A heparin-binding protein immune complex, its preparation method and application
Heparin-binding protein immune complexes were prepared by binding biotinylated heparin-binding protein with streptavidin immunomagnetic beads, which solved the problems of low titer and non-specific antibody production in the existing technology and achieved the preparation of high-titer and high-specificity heparin-binding protein antibodies.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- NANJING SANTA SCOTT BIOTECHNOLOGY CO LTD
- Filing Date
- 2025-05-27
- Publication Date
- 2026-08-04
AI Technical Summary
Existing technologies for preparing highly sensitive and high-affinity heparin-binding protein monoclonal antibodies suffer from insufficient titers when using Freund's adjuvant and water-soluble adjuvants, and the magnetic bead method exhibits poor binding performance to unlabeled antigens, resulting in reduced antibody purity and waste of magnetic beads.
An immune complex was formed by combining biotinylated heparin-binding protein with streptavidin immunomagnetic beads to immunize animals, which avoided the generation of non-specific antibodies by GST tags in vivo and improved antibody titers.
A higher titer heparin-binding protein antibody was obtained, which reduced the production of non-specific antibodies and improved the specificity and binding efficiency of the antibody.
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Figure CN120501853B_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of biomedicine, and in particular relates to a heparin-binding protein immune complex, its preparation method, and its application. Background Technology
[0002] Hybridoma technology was developed in 1975 by Georges. A revolutionary biotechnology invented by Cesar Milstein for the production of monoclonal antibodies (mAbs). This technology involves fusing B lymphocytes from immunized animals (such as mice) with myeloma cells (cancer cells) to form hybridoma cells, thereby obtaining cell lines that can proliferate indefinitely and continuously secrete a single specific antibody.
[0003] Hybridoma antibody technology comprises five steps: animal immunization, cell fusion, screening of hybridoma cells, clonal culture, and antibody production and purification. Its principle is as follows: Animal immunization: Experimental animals (usually mice) are injected with a target antigen, stimulating their immune system to produce specific B lymphocytes (plasma cells). These B cells can secrete antibodies against the antigen. Cell fusion: B cells are isolated from the spleen of the immunized animal and fused with myeloma cells (which have unlimited proliferative capacity but cannot secrete antibodies) using a fusion agent (such as polyethylene glycol PEG or electrofusion) to form hybridoma cells. Hybridoma cell screening: Hybridoma cells are cultured using a selective medium (such as HAT medium). Unfused B cells cannot survive long-term (natural death), and unfused myeloma cells die in HAT medium due to the lack of HGPRT enzyme (hypoxanthine-guanine phosphoribosyltransferase). Hybridoma cells, possessing both the antibody-secreting capacity of B cells and the unlimited proliferative capacity of myeloma cells, can survive in HAT medium. Clonal culture involves isolating and culturing individual hybridoma cells using limiting dilution or flow cytometry to form monoclonal cell lines, ensuring that all cells secrete the same antibody. Antibody production and purification follow by the expansion of hybridoma cells through in vitro culture or injection into the peritoneum of mice (ascites method). The culture medium or ascites fluid is then collected and purified to obtain high-purity monoclonal antibodies.
[0004] Animal immunization is a crucial step in hybridoma antibody technology, significantly impacting the sensitivity, affinity, and stability of hybridoma antibodies. Mice are typically chosen as experimental animals. Immunogens can be proteins, peptides, cells, viruses, etc., and their purity must be ensured (to avoid impurities interfering with the immune response). The immunization method usually involves emulsifying the immunogen with Freund's adjuvant to form a stable water-in-oil emulsion structure, followed by subcutaneous, intraperitoneal, or footpad injection. Alternatively, the immunogen may be mixed with a water-soluble adjuvant; in this case, emulsification is unnecessary, and the mixture can be directly injected subcutaneously, intraperitoneally, or into the footpad.
