Natural neutrophil apolipoprotein dimer and preparation method thereof

Abstract

The invention discloses a natural neutrophil apolipoprotein dimer and a preparation method thereof, and belongs to the technical field of biomolecule preparation and biomedical detection. The problems that the recombinant protein dimer prepared by a genetic engineering recombination technology is low in yield, the structure of the recombinant protein dimer is different from that of natural protein, so that the development of antibodies and the detection effect of related kits are influenced, a method for extracting the recombinant protein dimer from peripheral blood is complicated, raw materials are not easy to obtain, and blood resources are wasted are solved. According to the preparation method disclosed by the invention, a leukocyte filter or a leukocyte layer generated in a component blood preparation process is taken as a raw material, a leukocyte lysis solution is adopted for lysis, and the obtained granulocyte lysis solution is combined with anion exchange and affinity chromatography methods to obtain the natural neutrophil apolipoprotein dimer. The preparation method can be used for preparing the natural neutrophil apolipoprotein dimer in batches, is simple and convenient to operate and high in protein yield, and can realize comprehensive utilization of blood resources and turn waste into wealth.

Description

Technical Field

[0001] This invention belongs to the field of biomolecule preparation and biomedical detection technology, specifically relating to a natural neutrophil apolipoprotein dimer and its preparation method. Background Technology

[0002] Human neutrophil lipoprotein (HNL, also known as Lipocalin-2 or NGAL) is a protein secreted by neutrophils. It is involved in lipid metabolism, bacterial growth inhibition, and tissue repair, and has the potential to act as a biomarker in diseases such as infection, acute kidney injury, and cancer. HNL is a secretory glycosylated protein composed of 198 amino acids, with a 20-amino acid signal peptide sequence at its N-terminus. The mature protein contains 178 amino acids. HNL exists in various molecular forms, including monomers, homodimers, and heterodimers. Different molecular forms of HNL are associated with different pathological processes. For example, monomers may have a relatively high correlation with acute kidney injury, homodimers have high specificity and sensitivity in distinguishing between bacterial and viral infections, and heterodimers may be associated with tumor invasion.

[0003] Existing technologies have developed kits for detecting various diseases based on human neutrophil apolipoproteins as biomarkers. However, the yield of recombinant protein dimers prepared using genetic engineering recombination technology is low, and the structure of the obtained recombinant protein dimers differs from that of the natural protein. This leads to significant deviations in the development of neutrophil apolipoprotein detection kits, including antibody preparation, calibrators, and quality control materials, resulting in low clinical concordance. Furthermore, methods that obtain protein dimers by isolating and lysing leukocytes from human peripheral blood, followed by ion chromatography and molecular sieve chromatography, involve complex extraction and purification processes, have limited access to raw blood, and result in wasted blood resources. Summary of the Invention

[0004] This invention addresses the technical problems of low yield of recombinant protein dimers prepared by genetic engineering recombination technology, structural differences from natural proteins affecting antibody development and related reagent kit detection effects, as well as the complexity of methods for extracting natural proteins from peripheral blood, the difficulty in obtaining raw materials, and the waste of blood resources. It provides a natural neutrophil apolipoprotein dimer and its preparation method.

[0005] The technical solution adopted by the present invention to solve the above-mentioned technical problems is as follows.

[0006] In a first aspect, the method for preparing natural neutrophil apolipoprotein dimers of the present invention includes the following steps: S1, the waste containing granulocytes generated during the component blood preparation process is first depleted of red blood cells to obtain granulocytes, and then the obtained granulocytes are lysed using leukocyte lysis buffer to obtain granulocyte lysis buffer; The leukocyte lysis buffer includes a surfactant, a protease inhibitor at a final concentration of 1 mM to 10 mM, a preservative at a final concentration of 0.01 wt% to 0.2 wt%, and a buffer solution. S2, the granulocyte lysate was subjected to anion exchange chromatography to obtain the crude target protein; S3, affinity chromatography was performed on the crude target protein to obtain natural neutrophil apolipoprotein dimers.

