Heparin binding protein immune complex as well as preparation method and application thereof

Abstract

The invention relates to the field of biological medicine, and discloses a heparin-binding protein immune complex and a preparation method and application thereof, and the preparation method of the heparin-binding protein immune complex comprises the following steps: biotinylating heparin-binding protein, and then combining the biotinylated heparin-binding protein with streptavidin immunomagnetic beads to form the immune complex to stimulate an organism to generate stronger immune response. Compared with the traditional Freund's adjuvant and water-soluble adjuvant immunization, the coupled heparin-binding protein magnetic beads can enable the immunized mouse to generate a heparin-binding protein antibody with higher titer, and the output of a non-specific antibody is lower. More possibilities are provided for developing and obtaining the heparin binding protein monoclonal antibody with high sensitivity and high affinity, and a high-quality raw material is provided for developing a clinical diagnostic reagent.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and in particular relates to a heparin-binding protein immune complex and a preparation method and application thereof. Background Art

[0002] Hybridoma technology was invented by Georges A revolutionary biotechnology invented by Dr. Robert H. Miller and Cesar Milstein for the production of monoclonal antibodies (mAbs). This technology fuses B lymphocytes from immune animals (such as mice) with myeloma cells (cancer cells) to form hybridoma cells, thereby obtaining a cell line that can proliferate indefinitely and continuously secrete a single specific antibody.

[0003] Hybridoma antibody technology involves five steps: animal immunization, cell fusion, hybridoma cell screening, cloning and culture, and antibody production and purification. The principle is as follows: Immunization involves injecting a target antigen into a laboratory animal (usually a mouse), stimulating its immune system to produce specific B lymphocytes (plasma cells), which secrete antibodies against the antigen. Cell fusion involves isolating B cells from the spleen of the immunized animal and fusing them with myeloma cells (which have the capacity to proliferate indefinitely but cannot secrete antibodies) using a fusing agent (such as polyethylene glycol or electrofusion) to form hybridoma cells. Hybridomas are screened and cultured in a selective medium (such as HAT medium). Unfused B cells cannot survive long-term (natural death). Unfused myeloma cells, lacking the enzyme HGPRT (hypoxanthine-guanine phosphoribosyltransferase), die in HAT medium. Hybridoma cells, however, possess both the antibody-secreting capacity of B cells and the indefinite proliferative capacity of myeloma cells and can survive in HAT medium. Clonal culture involves isolating and culturing individual hybridoma cells through limiting dilution or flow cytometry to form a monoclonal cell line, ensuring that all cells secrete the same antibody. For antibody production and purification, hybridoma cells can be cultured in vitro or injected into the peritoneal cavity of mice (ascites method) to proliferate in large quantities. The culture fluid or ascites fluid is then collected and purified to obtain high-purity monoclonal antibodies.

[0004] Animal immunization, a key step in hybridoma antibody technology, has a crucial impact on the sensitivity, affinity, and stability of hybridoma antibodies. Mice are typically used as experimental animals. Immunogens can include proteins, peptides, cells, viruses, and other materials, and purity must be guaranteed to prevent impurities from interfering with the immune response. Immunization is typically performed by emulsifying the immunogen with Freund's adjuvant to form a stable oil-in-water structure, followed by subcutaneous, intraperitoneal, or footpad injection. Alternatively, the immunogen may be mixed with a water-soluble adjuvant; these adjuvants do not require emulsification and can be directly injected subcutaneously, intraperitoneally, or in the footpad.

[0005] Heparin-binding protein (HBP) is primarily synthesized by various cells and is widely present in blood and tissues. Following infection and tissue damage, HBP levels rapidly increase, making it a key marker of inflammatory responses. HBP plays a crucial role in the diagnosis of conditions such as sepsis, severe pneumonia, and acute appendicitis. While developing highly sensitive, high-affinity monoclonal antibodies against HBP using Freund's adjuvant and water-soluble adjuvant immunization methods, we discovered that mouse serum antibody titers were suboptimal, making it difficult to obtain antibodies with optimal performance, hindering the development of clinical diagnostic reagents.