[0005] Heparin-binding protein (HBP) is mainly synthesized by various cells and is widely distributed in blood and tissues. HBP levels rise rapidly after infection and tissue damage, making it an important biomarker reflecting inflammatory responses. Heparin-binding protein plays a crucial role in the diagnosis of diseases such as sepsis, severe pneumonia, and acute appendicitis. In developing highly sensitive, high-affinity monoclonal antibodies against heparin-binding protein, we used Freund's adjuvant and water-soluble adjuvant immunization methods. However, we found that the antibody titer in mouse serum was not ideal, making it difficult to obtain antibodies with satisfactory performance, which is detrimental to the development of clinical diagnostic reagents.
[0006] Magnetic beads consist of two parts: a carrier microsphere and an immunoligand. The core component is iron(III) oxide (Fe3O4), coated with a superparamagnetic polymer. The outermost layer consists of functional groups (amino, carboxyl, hydroxyl, etc.). These functional groups are coupled to corresponding immunoligands (such as proteins and nucleic acids), and under the attraction of an external magnetic field, they can move directionally, thus achieving separation, detection, and purification. Currently, immunomagnetic beads are widely used in diagnostic reagents. Utilizing the antigen-antibody interaction principle combined with the properties of magnetic beads, they can more effectively, sensitively, and rapidly detect and separate specific pathogens. Magnetic beads can also be used as novel immunoadjuvants. Antibodies against GST-tagged proteins are coupled to the surface of modified magnetic beads to prepare immunomagnetic beads coated with GST antibodies. The GST antibodies on the magnetic beads then capture antigen fusion proteins carrying the GST tag. Finally, the antigen-magnetic bead complex is used to immunize experimental animals to obtain the corresponding antibodies. However, this method requires the antigen expression to carry a GST tag; it cannot bind to untagged antigens. Furthermore, GST tags and GST antibodies have high immunogenicity in animals, producing non-specific antibodies (antibodies against GST and antibodies against GST antibodies), leading to reduced antibody purity. One magnetic bead can only carry one antibody, and one antibody can bind to a maximum of two antigens, resulting in wasted magnetic beads and poor immunization efficacy. Summary of the Invention
[0007] This invention aims to provide a heparin-binding protein immune complex, its preparation method, and its application. Animal immunization with the heparin-binding protein immune complex prepared using this method yields heparin-binding protein antibodies with higher serum titers compared to Freund's adjuvant and water-soluble adjuvants. This method involves biotinylating heparin-binding protein and binding it to streptavidin immunomagnetic beads to form an immune complex, stimulating a stronger immune response, enhancing the immunogenicity of heparin-binding protein, and obtaining antibodies with higher titers. Furthermore, it avoids the generation of non-specific antibodies by the GST tag in animals. One streptavidin-bound streptavidin bead can bind four biotinylated heparin-binding proteins, resulting in higher antibody titers under the same number of beads.
[0008] To address the above problems, this invention provides a method for preparing heparin-binding protein immune complexes, comprising the following steps:
[0009] S1: Biotin was dissolved and added to the coating buffer, then heparin-binding protein was added and incubated at room temperature in the dark to obtain biotinylated heparin-binding protein.
[0010] S2: Place streptavidin immunomagnetic beads in a centrifuge tube and resuspend the beads in binding buffer;
[0011] S3: Biotinylated heparin-binding protein is added to streptavidin immunomagnetic beads, binding buffer is added, and incubation is performed to form heparin-binding protein immune complexes.
[0012] This invention utilizes biotinylated heparin-binding protein to bind with streptavidin immunomagnetic beads to form a heparin-binding protein immune complex, which is then injected subcutaneously at multiple points and into the paws of mice for immunization. After three immunizations, mouse serum with high antibody titers is obtained.
[0013] Preferably, in step S1, the biotin is dissolved in dimethyl sulfoxide to obtain a concentration of 3-7 mg / mL, and the volume ratio of biotin to coating buffer is 1:(80-120).
[0014] Preferably, in step S1, the concentration of heparin-binding protein is 0.5-1.5 mg / mL, the molar ratio of heparin-binding protein to biotin is 1:(5-15), and the incubation time at room temperature in the dark is 1-3 h.