[0007] Preferably, the waste containing granulocytes generated during the preparation of the component blood is a leukocyte layer. S1 includes: mixing the leukocyte layer with a buffer containing dextran, removing red blood cells by sedimentation, centrifuging the obtained plasma, removing the supernatant and resuspending it, centrifuging again to remove the supernatant to obtain granulocytes, suspending the granulocytes in leukocyte lysis buffer, letting it stand and then centrifuging to remove the precipitate to obtain granulocyte lysis buffer. More preferably, the volume ratio of the leukocyte layer to the buffer solution containing dextran is 1:1 to 1:5; More preferably, the concentration of dextran in the dextran-containing buffer solution is 1 wt% to 5 wt%. More preferably, the buffer solution in the dextran-containing buffer solution is 0.01M phosphate or 0.1M Tris-hydrochloric acid, with a pH of 6.5 to 7.5; More preferably, the settling time is 20 min to 1 h; More preferably, the plasma is first centrifuged at 400×g for 10 min, the supernatant is removed and the plasma is resuspended in PBS buffer, then centrifuged at 400×g for 10 min, the supernatant is removed and granulocytes are obtained. Particularly preferred is that the concentration of the PBS buffer is 0.01M and the pH is 6.5~7.5; More preferably, the granulocytes are suspended in leukocyte lysis buffer, allowed to stand for more than 10 minutes, and then centrifuged at 10000×g for 10 minutes.

[0008] Preferably, the waste containing granulocytes generated during the preparation of the component blood is a leukocyte filter, and S1 includes: S1-1, Take N white blood cell filters, fill them with air to remove residual red blood cells, N≥1; S1-2, wash N white blood cell filters with PBS buffer and purge with air to remove the liquid from the white blood cell filters; S1-3, process each white blood cell filter separately or process N white blood cell filters in series. First, wash N white blood cell filters with PB buffer and then introduce air to remove the liquid from the white blood cell filters. S1-4, then wash N white blood cell filters with pre-cooled white blood cell lysis buffer, lyse, after lysis, purge with air to remove the liquid in the white blood cell filters, and collect; S1-5, then wash N white blood cell filters with pre-cooled PBS buffer, purge air to remove liquid from the white blood cell filters, and collect; S1-6: Mix the liquids collected in S1-4 and S1-5 to obtain granulocyte lysis buffer; More preferably, in S1-2, the concentration of the PBS buffer is 0.01M and the pH is 7.2; More preferably, in S1-3, the concentration of PB buffer is 0.01M and the pH is 7.2; More preferably, in S1-4 and S1-5, the pre-cooling temperature is independently 2~8°C; More preferably, in S1-4, each leukocyte filter is lysed for more than 4 minutes, or N leukocyte filters are processed in series for more than 20 minutes; More preferably, in S1-4, the amount of leukocyte lysis buffer used is as follows: each leukocyte filter is treated separately, with 30 mL for each leukocyte filter; N leukocyte filters are connected in series, with 30 mL for each leukocyte filter, and an additional 15 mL of leukocyte lysis buffer is added for each additional leukocyte filter. More preferably, in S1-5, the amount of PBS buffer used is as follows: each leukocyte filter is treated separately, with 30 mL for each leukocyte filter; N leukocyte filters are connected in series, with 30 mL for each leukocyte filter, and 15 mL of PBS buffer is added for each additional leukocyte filter.

[0009] Preferably, in S1, the surfactant is one or more of the following: sodium dodecyl sulfate (SDS) with a final concentration of 0.05wt% to 5wt%, dodecyltrimethylammonium chloride with a final concentration of 0.05wt% to 5wt%, tetradecyltrimethylammonium chloride with a final concentration of 0.05wt% to 5wt%, hexadecyltrimethylammonium bromide with a final concentration of 0.05wt% to 5wt%, Tx-100 with a final concentration of 0.5wt% to 2wt%, Tween 20 / 80 with a final concentration of 0.5wt% to 2wt%, and NP40 with a final concentration of 0.5wt% to 5wt%.

[0010] Preferably, in S1, the protease inhibitor is one or more of benzyl sulfonyl fluoride (PMSF), aprotinin, and leupeptin.

[0011] Preferably, the preservative is one or more of sodium azide, Proclin 300, thimerosal, and isothiazolinone.

