity by immunomagnetic separation combined with asparagine using LC-MS / MS according to claim 1, characterized in that, Includes the following steps: (a) Ferric oxide magnetic nanoparticles were prepared by hydrothermal method, and then an agarose molecular layer was introduced in situ on the surface of the magnetic nanoparticles to further modify the NHS activated ester groups, thus preparing NHS magnetic beads. (b) Take the NHS-activated magnetic nanoparticle dispersion, remove the supernatant by magnetic separation, quickly add pre-cooled hydrochloric acid solution, mix and wash, and remove the supernatant by magnetic separation. (c) Add asparaginase monoclonal antibody, mix and react at 4°C in the dark by rotating for 12 h, and remove the supernatant by magnetic separation. (d) Under an external magnetic field, wash the sample four times with 100mM Tris-HCl buffer, redisperse it in 100mM Tris-HCl, and seal it at room temperature in the dark for 2 hours by rotation. Remove the supernatant by magnetic separation. (e) The magnetic beads were washed four times by blowing and mixing with 10mM PBS containing 5% BSA, and then redispersed in 10mM PBS containing 5% BSA. The mixture was then sealed at 4°C in the dark for 12 hours. (f) Remove the supernatant by magnetic separation, wash repeatedly with PBS containing 0.1% BSA 4 times and then disperse to obtain antibody-immunofunctionalized magnetic beads. (g) Take the prepared antibody immunofunctionalized magnetic bead suspension, separate it with an external magnetic field to remove the supernatant, and dilute it with 5 times the volume of 2% BSA in PBS; (h) Add the test sample or standard sample containing asparaginase and mix and react at room temperature for 2 hours; (i) Remove the supernatant by applying an external magnetic field, wash three times with washing buffer, and then remove the supernatant by applying an external magnetic field; (j) Add asparagine phosphate buffer at pH 8.0, shake well at 37°C for 15 min; add 4 times the volume of acetonitrile containing internal standard, vortex thoroughly for 1 min, centrifuge to quantitatively collect the supernatant, dilute 600 times with 85% acetonitrile-water mixed solution, vortex well, and transfer to a vial. (k) Analyze and detect using LC-MS / MS method.

3. The method for specific detection of in vivo asparaginase activity by immunomagnetic separation combined with asparagine using LC-MS / MS according to claim 2, characterized in that, In step c, add 1 volume of 0.5 mg / mL asparaginase monoclonal antibody.

4. The method for specific detection of in vivo asparaginase activity by immunomagnetic separation combined with asparagine using LC-MS / MS according to claim 2, characterized in that, In step d, the Tris-HCl buffer contains 150 mM NaCl and has a pH of 8.

0.

5. The method for specific detection of in vivo asparaginase activity by immunomagnetic separation combined with asparagine using LC-MS / MS according to claim 2, characterized in that, In step e, the PBS contains 137 mM NaCl and has a pH of 7.

4.

6. The method for specific detection of in vivo asparaginase activity by immunomagnetic separation combined with asparagine using LC-MS / MS according to claim 2, characterized in that, In step j, the concentration of asparagine phosphate buffer is 5.0 mg / mL.

7. The method for specific detection of in vivo asparaginase activity by immunomagnetic separation combined with asparagine using LC-MS / MS according to claim 2, characterized in that, In step j, the volume ratio of acetonitrile to water in the acetonitrile-water mixed solution is 85:

15.

8. The method for specific detection of in vivo asparaginase activity by immunomagnetic separation combined with asparagine using LC-MS / MS according to claim 2, characterized in that, The method for analysis and detection using the LC-MS / MS method includes the following steps: (a) Chromatographic conditions: Shimadzu GLInertsil HILIC column, inner diameter 2.1 mm, column length 75 mm, particle size 3 μm, mobile phase: 0.05% formic acid water - 0.05% formic acid acetonitrile volume ratio 15:85, isocratic elution, flow rate 0.3 mL / min, column temperature 35℃, injection volume 1 μL, analysis time 2 min; (b) Mass spectrometry conditions: Shimadzu 8045 triple quadrupole mass spectrometer, ESI ion source, multiple reaction monitoring, detection in positive ion mode. The m / z of the aspartic acid precursor ion and daughter ion were 134.0 and 74.0, respectively, with a collision energy of -30 eV. The m / z of the asparagine precursor ion and daughter ion were 133.0 and 74.0, respectively, with a CE of -35 eV.

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