Preparation method of ferritin blank serum matrix

Abstract

The invention discloses a preparation method of a ferritin blank serum matrix, which comprises the following steps: defibrating: adjusting the pH value of plasma to 7.5 + / -0.2, adding a calcium chloride solution, stirring and centrifuging to obtain defibrated supernate; degreasing: adding a calcium chloride solution and a dextran sulfate sodium salt solution into the defibrated supernate, stirring, standing, centrifuging, removing sediment, adding a barium chloride solution, stirring, and centrifuging to obtain degreased supernate; immunoadsorption: mixing immunomagnetic beads coated with a mouse anti-human ferritin antibody with the degreased supernatant to carry out immunoadsorption reaction, and carrying out magnetic separation to obtain an adsorbed supernatant; and dialysis: dialyzing the supernatant after adsorption to obtain the ferritin blank serum matrix. According to the method, the clean blank matrix liquid can be obtained on the basis of reserving plasma protein as much as possible.

Description

TECHNICAL FIELD

[0001] The application relates to a preparation method of an apoferritin blank serum matrix and belongs to the technical field of biological product preparation. BACKGROUND

[0002] Ferritin (Ferr) is a high molecular weight iron-containing protein that plays a role as an iron storage complex in the body. It is a core indicator for evaluating the body's iron status and a sensitive marker for inflammation / tissue damage, and is widely used in clinical applications. The clinical blank ferritin serum has very important significance. It has similar biological properties to the blood sample to be tested and does not contain the target ferritin. It is used as a blank control and blank diluent or sample diluent. Since the ferritin content in normal human serum samples is several times the clinical iron deficiency anemia determination index, it is very difficult to directly prepare a blank ferritin serum matrix from a serum sample. The residual content of the test substance in the blank serum matrix directly affects the accuracy of the prepared reagent. Therefore, the removal degree of the test substance in the blank serum matrix is crucial.

[0003] Currently, there is no method for removing ferritin from plasma to form a blank ferritin serum matrix. Other project blank matrix preparations basically use physical adsorbent methods to remove vitamins, hormones, etc. (such as CN112014186B), or directly ammonium sulfate precipitation and physical adsorbent to remove proteins and lipids in the matrix (such as CN113176133B).

[0004] A major disadvantage of physical adsorption is that while removing the target substance, other proteins are excessively removed, and the total protein content is extremely low (such as CN113176133B≤6 g / L). Excessive removal of matrix components makes the sample "too clean" and loses the matrix properties. In actual clinical test applications, most in vitro diagnostic reagent preparation calibration and quality control products require a certain protein concentration of the matrix serum (generally≥40 g / L). Therefore, how to effectively remove interfering substances and retain as much matrix protein as possible can meet the true business needs. SUMMARY

[0005] The purpose of the present application is to provide a preparation method of an apoferritin blank serum matrix, which obtains a clean blank matrix liquid on the basis of retaining as much plasma protein as possible.

[0006] In order to achieve the above purpose, the technical scheme adopted by the present application is as follows: A preparation method of an apoferritin blank serum matrix, comprising the following steps: S1, defibrination: adjusting the pH of the plasma to 7.5±0.2, then adding calcium chloride solution, stirring and centrifuging to obtain defibrination supernatant; S2, defatting: adding calcium chloride solution and sodium dextran sulfate solution to the defibrillation supernatant, stirring and standing, centrifuging to remove the precipitate, then adding barium chloride solution, stirring and centrifuging to obtain defatted supernatant; S3, immunoadsorption: mixing immunomagnetic beads coated with mouse anti-human ferritin antibody with the defatted supernatant to perform immunoadsorption reaction, and obtaining adsorbed supernatant after magnetic separation; S4, dialysis: dialyzing the adsorbed supernatant to obtain ferritin-free serum matrix.

[0007] Preferably, in step S1, the concentration of calcium chloride solution is 200-500 g / L, and the amount added is 0.5-2 mL per 100 mL of plasma; the stirring conditions are 20-30°C, 100-300 rpm, and 10-20 h; and the centrifugation conditions are 30,000-45,000 rpm and 15-30 min.