[0012] It should be noted that the leukocyte layer comes from the preparation of blood components in a multi-bag system. After the first centrifugation, the plasma is transferred to the first transfer bag, and an appropriate amount of plasma and leukocyte layer are transferred to the second transfer bag. The red blood cell preservation solution in the red blood cell preservation solution bag (first transfer bag) is transferred to the red blood cell bag and mixed thoroughly to form suspended red blood cells. The leukocyte component bag (second transfer bag) and an empty bag are lightly centrifuged together, and the platelet-rich plasma (upper layer) is transferred to the empty bag to form concentrated platelets. The platelets are then heat-sealed and separated, and the leukocyte component bag is discarded. The substance in the discarded leukocyte component bag is the leukocyte layer. The leukocyte filter is made from blood components prepared in a multi-bag system. After the first centrifugation, as much plasma as possible is transferred to the transfer bag. The red blood cell preservation solution in the red blood cell preservation bag (transfer bag) is transferred to the red blood cell bag and mixed thoroughly to obtain suspended red blood cells. The filtration operation is performed according to the instructions of the leukocyte filter. The leukocyte filtration technology is used to remove leukocytes from the suspended red blood cells to obtain the leukocyte filter (medical waste).

[0013] Preferably, in S2, the granulocyte lysate after G-25 desalting or dialysis is loaded into a DE-52 anion exchange column, equilibrated with 0.01~0.02M, pH 8.0 PB buffer at a volume of at least 4 times the column bed volume, and eluted with a gradient of 2M NaCl solution at a flow rate of 0.5~1ml / min. The flow-through is collected to obtain the crude target protein. More preferably, the dialysis method is as follows: granulocyte lysis buffer is introduced into a dialysis bag and dialyzed overnight at 4°C in PB buffer with a concentration of 0.01~0.02M NaH2PO4 and pH 8.0.

[0014] Preferably, in S3, the crude target protein is loaded onto an affinity chromatography column and repeated once. After loading is stopped, the column is equilibrated with 0.01~0.02M PB buffer at pH 8.0, and then the target protein is eluted with a gradient elution buffer at pH 2.5~4.0. More preferably, the target protein is collected using Tris-HCl buffer; More preferably, the gradient elution buffer is a glycine-hydrochloric acid buffer or a citrate buffer; More preferably, the affinity medium of the affinity chromatography column is a cyanogen bromide activated agarose gel 4B immobilized with an anti-neutrophil apolipoprotein monoclonal antibody (obtained by referring to international patent application WO2024EP64616, SPECIFIC DETECTION OF HOMODIMERICHNL).

[0015] Secondly, the present invention also provides natural neutrophil apolipoprotein dimers prepared by the above-mentioned method for preparing natural neutrophil apolipoprotein dimers.

[0016] The principle of this invention is as follows: The method for preparing natural neutrophil apolipoprotein dimers of this invention uses a suitable leukocyte lysis buffer to lyse the leukocyte layer and granulocytes in the leukocyte filter generated during the preparation of component blood. Combined with protein purification chromatography technology, natural neutrophil apolipoprotein dimers are obtained. These dimers can be used in the development of neutrophil apolipoprotein detection kits, including antibody preparation, calibrators, and quality control products. At the same time, the leukocyte layer and the leukocyte-free filter are fully utilized, saving blood resources.

[0017] Compared with the prior art, the beneficial effects of the present invention are as follows: The method for preparing natural neutrophil apolipoprotein dimers of the present invention can prepare natural neutrophil apolipoprotein dimers.

[0018] The method for preparing natural neutrophil apolipoprotein dimers of the present invention is simple to operate. By treating the leukocyte filter or leukocyte layer with leukocyte lysis buffer, the protein yield is high and a large amount of target protein can be obtained.

[0019] The method for preparing natural neutrophil apolipoprotein dimers of the present invention enables the comprehensive utilization of blood resources, turning waste into treasure. Attached Figure Description

[0020] Figure 1 The figures show the SDS-PAGE electrophoresis detection and analysis results of Example 5 of this invention. In the figures, 1. Sample 1 collected by elution at pH=2.5; 2. Sample 2 collected by elution at pH=2.5; 3. Sample 1 collected by elution at pH=3.0; 4. Sample 2 collected by elution at pH=3.0; 5. Sample 1 collected by elution at pH=3.5; 6. Sample 2 collected by elution at pH=3.5; 7. Sample 1 collected by elution at pH=4.0; 8. Sample 2 collected by elution at pH=4.0; 9. Affinity chromatography flow-through buffer; Marker indicates molecular marker.

[0021] Figure 2 This is the correlation curve for the interchangeability verification of enterprise product calibrators in Embodiment 6 of the present invention. Detailed Implementation

[0022] To enable those skilled in the art to better understand the technical solutions of the present invention, the present invention will be further described in detail below with reference to embodiments.