[0015] Preferably, in step S1, the coating buffer is a 0.1M potassium phosphate buffer with a pH of 7.3-7.7.
[0016] Preferably, in step S2, the ratio of binding buffer to streptavidin immunomagnetic beads added in step S2 is 1 mL:(15-25 mg).
[0017] Preferably, in step S2, the binding buffer components are: PBS with pH 7.2-7.6, Tween-20 with a volume fraction of 0.03-0.07%, and BSA with a mass fraction of 0.05-0.15%.
[0018] Preferably, in step S3, the mass ratio of the streptavidin immunomagnetic beads to the biotinylated heparin-binding protein is (50-150):1.
[0019] Preferably, in step S3, the incubation temperature is 30-40°C and the incubation time is 30-60 minutes.
[0020] The present invention also provides a heparin-binding protein immune complex, which is obtained by the above preparation method.
[0021] The present invention also provides the application of the above-described heparin-binding protein immune complex in animal immunization.
[0022] Compared with the prior art, the beneficial effects of the present invention are reflected in:
[0023] 1. This invention involves biotinylating heparin-binding protein and binding it to streptavidin immunomagnetic beads to form a heparin-binding protein immune complex, thus changing the existing method of using tagged antibodies to modify magnetic beads and bind tagged antigens. The method of this invention does not require the antigen to carry a GST tag, reducing the difficulty of antigen production. It also avoids the generation of non-specific antibodies by GST tags and GST antibodies in immunized animals, increasing the specificity of heparin-binding protein antibodies.
[0024] 2. This invention uses streptavidin immunomagnetic beads to bind biotinylated heparin-binding proteins. One streptavidin immunomagnetic bead can bind four biotinylated heparin-binding proteins, which significantly improves the binding efficiency and can produce higher-titer antibodies in immunized animals. Compared with immunization using traditional Freund's adjuvant and water-soluble adjuvants, this method produces higher antibody titers. Attached Figure Description
[0025] Figure 1 This is a flowchart of a method for preparing a heparin-binding protein immune complex.
[0026] Figure 2 This is a schematic diagram illustrating the principle of a method for preparing a heparin-binding protein immune complex. Detailed Implementation
[0027] The following embodiments are only used to illustrate the technical solutions of the present invention more clearly, and should not be used to limit the scope of protection of the present invention.
[0028] The main compounds used in the examples and comparative examples were all commercially available products and were not subjected to any further purification treatment.
[0029] Example 1
[0030] A method for preparing heparin-binding protein immune complexes includes the following steps:
[0031] S1: Add 10 μL of 5 mg / mL biotin to 1 mL of coating buffer (0.1 M potassium phosphate buffer, pH 7.5) and vortex to mix; add 500 μL of 1 mg / mL heparin-binding protein (Suzhou Nearshore Protein Technology Co., Ltd., catalog number: DC430), incubate at room temperature in the dark for 2 h, vortexing every 15 min to obtain biotinylated heparin-binding protein;
[0032] S2: Take 5 mL of 10 mg / mL streptavidin immunomagnetic beads (Yisheng Biotechnology (Shanghai) Co., Ltd., catalog number: 47503ES), place them in a centrifuge tube, wash twice with 10 mL of binding buffer (PBS at pH 7.4, Tween-20 at a volume fraction of 0.05%, and BSA at a mass fraction of 0.1%), centrifuge, discard the supernatant, add 2.5 mL of binding buffer, and resuspend the magnetic beads in the binding buffer;
[0033] S3: Biotinylated heparin-binding protein is added to the streptavidin immunomagnetic beads described in S2, and binding buffer is added to a final volume of 10 mL. The mixture is incubated at 37°C for 40 min, with gentle shaking to prevent bead deposition, to form a heparin-binding protein immune complex for later use. The principle of heparin-binding protein binding by magnetic beads is as follows... Figure 2 As shown.