[0008] Preferably, in step S2, the concentration of calcium chloride solution is 200-500 g / L, and the amount added is 8-15 mL per 100 mL of plasma; the concentration of sodium dextran sulfate solution is 100-150 g / L, and the amount added is 3-10 mL per 100 mL of plasma.

[0009] Preferably, in step S2, after adding the sodium dextran sulfate solution, stirring for 5-8 h, and standing at 1-5°C for 20-30 h; the centrifugation conditions for removing the precipitate are 5,000-10,000 rpm and 15-30 min.

[0010] Preferably, in step S2, the concentration of barium chloride solution is 100-300 g / L, and the amount added is 3-8 mL per 100 mL of plasma; after adding the barium chloride solution, stirring at 20-30°C and 100-300 rpm for 3-7 h, and then centrifuging at 6,000-10,000 rpm for 10-30 min.

[0011] Preferably, in step S3, the preparation method of immunomagnetic beads includes the following steps: Activation: mixing the magnetic beads with MES buffer and EDC or CMC solution, and rotating and mixing uniformly at 15-30°C for 15-30 min; Antibody coupling: after removing the supernatant by magnetic separation, resuspending with MES buffer, adding mouse anti-human ferritin antibody, and rotating and mixing uniformly for 1-3 h; Blocking: adding blocking agent, and rotating and mixing uniformly for 1-3 h; Washing: washing with PBS buffer containing Tween 20, removing the supernatant, and repeating multiple times; Storage: adding a solution containing NaCl and BSA, and mixing uniformly.

[0012] Preferably, in the activation step, a NHS solution is also added; The blocking agent is one or more of BSA, casein, gelatin, glycine, Tween 20, CE210, and CE510.

[0013] Preferably, the concentration of the magnetic beads is 5-20 mg / mL; the concentration of the EDC solution is 1-200 mg / mL; and the concentration of the CMC solution is 1-200 mg / mL. The volume ratio of the magnetic beads to the EDC solution or the CMC solution is (15-25):(1-5) or (8-12):(2-3).

[0014] Preferably, in step S3, the volume ratio of the immunomagnetic beads to the defatted supernatant is 1:(90-100); and the conditions of the immunoadsorption reaction are 15-30°C, 50-200 rpm, and 10-60 min.

[0015] Preferably, in step S4, the dialysis treatment is performed in a sodium chloride dialysis solution at 1-5°C using a dialysis bag with a molecular weight cut-off of 10 KDa; the volume ratio of the dialysate to the dialysis solution is 1:50, and the dialysis solution is replaced every 4 hours.

[0016] The present application has the following advantages: By adding calcium ions to the negative plasma, calcium ions are essential cofactors in multiple key steps of the coagulation cascade, especially the common pathway. Prothrombin activators (produced by the endogenous or exogenous pathway) activate prothrombin into thrombin in the presence of calcium ions, which can convert fibrinogen into fibrin, and the fibrin is cross-linked into a clot. Then, through physical stirring, the formed fibrin clot (containing consumed coagulation factors and platelets) is separated from the blood, obtaining a serum that does not contain fibrinogen and does not coagulate. Defibrination can improve stability.

[0017] By adding an appropriate amount of calcium ions and sodium dextran sulfate to the defibrinated negative serum, sodium dextran sulfate binds Ca 2+ Based on charge neutralization, hydrophobic aggregation, and metal ion bridging, insoluble complexes are formed to precipitate cholesterol and triglycerides (TG), achieving the effect of defatting. Blood lipids, especially CM / LDL / VLDL, are stored for a long time and will separate, making the serum matrix turbid, affecting appearance and use, and defatting can improve stability.