[0023] In the following embodiments, various processes and methods not described in detail are conventional methods known in the art. Unless otherwise specified, the materials, reagents, apparatus, instruments, equipment, etc., used in the following embodiments are commercially available.

[0024] In this embodiment, the enterprise working calibrators, the enterprise's standing measurement procedures, and the user's routine measurement procedures were obtained from Changchun Bode Biotechnology Co., Ltd.; the enterprise's standing measurement procedures and the user's routine measurement procedures were all performed using a TECAN F50 microplate reader and a neutrophil apolipoprotein (HNL) detection kit (enzyme-linked immunosorbent assay).

[0025] Example 1 Prepare leukocyte lysis buffer 1, leukocyte lysis buffer 2 and leukocyte lysis buffer 3 according to Tables 1-3 respectively.

[0026] Table 1. Formula of Leukocyte Lysis Buffer 1

[0027] Table 2 Formula of Leukocyte Lysis Buffer 2

[0028] Table 3. Formula of Leukocyte Lysis Buffer 3

[0029] Example 2 The hemolytic effect of leukocyte lysis buffer 1, leukocyte lysis buffer 2 and leukocyte lysis buffer 3 prepared in Example 1 was verified.

[0030] 2.1 Visual observation 100µl of whole blood sample from healthy individuals was taken and 400µl of leukocyte lysis buffer was added. The mixture was allowed to react for 5 min, and the hemolysis effect was observed visually. The mixture was then centrifuged at 10,000 rpm for 5 min, and the supernatant and precipitate were observed. The results are shown in Table 4.

[0031] Table 4. Hemolytic effects of leukocyte lysate 1, leukocyte lysate 2, and leukocyte lysate 3

[0032] 2.2 Verification of lysis effect using a blood cell analyzer 100 µl of whole blood sample was taken from a healthy person, and 400 µl of leukocyte lysis buffer was added. After incubation for 5 min, the number of cells in the lysed whole blood sample was determined using a blood cell analyzer (pre-dilution mode). The results are shown in Table 5.

[0033] Table 5 Hemolytic effects of leukocyte lysate 1, leukocyte lysate 2, and leukocyte lysate 3

[0034] 2.3 Determination of neutrophil apolipoprotein (HNL) in leukocyte lysate (immunochromatography) The supernatant obtained after centrifugation in step 2.1 was diluted 1000 times, and the immunochromatographic detection results are as follows: Table 6 Hemolytic effects of leukocyte lysate 1, leukocyte lysate 2, and leukocyte lysate 3

[0035] As can be seen from Tables 4-6, after 5 minutes of treatment, the cells in the leukocyte lysis buffer sample were completely dissolved and basically clear by visual observation; the number of leukocytes, erythrocytes, platelets and granulocytes decreased significantly; granulocytes were fully lysed and neutrophil apolipoproteins were successfully released into the leukocyte lysis buffer sample.

[0036] Example 3 Methods for preparing natural neutrophil apolipoprotein dimers include: S1. First, take 5 white blood cell filters for filtering out white blood cells from red blood cells, connect them to the peristaltic pump, introduce air to remove the red blood cells remaining in the white blood cell filters, until the white blood cell filters are full of air and no more red blood cells are discharged. S2, wash the leukocyte filter with 200 mL of 0.01 M PBS buffer (pH 7.2) and purge with air to remove the liquid from the leukocyte filter; S3. Connect the 5 white blood cell filters in series and wash the white blood cell filters with 0.01M PB buffer (pH 7.2) to lyse the residual red blood cells. Stop when the outflow is basically colorless and then introduce air to remove the liquid from the white blood cell filters. S4. Five leukocyte filters were lysed using 90 mL of pre-cooled leukocyte lysis buffer for 20 min. During the lysis process, the container containing the pre-cooled lysis buffer was placed in an ice-water mixture, and air was introduced to remove the liquid from the leukocyte filters. The lysate was collected and stored at -20°C for later use. S5. Wash the leukocyte filter with 90 mL of pre-cooled 0.01 M PBS buffer (pH 7.2), then purge the liquid from the leukocyte filter with air, collect the cells, and freeze at -20°C for later use. S6, combined with the liquids collected in S4 and S5, diluted 1000 times, and used for neutrophil apolipoprotein (HNL) assay (immunochromatography). The results are shown in Table 7.