[0034] A method for preparing polyclonal antibodies from heparin-binding protein immune complexes includes the following steps:
[0035] One ml of the prepared heparin-binding protein immune complex was drawn into a syringe and injected subcutaneously at multiple sites in 6-8 week old Balb / c female mice. Four sites were evenly selected on the abdomen and one site was selected on the back. After the injection, another 1 ml of immunogen was drawn and injected into the paw pads of the mice. Five mice were immunized in each group and numbered 1-5. Each mouse was immunized with a total of 2 ml. This was the first immunization. Two weeks later, the second and third immunizations were performed using the same immunogen and the same immunization method as the first immunization. Serum was collected from the mice on the seventh day after the third immunization.
[0036] Example 2
[0037] A method for preparing heparin-binding protein immune complexes includes the following steps:
[0038] S1: Add 10 μL of 3 mg / mL biotin to 0.8 mL of coating buffer (0.1 M potassium phosphate buffer, pH 7.3) and vortex to mix; add 420 μL of 0.5 mg / mL heparin-binding protein (Suzhou Nearshore Protein Technology Co., Ltd., catalog number: DC430), incubate at room temperature in the dark for 1 h, vortexing every 15 min to obtain biotinylated heparin-binding protein;
[0039] S2: Take 3.15 mL of 10 mg / mL streptavidin immunomagnetic beads (Yisheng Biotechnology (Shanghai) Co., Ltd., catalog number: 47503ES), place them in a centrifuge tube, wash twice with 10 mL of binding buffer (PBS pH 7.2, Tween-20 volume fraction 0.03%, BSA mass fraction 0.05%), centrifuge, discard the supernatant, add 2.1 mL of binding buffer, and resuspend the magnetic beads in the binding buffer;
[0040] S3: Biotinylated heparin-binding protein is added to the streptavidin immunomagnetic beads described in S2, and binding buffer is added to a final volume of 10 mL. The mixture is incubated at 30°C for 30 min, with gentle shaking to prevent bead deposition, to form a heparin-binding protein immune complex for later use. The principle of heparin-binding protein binding by magnetic beads is as follows: Figure 2 As shown.
[0041] A method for preparing polyclonal antibodies from heparin-binding protein immune complexes includes the following steps:
[0042] One ml of the prepared heparin-binding protein immune complex was drawn into a syringe and injected subcutaneously at multiple sites in 6-8 week old Balb / c female mice. Four sites were evenly selected on the abdomen and one site was selected on the back. After the injection, another 1 ml of immunogen was drawn and injected into the paw pads of the mice. Five mice were immunized in each group and numbered 1-5. Each mouse was immunized with a total of 2 ml. This was the first immunization. Two weeks later, the second and third immunizations were performed using the same immunogen and the same immunization method as the first immunization. Serum was collected from the mice on the seventh day after the third immunization.
[0043] Example 3
[0044] A method for preparing heparin-binding protein immune complexes includes the following steps:
[0045] S1: Add 10 μL of 7 mg / mL biotin to 1.2 mL of coating buffer (0.1 M potassium phosphate buffer, pH 7.7) and vortex to mix; add 933 μL of 1.5 mg / mL heparin-binding protein (Suzhou Nearshore Protein Technology Co., Ltd., catalog number: DC430), incubate at room temperature in the dark for 3 h, vortexing every 15 min to obtain biotinylated heparin-binding protein;
[0046] S2: Take 7 mL of 10 mg / mL streptavidin immunomagnetic beads (Yisheng Biotechnology (Shanghai) Co., Ltd., catalog number: 47503ES), place them in a centrifuge tube, wash twice with 10 mL of binding buffer (PBS at pH 7.6, Tween-20 at a volume fraction of 0.07%, and BSA at a mass fraction of 0.15%), centrifuge, discard the supernatant, add 2.8 mL of binding buffer, and resuspend the magnetic beads in the binding buffer;
[0047] S3: Biotinylated heparin-binding protein is added to the streptavidin immunomagnetic beads described in S2, and binding buffer is added to a final volume of 10 mL. The mixture is incubated at 40°C for 60 min, with gentle shaking to prevent bead deposition, to form a heparin-binding protein immune complex for later use. The principle of heparin-binding protein binding by magnetic beads is as follows: Figure 2 As shown.