[0018] By adding magnetic bead-coated specific antibodies to the defibrinated and defatted negative serum, the antibodies bind to specific antigens in the serum to form antigen-antibody complexes, which can be removed by magnetic separation. Through dialysis, the plasma proteins are preserved as much as possible to obtain a clean blank blood matrix liquid, which can be used for the production and application of blank samples in ferritin kits. DETAILED DESCRIPTION

[0019] Example 1: This embodiment provides a preparation method of ferritin blank serum matrix, the specific steps are as follows.

[0020] 1. Defibrination.

[0021] Mix 100 mL of negative human plasma raw material, and adjust the pH value to 7.5 ± 0.2 by using 0.1 M HCl or NaOH solution according to the pH of the plasma raw material.

[0022] Add 1 mL of 300 g / L calcium chloride solution to 100 mL of plasma, and stir at 25°C and 200 rpm for 16 h.

[0023] Centrifuge at 39000 r / min for 20 min to obtain the supernatant.

[0024] 2. Defatting.

[0025] Add 10 mL of 300 g / L calcium chloride solution to the supernatant after defibrination, stir for 5 min, then add 5 mL of 120 g / L 500 kDa sodium sulfate dextran salt solution, stir for 6 h, and then stand overnight at 4°C.

[0026] Slowly pour out the supernatant, then centrifuge at 9000 r / min for 20 min to remove lipoprotein, add 5 mL of 200 g / L barium chloride solution to the supernatant, stir at 25°C and 200 rpm for 6 h, and then centrifuge at 9000 r / min for 20 min to obtain the supernatant.

[0027] 3. Magnetic bead coated antibody.

[0028] Take 100 μL of 1 mm magnetic beads (from commercially available 10 mg / mL 1 mm carboxyl magnetic beads), add 1 mL of 50 mM MES (pH=6.1) buffer, 12 μL of 10 mg / mL EDC solution, and 24 μL of 5 mg / mL NHS solution, mix well at 25°C and 30 rpm for 30 min.

[0029] Remove the supernatant by magnetic separation, add 1 mL of 50 mM MES (2-morpholinoethanesulfonic acid, pH=6.1) buffer, mix well, and add 150 μg of mouse anti-human ferritin antibody (Ferr). Mix well at 25°C and 30 rpm for 2 h.

[0030] Add 100 μL of 100 mg / mL BSA (human serum albumin) blocking agent, mix well at 25°C and 30 rpm for 1 h.

[0031] Magnetic separation to remove supernatant. Add 1 mL of 10 mM PBS (pH = 7.4) + 0.05% (V / V) Tween 20 (the amount of Tween 20 involved in the subsequent steps is all volume ratio) to wash once. Remove supernatant for 5 min each time. Repeat this step 4 times.

[0032] Add 1 mL of 150 mM NaCl + 10 g / L BSA. Mix well, and the magnetic bead antibody is obtained.

[0033] 4. Magnetic bead antibody immunoadsorption.

[0034] Within 2 days, add the coated magnetic bead antibody to the supernatant after degreasing at a volume ratio of 100:1, stir at 25°C and 200 rpm for 20 min, magnetically separate, and obtain the supernatant.

[0035] The above supernatant is dialyzed in a dialysis bag with a molecular weight cutoff of 10 KDa at an indoor temperature of 15°C, and the dialysis fluid is 0.9% sodium chloride. The volume ratio of dialysate to dialysate is 50:1, and the dialysate is replaced every 4 hours. Replace the dialysate 3 times. Finally, the dialyzed serum is obtained.

[0036] Example 2: The difference from Example 1 is that the carboxyl magnetic bead activator is different. Replace the 10 mg / mL 1-(3-dimethylaminopropyl)-3-ethyl carbodiimide hydrochloride (EDC) solution with a 10 mg / mL 1-cyclohexyl-2-morpholinoethyl carbodiimide p-toluenesulfonate (CMC) solution. The proportions of other components and the operation steps remain unchanged.

[0037] Application Example 1: Determination of the content of related substances in human negative plasma before and after treatment.

[0038] The negative plasma is treated by the method in Example 1 and Example 2, and the samples before and after treatment are measured by the corresponding kit.