[0037] Table 7. Concentration of neutrophil apolipoproteins in leukocyte lysates from Example 3

[0038] Comparative analysis of the results demonstrates that granulocytes were fully lysed and neutrophil apolipoproteins were successfully released into the leukocyte lysate.

[0039] Example 4 Preparation method of natural neutrophil apolipoprotein dimers: S1, In a graduated cylinder, mix the remaining leukocyte layer in the leukocyte component bag with PBS buffer containing dextran (3wt% dextran) at a volume ratio of 1:2. Let it stand for 20 min to allow the erythrocytes to settle. Transfer the resulting plasma containing granulocytes to a 50 mL centrifuge tube, centrifuge at 400×g for 10 min, remove the supernatant, resuspend the cells in 0.01M PBS buffer (pH 7.2), centrifuge at 400×g for 10 min, remove the supernatant, retain the granulocytes at the bottom of the centrifuge tube, suspend the granulocytes in the leukocyte lysis buffer sample, let it stand for 10 min, centrifuge at 10000×g for 10 min, remove the precipitate, and obtain the granulocyte lysis buffer for later use.

[0040] S2, the granulocyte lysate obtained in S1 was diluted 1000 times and neutrophil apolipoprotein (HNL) was measured (immunochromatographic method). The results are shown in Table 8.

[0041] Table 8. Concentration of neutrophil apolipoproteins in leukocyte lysates from Example 4

[0042] Comparative analysis of the results demonstrates that granulocytes were fully lysed and neutrophil apolipoproteins were successfully released into the leukocyte lysate.

[0043] Example 5 Neutrophil apolipoprotein (HNL) affinity chromatography purification: 5.1 Treatment of pyrolysis solution Granulocyte lysis buffer was transferred into a dialysis bag and dialyzed overnight at 4°C in 0.02M NaH2PO4, pH 8.0 PB buffer.

[0044] 5.2 Anion exchange column DE52 purification S1, remove the anion exchange column, let it stand at room temperature for 30 min, and equilibrate 5 column volumes with 0.02M NaH2PO4, pH 8.0 PB buffer.

[0045] S2, load the sample, elute with 2M NaCl solution, control the flow rate at 0.5~1ml / min, collect the anion-flowing solution to obtain the crude target protein.

[0046] 5.3 Purification of 4B-anti-HNL monoclonal antibody using cyanogen bromide-activated affinity chromatography column Preparation of S1, cyanogen bromide activated 4B-anti-HNL monoclonal antibody affinity chromatography column: Based on the volume of the pseudo-coupling packing material, add 5 mg of monoclonal antibody per 1 ml of packing material. Take an appropriate amount of monoclonal antibody and centrifuge it at 6000 rpm for 6-10 min using an ultrafiltration concentration tube to concentrate it to a protein concentration of 5-10 mg / mL. Then, add 3 times the volume of the concentration solution of 0.1 M NaHCO3 / 0.5 M NaCl, pH 8.5 coupling buffer to resuspend the antibody in the concentration tube, centrifuge it at 6000 rpm for 6-10 min, and concentrate it to a protein concentration of 5-10 mg / mL. Determine the protein content using ultraviolet spectrophotometry, with a concentration of 5-10 mg / mL.

[0047] The steps for preparing the monoclonal antibody are as follows: Accurately weigh 1g of cyanogen bromide-activated 4B resin powder into a 100mL beaker, add 50mL of pre-cooled 1mM HCl solution to swell the cyanogen bromide-activated 4B resin powder for 30min; remove the supernatant, and wash three times with 50mL of pre-cooled 1mM HCl solution to obtain 4-5mL of swollen gel. Wash with 50mL of water for injection, repeat twice, discard the water for injection, and then wash once with 5mL of 0.1M NaHCO3 / 0.5M NaCl, pH 8.5 coupling buffer to obtain the washed resin. Add the buffer containing anti-neutrophil apolipoprotein monoclonal antibody to the washed resin and mix at room temperature for 2h (or mix overnight at 2-8℃). Wash away unreacted monoclonal antibody with 5mL of 0.1M NaHCO3 / 0.5M NaCl, pH 8.5 coupling buffer, repeat twice, to construct the filler resin. Add 5 mL of 0.2 M glycine, pH 8.0, and mix at room temperature for 2 h (or mix overnight at 2–8 °C). Wash the constructed resin packing material with 25 mL of 0.1 M NaHCO3 / 0.5 M NaCl, pH 8.5 coupling buffer, and then wash with 25 mL of 0.1 M HAc / 0.5 M NaCl buffer, pH 4.0. Repeat the washing cycle 4 times.