[0048] A method for preparing polyclonal antibodies from heparin-binding protein immune complexes includes the following steps:
[0049] One ml of the prepared heparin-binding protein immune complex was drawn into a syringe and injected subcutaneously at multiple sites in 6-8 week old Balb / c female mice. Four sites were evenly selected on the abdomen and one site was selected on the back. After the injection, another 1 ml of immunogen was drawn and injected into the paw pads of the mice. Five mice were immunized in each group and numbered 1-5. Each mouse was immunized with a total of 2 ml. This was the first immunization. Two weeks later, the second and third immunizations were performed using the same immunogen and the same immunization method as the first immunization. Serum was collected from the mice on the seventh day after the third immunization.
[0050] Comparative Example 1
[0051] A method for preparing polyclonal antibodies using heparin-binding protein in combination with Freund's adjuvant includes the following steps:
[0052] Take 500 μL of PBS solution containing heparin-binding protein (total 250 μg) into a 1.5 ml EP tube. Add an equal volume of Freund's adjuvant to the EP tube. Using a 2 ml syringe, draw up the mixture from the EP tube and emulsify it, repeatedly aspirating and retracting approximately 30-50 times until a stable emulsion is obtained (a drop of the emulsion forms a spherical shape without dispersing when dropped into water). This emulsion is to be used as the immunogen. Draw 1 ml of the prepared immunogen into a syringe and inject it subcutaneously at multiple sites in 6-8 week old B / c female mice. Select four sites evenly on the abdomen and one site on the back. After each injection, draw up another 1 ml of immunogen and inject it into the paw pads of the mice. Immune five mice per group, numbered 1-5. Each mouse receives a total of 2 ml of immunogen; this is the first immunization. Two weeks later, administer the second and third immunizations using the same immunogen and the same immunization method as the first immunization. Collect mouse serum on the seventh day after the third immunization.
[0053] Comparative Example 2
[0054] A method for preparing polyclonal antibodies using heparin-binding protein in combination with a water-soluble adjuvant includes the following steps:
[0055] Transfer 950 μL of PBS solution containing heparin-binding protein (total 250 μg) to a 1.5 mL EP tube. Add 50 μL of water-soluble adjuvant to the EP tube and vortex to mix. This will serve as the immunogen. Using a syringe, inject 1 mL of the prepared immunogen subcutaneously at multiple sites in 6-8 week old Balb / c female mice. Select four sites evenly on the abdomen and one site on the back. After each injection, inject another 1 mL of immunogen into the paw pads of the mice. Immune five mice per group, numbered 1-5. Each mouse receives a total of 2 mL of immunogen; this is the first immunization. Two weeks later, administer the second and third immunizations using the same immunogen and the same immunization method as the first immunization. Collect mouse serum on day seven after the third immunization.