[0039] Main instruments: Hitachi 7180 full-automatic biochemical analyzer, Mindray CL-2000i full-automatic chemiluminescence immunoassay analyzer.

[0040] Total protein (TP), albumin (ALB), total cholesterol (TC), and triglyceride (TG) kits were purchased from Beijing Jiujiang Bioengineering. Ferritin (Ferr) kit (chemical immunoassay method) was purchased from Shenzhen Mindray.

[0041] The test operation is in accordance with the relevant steps in the kit instruction manual.

[0042] Table 1: Content of related substances in blank serum of each example before and after treatment

[0043] The results show that the method provided by the application can effectively remove lipid substances in blood plasma, specifically remove Ferr, and retain serum matrix proteins (≥40 g / L), which meets the actual clinical requirements.

[0044] Application Example 2: Specific immunoadsorption effect comparison between defatting and non-defatting.

[0045] Comparison of the influence of defatting and non-defatting on the residual amount of ferritin in blank serum.

[0046] Take 40 mL of negative blood plasma, normally defibrate, adjust pH to 7.5, add 0.4 mL of 300 g / L calcium chloride solution, stir at 25°C and 200 rpm for 16 h, centrifuge at 9000 r / min for 20 min to take the supernatant. Divide the obtained supernatant into two equal parts, each 18 mL. One group is normally defatted, and the other group is not defatted.

[0047] Add 2 mL of 300 g / L calcium chloride solution to one group of supernatant, stir for 5 min, then add 1 mL of 120 g / L 500 kDa sodium sulfate dextran salt solution, stir for 6 h, and then stand overnight at 4°C. Slowly pour out the supernatant, then centrifuge at 9000 r / min for 20 min to remove lipoprotein, then add 1 mL of 200 g / L barium chloride solution to the supernatant, stir at 25°C and 200 rpm for 6 h, centrifuge at 9000 r / min for 20 min to take the supernatant, and obtain 18.5 mL of defatted supernatant.

[0048] Take the magnetic beads-Ferr antibody coated according to the process in Example 1. Add 185 uL and 180 uL of the coated magnetic beads-Ferr antibody to the 18.5 mL of defatted supernatant and the 18 mL of non-defatted supernatant, respectively, stir at 25°C and 200 rpm for 20 min, magnetically separate, and obtain supernatant 1 and supernatant 2.

[0049] Dialyze supernatant 1 and supernatant 2 in a room temperature of 15°C using a dialysis bag with a molecular weight cutoff of 10 KDa. The dialysis fluid is 0.9% sodium chloride, and the volume ratio of dialysate to dialysate is 50:1. The dialysis fluid is replaced every 4 h, and the dialysis fluid is replaced 3 times. Finally, dialyzed serum 1 (defatted serum) and serum 2 (non-defatted serum) are obtained.

[0050] Measure the defatted and non-defatted supernatants using related kits. The instruments and reagents used are the same as in Application Example 1.

[0051] Table 2: Comparison of specific immunoadsorption effect between defatting and non-defatting

[0052] Ferritin removal rate = 1-(post-treatment ferritin content / pre-treatment ferritin content) * 100%.

[0053] The results are shown in Table 2. According to the normal process of fiber removal + degreasing + immunoadsorption, the Ferr removal rate is more than 99.7%; while without degreasing, only fiber removal + immunoadsorption, the Ferr removal rate is only 79.6%, less than 80%, and the serum lipid will physically adsorb a part of the magnetic bead antibody, resulting in the weakening of the specific immunoadsorption effect. Therefore, degreasing is the key to the complete specific adsorption of the magnetic bead antibody.

[0054] Application Example 3: Influence of washing times of magnetic bead coated antibody and change of washing time on immunoadsorption effect.

[0055] The influence of washing times of magnetic bead coated antibody and change of washing time on ferritin removal rate and mouse anti-human Ferr residual amount was studied.