[0048] If to be used immediately, equilibrate with 50 mL of 0.01 M PBS buffer (pH 7.2); otherwise, store for long-term preservation in a solution containing 0.01 M PBS buffer (pH 7.2) and 0.1% NaN3.

[0049] S2, remove the affinity chromatography column, let it stand at room temperature for 30 min, and equilibrate 5 column volumes with 0.02M NaH2PO4, pH 8.0 solution.

[0050] S3. Load the collected anion flow-through solution onto the column. After loading, equilibrate with 0.02M NaH2PO4 and PB buffer (pH 8.0) to obtain the affinity chromatography column flow-through solution.

[0051] S4. After equilibration, elute sequentially with pH 4.0 elution buffer, pH 3.5 elution buffer, pH 3.0 elution buffer, and pH 2.5 elution buffer, using glycine-hydrochloric acid buffer as the elution buffer, and collect the target protein.

[0052] S5. The target protein obtained by eluting with elution buffers of different pH values ​​was adjusted to neutral pH and the concentration was measured. The results are shown in Table 9.

[0053] Table 9. Concentration of neutrophil apolipoproteins in the samples obtained in Example 5

[0054] Step 6: SDS-PAGE electrophoresis analysis results Affinity chromatography collected samples, SDS-PAGE electrophoresis image as shown below Figure 1 As shown, by Figure 1 Analysis revealed that the protein obtained by elution with pH=3.0 was the target protein: natural neutrophil apolipoprotein dimer.

[0055] Example 6 Determination of neutrophil apolipoprotein (HNL) levels and preparation and traceability of enterprise product calibrators: 6.1 Determination of neutrophil apolipoprotein (HNL) levels 6.1.1 Take a small amount of natural neutrophil apolipoprotein dimer from Example 5 (eluted with pH=3.0 elution buffer), accurately dilute it 250-fold with the sample diluent in the test kit, and then dilute it at two gradients (50-fold and 100-fold dilution) as samples. Assign values ​​to them using the selected measurement program and reagents. The specific method is as follows: Select a clinical laboratory that uses the selected measurement program and reagents (TECAN F50 microplate reader, neutrophil apolipoprotein (HNL) test kit (ELISA)) to calibrate the selected measurement program and perform internal quality control. After all requirements are met, measure the serially diluted samples. Each sample is tested 4 times. Select two samples (defined as Sample1 and Sample2) whose mean values ​​fall in the middle of the linear range of the selected measurement program and test reagents, and repeat the test 5 times independently. Each sample is tested 3 times in each test. The results are shown in Table 10.

[0056] Table 10 Results of Repeated Experiments

[0057] Note: Formula for calculating the uncertainty of the absolute deviation assignment:

[0058] 6.1.2 The average value of 40 concentration values ​​obtained from 5 experiments at 2 concentration points, 0.1998 mg / mL, was taken as the concentration value of the calibrator concentrate (natural neutrophil apolipoprotein dimer from Example 5). Wherein, Represents uncertainty. Represents the number of tests. Representing the 1st to The results of this test .

[0059] 6.1.3 Calculate the uncertainty of antigen determination Its expanded uncertainty is ( k =2), U A The uncertainty is 0 for certified reference materials. Neutrophil apolipoprotein has no certified reference material. U B To assign uncertainty, U C This represents the system uncertainty (primarily the preparation uncertainty, typically defined as 0.05%). U 1 =0.2907%, therefore The concentration of the neutrophil apolipoprotein antigen concentrate was 0.1998 ± 0.0012 (mg / mL).

[0060] 6.2 Preparation and Traceability of Enterprise Product Calibrators 6.2.1 Dilute the stock solution of neutrophil apolipoprotein (with a concentration of 0.1998 mg / mL assigned by the selected measurement program) with detection buffer to prepare calibrators at various concentration points S1 (1000 ng / mL), S2 (500 ng / mL), S3 (250 ng / mL), S4 (125 ng / mL), S5 (63 ng / mL), and S6 (31 ng / mL) as calibrators for the company's products.

[0061] 6.2.2 Traceability of Enterprise Product Calibration Materials The company's product calibrators were tested using the company's standard measurement procedures. The curves of the company's working calibrators were used to read the calibrators. Each product calibrator was measured five times. The deviation of the fitted concentration was required to be within ±10%, and the coefficient of variation (CV) should not exceed 12.0%. The test results are shown in Table 11.