[0056] Comparative Example 3
[0057] A method for preparing polyclonal antibodies by magnetically conjugating anti-GST-tagged antibodies with magnetic beads and then capturing HBP-GST fusion protein includes the following steps:
[0058] The amino acid sequence of the heparin-binding protein shown in SEQ ID NO: 1 was optimized to obtain the coding gene sequence of heparin-binding protein (HBP). This sequence was constructed into the multiple cloning site of the pGEX-4T-1 vector. The plasmid was transformed into E. coli BL21 and fermented in shake flasks on LB medium. Prokaryotic expression was induced by IPTG, and the expression product was purified to obtain the GST-HBP fusion protein. Commercially available carboxyl magnetic beads were washed with MES buffer (pH 6.0) to remove preservatives from the storage solution. The magnetic beads were separated using a magnetic rack, and the supernatant was discarded. The magnetic beads were resuspended in a buffer containing EDC / NHS and incubated at room temperature for 30 minutes to activate the carboxyl groups and form the NHS ester intermediate. Anti-GST tag antibody (buffered by PBS, pH 7.4) was prepared at a ratio of 10 μg antibody / mg magnetic beads. 5 mL of activated magnetic beads with a concentration of 5 mg / mL were mixed with the antibody and incubated at room temperature for 1 hour, with gentle shaking to prevent sedimentation. Wash twice with 10 mL binding buffer, and resuspend the magnetic beads in 2.5 mL binding buffer. Add 250 μL of 1 mg / mL GST-HBP fusion protein to the magnetic bead complex, and bring the binding buffer to 5 mL. Incubate at 37°C for 40 min, gently shaking to prevent sedimentation, to form heparin-binding protein immune complexes for later use. Using a syringe, draw 1 mL of the prepared immunogen and inject it subcutaneously at multiple sites in 6-8 week old Balb / c female mice. Select four sites evenly on the abdomen and one site on the back. After each injection, draw 1 mL of immunogen again and inject it into the paw pads of the mice. Five mice per group are immunized, numbered 1-5, with a total of 2 mL immunized per mouse; this is the first immunization. Two weeks later, administer the second and third immunizations using the same immunogen and the same immunization method as the first immunization. Collect mouse serum on the seventh day after the third immunization.
[0059] Experimental Example 1
[0060] The method for detecting serum heparin-binding protein antibody titer includes the following steps:
[0061] (1) Design the number of enzyme-labeled plates to be coated according to the experimental needs, and mark the plates.
[0062] (2) Dilute heparin-binding protein to a concentration of 0.5ug / ml with PBS coating solution, mix well and add to the enzyme-labeled strip, 100ul per well, and incubate overnight at 4°C.
[0063] (3) After coating, discard the coating solution, wash the plate once, add 200 μL of blocking solution (1% casein) to each well, incubate at 37°C for 2 hours, remove the microplate, discard the inner solution, and pat dry for later use.
[0064] (4) The mouse serum of Examples 1-3 and Comparative Examples 1-3 were diluted 1:100 to form the first well, and the remaining wells were serially diluted 5 times in sequence, with 11 gradients, 100 μL per well, and incubated in a constant temperature incubator at 37°C for 40 min.
[0065] (5) Remove the microplate, discard the internal liquid, wash the plate 4 times, add 100 μL of diluted enzyme-labeled secondary antibody to each well. The enzyme-labeled secondary antibody is goat anti-mouse-HRP with a working concentration of 1:5000. Incubate at 37°C for 40 min.
[0066] (6) Remove the microplate, discard the inner liquid, wash the plate 4 times, add 100ul TMB colorimetric solution to each well, and let it stand at room temperature for 5min.
[0067] (7) Add 50 μL of stop solution (dilute sulfuric acid) to each well to stop the reaction. Immediately take a reading at 450 nm on the microplate reader. The dilution corresponding to the well with an OD value greater than 2.1 times the set negative control OD value is defined as the titer of mouse serum heparin-binding protein antibody.
[0068] The serum heparin-binding protein antibody titer test results for the examples and comparative examples are shown in Tables 1-6 below.
[0069] Table 1: Serum Heparin-Binding Protein Antibody Titer Detection Results in Example 1
[0070]
[0071] Table 2: Serum Heparin-Binding Protein Antibody Titer Detection Results in Example 2
[0072]
[0073]
[0074] Table 3: Serum Heparin-Binding Protein Antibody Titer Detection Results in Example 3
[0075]
[0076] Table 4: Serum titer test results of Comparative Example 1
[0077]
[0078] Table 5: Serum titer test results of Comparative Example 2
[0079]
[0080]
[0081] Table 6: Serum titer test results of Comparative Example 3
[0082]
[0083] As shown in Tables 1-6, in Examples 1-3, heparin-binding protein immune complexes formed by biotinylated heparin-binding protein and streptavidin immunomagnetic beads were used to immunize experimental animals. The serum heparin-binding protein antibody titers of the prepared mice were all 1:1562500. In Comparative Example 1 (heparin-binding protein and Freund's adjuvant were mixed in equal volumes and then emulsified), the serum titers of the 5 mice were all 1:62500. In Comparative Example 2 (heparin-binding protein was mixed with water-soluble adjuvant), the serum titers of the other 4 mice were all 1:62500, except for mouse number 3 which had a serum titer of 1:12500. In Comparative Example 3, magnetic beads were used to conjugate anti-GST-tagged antibodies, and then HBP-GST fusion protein was captured. The serum heparin-binding protein antibody titers of the 5 immunized mice were all 1:312500. This method can obtain high-titer heparin-binding protein antibodies, providing a method for producing heparin-binding protein monoclonal antibodies using hybridoma cells and providing high-quality raw materials for the development of clinical diagnostic reagents.