[0056] The human negative plasma to be treated was normally fibered and degreased in the manner of Example 1. The washing of magnetic bead coated antibody used washing solution (10 mM PBS (pH = 7.4) + 0.05% (V / V) Tween 20) to set up experimental groups: no washing, 1 wash, 3 washes, 5 washes, 7 washes, 5 min each time; 5 washes, 10 min each time; 5 washes, 15 min each time, 3 washes, 30 min each time, 2 washes, 1 h each time; 1 wash, overnight 16 h. Other immunoadsorption was the same as Example 1. The corresponding serum and plasma without treatment in the manner of Example 1 were obtained, and the ferritin content and mouse anti-residual amount were measured.

[0057] Main instruments: Mindray CL-2000i full-automatic chemiluminescence immunoassay analyzer, Weigao semi-automatic chemiluminescence immunoassay analyzer JR-1.

[0058] Ferritin (Ferr) kit (chemical immunoassay method) was purchased from Shenzhen Mindray; mouse IgG enzyme-linked immunosorbent assay kit was purchased from Biyun Tian (plate luminometer) to measure whether there is mouse anti-human Ferr antibody residual in the serum.

[0059] The tests were performed according to the relevant steps of the kit instructions.

[0060] Table 3: Influence of washing times of magnetic bead coated antibody and change of washing time on immunoadsorption effect (washing solution 10 mM PBS (pH = 7.4) + 0.05% Tween 20)

[0061] Mouse anti-residual rate = (mouse anti content after treatment - mouse anti content in untreated plasma) / mouse anti content in untreated plasma * 100%.

[0062] The results are shown in Table 3. Using a washing solution of 10mM PBS (pH=7.4) + 0.05% Tween 20, with each wash lasting 5 minutes, increasing the number of washes decreased the content of mouse anti-human Ferrer antibodies in the serum. After 5-7 washes, the residual amount of mouse anti-antibody in the serum was basically equivalent to that in the original plasma, and the removal rate of Ferrer antigen in the serum remained at >99.8%. With the extension of the total washing time, the residual rate of mouse anti-antibody in the serum was relatively low, but the residual Ferrer antigen content in the serum gradually increased. Tween 20 can wash away the non-specifically adsorbed mouse anti-human Ferrer antibodies on the surface of the magnetic beads, but excessive washing time will displace the antibodies coupled to the surface of the magnetic beads, resulting in a decrease in the ability of the magnetic beads-Ferr antibody to capture Ferrer antigen. In summary, washing with 10mM PBS (pH=7.4) + 0.05% Tween 20 for 5 washes, 5 minutes each, is more appropriate.

[0063] Application Example 4: Ferritin blank serum dilution experiment.

[0064] Fresh high-value Ferritrile serum (1085 ng / mL) was selected. The high-value samples were diluted 2.5-fold, 5-fold, 10-fold, 20-fold, 40-fold, and 80-fold (all within the linear detection range) using blank ferritin serum prepared according to the method described in Example 1, and physiological saline, respectively. The diluted samples were tested three times, and the mean value (y) of each test was calculated. i ), to dilute the theoretical concentration (x) i ) is the independent variable, and the mean of the detection results (y) i Find the linear regression equation with the dilution concentration (x) as the dependent variable. i Substitute the values ​​into the linear regression equation, calculate the estimated values, and use the mean (y) i The estimated value is the absolute deviation, and the relative deviation is calculated as (absolute deviation / estimated value) * 100%. A deviation within ±10% is considered acceptable (no offset) for dilution test results, meeting the usage requirements. All tests were performed using Mindray reagent kits (chemical immunoassay).

[0065] Table 4: Linearity deviation of high-value diluted blank serum samples obtained using the method in Example 1 in Application Example 1 (unit: ng / mL)

[0066] The linear regression equation 1 is: y i =1.0255x i -1.3282, correlation coefficient r=0.999.

[0067] Table 5: Linearity deviation of high-value samples diluted with physiological saline (unit: ng / mL)

[0068] Linear regression equation 2 is: y i = 1.0444x i -19.9721, the correlation coefficient r = 0.999.