[0062] Table 11 Fitting Concentration Results of Calibrator Samples for Enterprise Products at Various Points

[0063] The deviations in the fitted concentrations of the company's product calibrators all meet the requirements, and the intra-batch variation at each point is no greater than 12.0%.

[0064] 6.2.3 Value setting of enterprise product calibrators The test was repeated twice using a detection system consisting of the company's working calibrator and the company's established measurement procedures. A curve was plotted using the company's working calibrator. Five replicates were performed for each test, and the company's product calibrator was tested a total of 10 times. The average value at each point was taken as the target value of the company's product calibrator. The results are shown in Table 12.

[0065] Table 12. Calibration Results of Enterprise Products Using Enterprise's Standard Measurement Procedures

[0066]

[0067]

[0068] 6.2.4 Verification of interchangeability of enterprise product calibrators A detection system consisting of the company's product calibrators and user-standard measurement procedures was used to test 25 clinical samples, covering the low, medium, and high values ​​within the kit's detection range. The results were compared with those from a detection system consisting of the company's working calibrators and standard measurement procedures. The linearity of the company's product calibrators was assessed based on the results, requiring a correlation coefficient greater than 0.975 and a slope of 1 ± 0.05. The test results are shown in Table 13.

[0069] Table 13 Interchangeability verification of enterprise product calibrators (results from 25 clinical samples)

[0070] The correlation between the detection results from the two systems described above was calculated using regression analysis. The correlation coefficient was obtained as R. 2 =0.967, the correlation curve is as follows Figure 2 As shown.

[0071] Depend on Figure 2 It can be seen that the correlation coefficient R between the two is... 2 A value exceeding 0.975 allows for analysis using linear regression, and the slope of the two test results is 0.966, indicating that their test results for the samples are very close. Calibration using the company's product calibrators makes the results of users' routine measurement procedures comparable to those of the company's established measurement procedures, thus achieving the transfer of measurement values.

[0072] Obviously, the above embodiments are merely examples for clear illustration and are not intended to limit the embodiments. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all embodiments here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. A method for preparing natural neutrophil apolipoprotein dimers, characterized in that, Includes the following steps: S1, the waste containing granulocytes generated during the component blood preparation process is first depleted of red blood cells to obtain granulocytes, and then the obtained granulocytes are lysed using leukocyte lysis buffer to obtain granulocyte lysis buffer; The leukocyte lysis buffer includes a surfactant, a protease inhibitor at a final concentration of 1 mM to 10 mM, a preservative at a final concentration of 0.01 wt% to 0.2 wt%, and a buffer solution. S2, the granulocyte lysate was subjected to anion exchange chromatography to obtain the crude target protein; S3, affinity chromatography was performed on the crude target protein to obtain natural neutrophil apolipoprotein dimers.

2. The method for preparing natural neutrophil apolipoprotein dimers according to claim 1, characterized in that, The waste containing granulocytes generated during the preparation of the component blood is a leukocyte layer. S1 includes: mixing the leukocyte layer with a buffer containing dextran, removing red blood cells by sedimentation, centrifuging the obtained plasma, removing the supernatant and resuspending it, centrifuging again to remove the supernatant, obtaining granulocytes, suspending the granulocytes in leukocyte lysis buffer, letting it stand and then centrifuging to remove the precipitate, obtaining granulocyte lysis buffer.

3. The method for preparing natural neutrophil apolipoprotein dimers according to claim 2, characterized in that, It possesses one or more of the following characteristics: The volume ratio of the leukocyte layer to the buffer solution containing dextran is 1:1 to 1:

5. The concentration of dextran in the buffer solution containing dextran is 1 wt% to 5 wt%. The buffer solution containing dextran uses 0.01M phosphate or 0.1M Tris-hydrochloric acid, with a pH of 6.5-7.5; The settling time is 20 minutes to 1 hour; The plasma was first centrifuged at 400×g for 10 min, the supernatant was removed, and the plasma was resuspended in PBS buffer. It was then centrifuged at 400×g for 10 min, the supernatant was removed, and granulocytes were obtained. The granulocytes were suspended in leukocyte lysis buffer and allowed to stand for more than 10 minutes before being centrifuged at 10000×g for 10 minutes.