[0084] Experimental Example 2
[0085] The method for detecting nonspecific antibody titers includes the following steps:
[0086] (1) Design the number of enzyme-labeled plates to be coated according to the experimental needs, and mark the plates.
[0087] (2) Dilute the GST antibody / GST protein to a concentration of 0.5 μg / ml with PBS coating solution, mix well and add to the enzyme-labeled strip, 100 μl per well, and incubate overnight at 4°C.
[0088] (3) After coating, discard the coating solution, wash the plate once, add 200 μl of blocking solution (1% casein) to each well, incubate at 37℃ for 2 h, remove the microplate, discard the inner solution, and pat dry for later use.
[0089] (4) Dilute the serum of Comparative Example 3 mice at a ratio of 1:100 to the first well, and then dilute the remaining wells 5 times sequentially, for 11 gradients, 100 μL per well, and incubate at 37°C for 40 min.
[0090] (5) Remove the microplate, discard the internal liquid, wash the plate 4 times, add 100 μL of diluted enzyme-labeled secondary antibody to each well. The enzyme-labeled secondary antibody is goat anti-mouse-HRP with a working concentration of 1:5000. Incubate at 37°C for 40 min.
[0091] (6) Remove the microplate, discard the inner liquid, wash the plate 4 times, add 100ul TMB colorimetric solution to each well, and let it stand at room temperature for 5min.
[0092] (7) Add 50 μL of stop solution (dilute sulfuric acid) to each well to stop the reaction. Immediately take a reading at 450 nm on the microplate reader. The dilution corresponding to the well with an OD value greater than 2.1 times the set negative control OD value is defined as the titer of nonspecific antibodies in mouse serum.
[0093] The results of nonspecific antibody titer detection in serum of Comparative Example 3 are shown in Table 7-8 below:
[0094] Table 7: Antibody titer results of serum GST antibodies in Comparative Example 3
[0095]
[0096] Table 8: Results of serum GST antibody titer detection in Comparative Example 3
[0097]
[0098] As shown in Table 7-8, the antibody titer of GST antibody in Comparative Example 3 was 1:1562500, and the antibody titer of GST was 1:312500. This indicates that the method in Comparative Example 3, in addition to producing HBP antibody, also produces GST antibody and GST antibody antibody, which to some extent affects the titer and specificity of HBP antibody in serum.
[0099] Experimental Example 3
[0100] The method for detecting nonspecific antibody titers includes the following steps:
[0101] (1) Design the number of enzyme-labeled plates to be coated according to the experimental needs, and mark the plates.
[0102] (2) Dilute SA protein to a concentration of 0.5 μg / ml with PBS coating solution, mix well and add to the enzyme-labeled strips, 100 μl per well, and incubate overnight at 4°C.
[0103] (3) After coating, discard the coating solution, wash the plate once, add 200 μl of blocking solution (1% casein) to each well, incubate at 37℃ for 2 h, remove the microplate, discard the inner solution, and pat dry for later use.
[0104] (4) Dilute the mouse serum from Example 1 at a ratio of 1:100 to the first well, and then dilute the remaining wells 5 times sequentially, for 11 gradients, 100 μL per well, and incubate at 37°C for 40 min.