[0069] Table 4 and Table 5 results show that when diluted with physiological saline, the 20-fold relative deviation is 16.84%, and the 80-fold relative deviation is 146.44%, which is different. When the blank serum obtained by the method in application example 1 in application example 1 is diluted to 80 times, the detection result deviation is 6.23%, which shows that the blank matrix serum prepared by the treatment method provided by the application is more reliable for blank diluent detection result.

[0070] Application Example 5: Comparison of the measured values of each blank serum sample diluted by the method provided by the application with different commercially available reagents.

[0071] Using the blank serum treated by the process in application example 1 in application example 1, dilute the fresh high-value Ferr serum (1085 ng / mL) 5 times, 10 times, 20 times, 40 times, and 50 times, respectively. Third-party quality control: multi-item liquid phase protein detection quality control level 1 (701LPC ZH 2026-06), level 2 (687LPC ZH 2025-12), level 3 (688LPC ZH 2025-12) are respectively used on Roche Cobas e801 (electrochemiluminescence), Siemens ADVIA Centaur XPT (direct chemiluminescence), Mindray CL-2000i (direct luminescence) three mainstream instruments and their respective ferritin detection of the above samples, each sample is measured twice, and the difference between the average values (shown in Table 6) is compared. The smaller the difference, the better the consistency of the blank serum diluted sample.

[0072] Table 6: Comparison of measured values of blank serum diluted sample in application example 1 treated by process in application example 1 on different reagents (unit: ng / mL)

[0073] The results show that the measured values of the high-value serum diluted by the blank serum of the application, the third-party quality control on Siemens ADVIA Centaur XPT and Mindray CL-2000i are consistent with Roche Cobas e801, with an overall deviation of less than 15%, which shows that the serum prepared by the application can be adapted to common models on the market, and can meet the actual needs.

[0074] Application Example 6: Sample (liquid) stability experiment of high-value sample diluted by blank serum prepared by the application.

[0075] The fresh mixed high-value Ferr serum (980 ng / mL) prepared by the process of Example 1 in Application Example 1 was diluted 5 times, 10 times, 20 times and 40 times with the blank serum prepared by the process of Example 1, and the stability of the obtained samples under different storage conditions of-80℃, (-20±5)℃, (2-8)℃, (20-25)℃ and 37℃ was investigated, and the samples were detected according to the specified period, 2 tubes were detected each time, and 2 times were detected each tube, and the average value was calculated. All were detected by Mindray kit (chemical immunization analysis method).

[0076] Table 7: Stability investigation plan

[0077] According to Table 7, the corresponding experiments were planned, and the diluted serum was stored in sufficient number of tubes, and the related concentration values were measured on the day.

[0078] Table 8: Stability experiment results of blank serum diluted high-value Ferr serum at 37℃ (average value, unit: ng / mL)

[0079] Table 9: Stability experiment results of blank serum diluted high-value Ferr serum at (20-25)℃ (average value, unit: ng / mL)

[0080] Table 10: Stability experiment results of blank serum diluted high-value Ferr serum at (2-8)℃ (average value, unit: ng / mL)

[0081] Table 11: Stability experiment results of blank serum diluted high-value Ferr serum at (-20±5)℃ (average value, unit: ng / mL)

[0082] Table 12: Stability experiment results of blank serum diluted high-value Ferr serum at-80℃ (average value, unit: ng / mL)

[0083] The results of Tables 8-12 show that the ferritin blank serum prepared by the present application as a diluent to dilute the high-value ferritin serum can be stable at-80℃ and (-20±5)℃ for at least 12 months, at (2-8)℃ for at least 1 month, at (20-25)℃ for at least 15 days, and at 37℃ for at least 7 days. The stability meets the requirements and can meet the actual measurement needs.