4. The method for preparing natural neutrophil apolipoprotein dimers according to claim 1, characterized in that, The waste containing granulocytes generated during the preparation of the component blood is a leukocyte filter, S1 includes: S1-1, Take N white blood cell filters, fill them with air to remove residual red blood cells, N≥1; S1-2, wash N white blood cell filters with PBS buffer and purge with air to remove the liquid from the white blood cell filters; S1-3, process each white blood cell filter separately or process N white blood cell filters in series. First, wash N white blood cell filters with PB buffer and then introduce air to remove the liquid from the white blood cell filters. S1-4, then wash N white blood cell filters with pre-cooled white blood cell lysis buffer, lyse, after lysis, purge with air to remove the liquid in the white blood cell filters, and collect; S1-5, then wash N white blood cell filters with pre-cooled PBS buffer, purge air to remove liquid from the white blood cell filters, and collect; S1-6: Mix the liquids collected in S1-4 and S1-5 to obtain granulocyte lysis buffer.

5. The method for preparing natural neutrophil apolipoprotein dimers according to claim 4, characterized in that, It possesses one or more of the following characteristics: In S1-2, the concentration of PBS buffer is 0.01M and the pH is 7.2; In S1-3, the concentration of PB buffer is 0.01M and the pH is 7.2; In S1-4 and S1-5, the pre-cooling temperature is independently 2~8℃; In S1-4, each white blood cell filter is lysed for more than 4 minutes, or N white blood cell filters are processed in series for more than 20 minutes; In S1-4, the amount of leukocyte lysis buffer used is as follows: each leukocyte filter is treated separately, with 30 mL for each leukocyte filter; N leukocyte filters are connected in series, with 30 mL for each leukocyte filter, and an additional 15 mL of leukocyte lysis buffer is added for each additional leukocyte filter. In S1-5, the amount of PBS buffer used is as follows: each leukocyte filter is treated separately, with 30 mL for each leukocyte filter; N leukocyte filters are connected in series, with 30 mL for each leukocyte filter, and 15 mL of PBS buffer is added for each additional leukocyte filter.

6. The method for preparing natural neutrophil apolipoprotein dimers according to claim 1, characterized in that, S1 possesses one or more of the following characteristics: The surfactant is one or more of the following: sodium dodecyl sulfate (final concentration 0.05wt%~5wt%), dodecyltrimethylammonium chloride (final concentration 0.05wt%~5wt%), tetradecyltrimethylammonium chloride (final concentration 0.05wt%~5wt%), hexadecyltrimethylammonium bromide (final concentration 0.05wt%~5wt%), Tx-100 (final concentration 0.5wt%~2wt%), Tween 20 / 80 (final concentration 0.5wt%~2wt%), and NP40 (final concentration 0.5wt%~5wt%). The protease inhibitor is one or more of benzyl sulfonyl fluoride, aprotinin, and leupeptin; The preservative is one or more of sodium azide, Proclin 300, thimerosal, and isothiazolinone; The buffer solution is 0.01M phosphate or 0.1M Tris-hydrochloric acid, with a pH of 6.5-7.

5.

7. The method for preparing natural neutrophil apolipoprotein dimers according to claim 1, characterized in that, In S2, the granulocyte lysate after desalting or dialysis using G-25 is loaded into a DE-52 anion exchange column and equilibrated with 0.01-0.02M, pH 8.0 PB buffer at a volume of at least 4 times the column bed volume. The column is then eluted with a gradient of 2M NaCl solution at a flow rate of 0.5-1 ml / min. The flow-through is collected to obtain the crude target protein.

8. The method for preparing natural neutrophil apolipoprotein dimers according to claim 1, characterized in that, In S3, the crude target protein is loaded onto the affinity chromatography column and repeated once. After loading is stopped, the column is equilibrated with 0.01-0.02M PB buffer at pH 8.0, and then the target protein is eluted with a gradient elution buffer at pH 2.5-4.

0.

9. The method for preparing natural neutrophil apolipoprotein dimers according to claim 8, characterized in that, The target protein was collected using Tris-HCl buffer; The gradient elution buffer is a glycine-hydrochloric acid buffer or a citrate buffer. The affinity medium of the affinity chromatography column is cyanogen bromide activated agarose gel 4B immobilized with anti-neutrophil apolipoprotein monoclonal antibody.

10. Natural neutrophil apolipoprotein dimers prepared by the method for preparing natural neutrophil apolipoprotein dimers according to any one of claims 1 to 9.

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