[0105] (5) Remove the microplate, discard the internal liquid, wash the plate 4 times, add 100 μL of diluted enzyme-labeled secondary antibody to each well. The enzyme-labeled secondary antibody is goat anti-mouse-HRP with a working concentration of 1:5000. Incubate at 37°C for 40 min.
[0106] (6) Remove the microplate, discard the inner liquid, wash the plate 4 times, add 100ul TMB colorimetric solution to each well, and let it stand at room temperature for 5min.
[0107] (7) Add 50 μL of stop solution (dilute sulfuric acid) to each well to stop the reaction. Immediately take a reading at 450 nm on the microplate reader. The dilution corresponding to the well with an OD value greater than 2.1 times the set negative control OD value is defined as the titer of nonspecific antibodies in mouse serum.
[0108] The results of the detection of non-specific antibody titers in serum in Example 1 are shown in Table 9 below.
[0109] Table 9: Serum GST Antibody Titer Detection Results in Example 1
[0110]
[0111] As shown in Table 9, the antibody titer of the SA antibody produced in Example 1 was 1:500. After biotinylation of heparin-binding protein, it was combined with streptavidin immunomagnetic beads to form a heparin-binding protein immune complex. The amount of non-specific antibody produced by animal immunization with this complex was low, far lower than that of the method of using magnetic beads to conjugate anti-GST tag antibody and then capturing HBP-GST fusion protein.
[0112] The method of biotinylating heparin-binding protein and conjugating it with streptavidin immunomagnetic beads produces higher-titer antibodies compared to Freund's adjuvant and water-soluble adjuvants. Compared to the method of conjugating anti-GST-tagged antibodies with magnetic beads and then capturing HBP-GST fusion protein, it produces higher-titer HBP antibodies and lower non-specific antibody levels. The antibodies prepared using the method described in this application have advantages such as high titer and high specificity, providing a method for obtaining high-quality polyclonal and monoclonal antibodies against heparin-binding protein, and providing high-quality raw materials for the development of diagnostic reagents.
[0113] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the technical principles of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.
Claims
1. A method for preparing a heparin-binding protein immune complex, characterized in that, Includes the following steps: S1: Biotin was dissolved and added to the coating buffer, then heparin-binding protein was added and incubated at room temperature in the dark to obtain biotinylated heparin-binding protein. S2: Place streptavidin immunomagnetic beads in a centrifuge tube and resuspend the beads in binding buffer; S3: Biotinylated heparin-binding protein was added to streptavidin immunomagnetic beads, binding buffer was added, and incubation was carried out to form heparin-binding protein immune complexes. In S1, the biotin is dissolved in dimethyl sulfoxide to obtain a concentration of 3-7 mg / mL, and the volume ratio of biotin to coating buffer is 1:(80-120); the concentration of heparin-binding protein is 0.5-1.5 mg / mL, and the molar ratio of heparin-binding protein to biotin is 1:(5-15); the incubation time at room temperature in the dark is 1-3 h. In S2, the ratio of the binding buffer to streptavidin immunomagnetic beads is 1 mL: (15-25 mg). In S3, the mass ratio of the streptavidin immunomagnetic beads to the biotinylated heparin-binding protein is (50-150):
1.
2. The method for preparing heparin-binding protein immune complexes according to claim 1, characterized in that, In S1, the coating buffer is 0.1M potassium phosphate buffer with a pH of 7.3-7.
7.
3. The method for preparing heparin-binding protein immune complexes according to claim 1, characterized in that, In S2, the binding buffer components are: PBS with a pH of 7.2-7.6, Tween-20 with a volume fraction of 0.03-0.07%, and BSA with a mass fraction of 0.05-0.15%.
4. The method for preparing heparin-binding protein immune complexes according to claim 1, characterized in that, In S3, the incubation temperature is 30-40℃ and the time is 30-60min.
5. A heparin-binding protein immune complex, characterized in that, It is obtained by the preparation method described in any one of claims 1-4.
6. The use of the heparin-binding protein immune complex according to claim 5 in the preparation of polyclonal antibodies.