Claims

1. A method for preparing a ferritin-free serum matrix, characterized in that, Includes the following steps: S1. Defibrinolysis: Adjust the pH of the plasma to 7.5±0.2, add calcium chloride solution, stir and centrifuge to obtain the defibrinolysis supernatant; S2, Degreasing: Add calcium chloride solution and sodium dextran sulfate solution to the defiber supernatant, stir and let stand, centrifuge to remove sediment, then add barium chloride solution, stir and centrifuge to obtain the degreasing supernatant; S3. Immunoadsorption: Immunomagnetic beads coated with mouse anti-human ferritin antibody are mixed with defatted supernatant for immunoadsorption reaction. After magnetic separation, the supernatant after adsorption is obtained. S4. Dialysis: Dialyze the supernatant after adsorption to obtain ferritin-free serum matrix.

2. The method for preparing ferritin-free serum matrix according to claim 1, characterized in that, In step S1, the concentration of calcium chloride solution is 200-500 g / L, and the amount added is 0.5-2 mL per 100 mL of plasma; the stirring conditions are: 20-30℃, 100-300 rpm, 10-20 h; the centrifugation conditions are: 30000-45000 rpm, 15-30 min.

3. The method for preparing ferritin-free serum matrix according to claim 1, characterized in that, In step S2, the concentration of calcium chloride solution is 200-500 g / L, and the amount added is 8-15 mL per 100 mL of plasma; the concentration of sodium dextran sulfate solution is 100-150 g / L, and the amount added is 3-10 mL per 100 mL of plasma.

4. The method for preparing ferritin-free serum matrix according to claim 1, characterized in that, In step S2, after adding sodium dextran sulfate solution, stir for 5-8 hours, and then let stand at 1-5℃ for 20-30 hours; the centrifugation conditions for removing sediment are: 5000-10000 rpm, 15-30 min.

5. The method for preparing ferritin-free serum matrix according to claim 1, characterized in that, In step S2, the concentration of barium chloride solution is 100-300 g / L, and the amount added is 3-8 mL per 100 mL of plasma. After adding barium chloride solution, stir at 20-30℃ and 100-300 rpm for 3-7 h, and then centrifuge at 6000-10000 rpm for 10-30 min.

6. The method for preparing ferritin-free serum matrix according to claim 1, characterized in that, In step S3, the preparation method of immunomagnetic beads includes the following steps: Activation: Mix the magnetic beads with MES buffer and EDC or CMC solution and then rotate to mix at 15-30℃ for 15-30 min; Antibody conjugation: After removing the supernatant by magnetic separation, resuspend in MES buffer, add mouse anti-human ferritin antibody, and mix by vortexing for 1-3 hours. Sealing: Add sealing agent and mix by swirl for 1-3 hours; Washing: Wash with PBS buffer containing Tween 20, then discard the supernatant. Repeat several times. Storage: Add a solution containing NaCl and BSA and mix well.

7. The method for preparing ferritin-free serum matrix according to claim 6, characterized in that, NHS solution was also added during the activation step; The blocking agent is one or more of BSA, casein, gelatin, glycine, Tween 20, CE210, and CE510.

8. The method for preparing ferritin-free serum matrix according to claim 7, characterized in that, The concentration of magnetic beads is 5-20 mg / mL; the concentration of EDC solution is 1-200 mg / mL; the concentration of CMC solution is 1-200 mg / mL. The volume ratio of magnetic beads to EDC solution or CMC solution is (15-25):(1-5) or (8-12):(2-3).

9. The method for preparing ferritin-free serum matrix according to claim 1, characterized in that, In step S3, the volume ratio of immunomagnetic beads to defatted supernatant is 1:(90-100); the conditions for the immunoadsorption reaction are: 15-30℃, 50-200rpm, 10-60min.

10. The method for preparing ferritin-free serum matrix according to claim 1, characterized in that, In step S4, the dialysis process is carried out using a dialysis bag with a molecular weight cutoff of 10 kDa at 1-5°C in sodium chloride dialysis solution; the volume ratio of dialysate to dialysis solution is 1:50, and the dialysis solution is changed every 4 hours.

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