[0006] Magnetic beads consist of two parts: carrier microspheres and immune ligands. Their core component is ferroferric oxide, and the surface is covered with a layer of superparamagnetic polymer. The outermost layer is functional groups (amino, carboxyl, hydroxyl, etc.). The functional groups on the surface are coupled with corresponding immune ligands (such as proteins and nucleic acids), and they can move in a directional manner under the attraction of an external magnetic field, thereby achieving the purposes of separation, detection, and purification. Currently, immunomagnetic beads are widely used in detection reagents. Utilizing the principle of antigen-antibody interaction and combining the characteristics of magnetic beads, they can more effectively, sensitively, and quickly detect and separate specific pathogens. Magnetic beads can also be used as a new type of immune adjuvant. Antibodies against GST-tagged proteins are coupled to the modified magnetic bead surface to produce immunomagnetic beads coated with GST antibodies. The GST antibodies on the magnetic beads are then used to capture the antigen fusion protein 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 that the antigen be expressed with a GST tag, and cannot bind to untagged antigens. Furthermore, the GST tag and GST antibodies are highly immunogenic in animals, producing nonspecific antibodies (antibodies to GST and antibodies to GST antibodies), which reduces the purity of the antibodies. 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 immune response. Summary of the Invention

[0007] The present invention aims to provide a heparin-binding protein immune complex, a preparation method and application thereof. When the heparin-binding protein immune complex prepared by the method is used for animal immunization, the obtained heparin-binding protein antibody has a higher serum titer than Freund's adjuvant and water-soluble adjuvant. The method forms an immune complex by biotinylating the heparin-binding protein and then combining it with streptavidin immunomagnetic beads, thereby stimulating the body to produce a stronger immune response, enhancing the immunogenicity of the heparin-binding protein, and obtaining a higher titer antibody. The method also avoids the generation of non-specific antibodies in the animal body by the GST tag. One streptavidin bound to the streptavidin immunomagnetic bead can bind to four biotinylated heparin-binding proteins, and can produce a higher titer antibody under the condition of the same number of magnetic beads.

[0008] In order to solve the above problems, the present invention provides a method for preparing a heparin-binding protein immune complex, comprising the following steps:

[0009] S1: After biotin is dissolved, it is added to the coating buffer, and heparin-binding protein is added. Incubate 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: Add biotinylated heparin-binding protein to streptavidin immunomagnetic beads, add binding buffer, and incubate to form heparin-binding protein immune complexes.

[0012] The invention combines biotinylated heparin binding protein with streptavidin immunomagnetic beads to form a heparin binding protein immune complex, which is then injected subcutaneously at multiple points and in the soles of the feet into mice for immunization. After three immunizations, mouse serum with high antibody titer is obtained.

[0013] Preferably, in S1, the biotin is dissolved in dimethyl sulfoxide to obtain a concentration of 3-7 mg / mL biotin, and the volume ratio of the biotin to the coating buffer is 1:(80-120).

[0014] Preferably, in S1, the concentration of the heparin-binding protein is 0.5-1.5 mg / mL, the molar ratio of the 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 S1, the coating buffer is 0.1 M potassium phosphate buffer with a pH of 7.3-7.7.

[0016] Preferably, in S2, the ratio of binding buffer to streptavidin immunomagnetic beads added to S2 is 1 mL: (15-25 mg).

[0017] Preferably, in S2, the binding buffer comprises: 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%.

[0018] Preferably, in S3, the mass ratio of the streptavidin immunomagnetic beads to the biotinylated heparin-binding protein is (50-150):1.

[0019] Preferably, in S3, the incubation temperature is 30-40° C. and the incubation time is 30-60 min.

[0020] The present invention also provides a heparin-binding protein immune complex, which is obtained by the above-mentioned preparation method.

[0021] The present invention also provides the use of the heparin-binding protein immune complex in animal immunization.

[0022] Compared with the prior art, the beneficial effects of the present invention are embodied in:

[0023] 1. The present invention biotinylates a heparin-binding protein and binds it to streptavidin immunomagnetic beads to form a heparin-binding protein immune complex. This method changes the existing art method of using labeled antibody-modified magnetic beads to bind to tagged antigens. The present method does not require the antigen to carry a GST tag, which reduces the difficulty of antigen production. It also avoids the production of nonspecific antibodies caused by the GST tag and GST antibody in the immunized animal, thereby increasing the specificity of the heparin-binding protein antibody.

[0024] 2. The present invention uses streptavidin immunomagnetic beads to bind to biotinylated heparin-binding proteins. One streptavidin immunomagnetic bead can bind to four biotinylated heparin-binding proteins, significantly improving the binding efficiency and producing higher titer antibodies in the immunized animal. Compared with immunization with traditional Freund's adjuvant and water-soluble adjuvant, this method produces higher antibody titers. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 The present invention is a flow chart of a method for preparing a heparin-binding protein immune complex.

[0026] Figure 2 Schematic diagram of the principle of a method for preparing a heparin-binding protein immune complex. DETAILED DESCRIPTION

[0027] The following examples are only used to more clearly illustrate the technical solutions of the present invention and are not intended 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] The method for preparing a heparin-binding protein immune complex comprises 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 thoroughly. Add 500 μL of 1 mg / mL heparin-binding protein (Suzhou Nearshore Protein Technology Co., Ltd., Cat. No. DC430) and incubate at room temperature in the dark for 2 h. Vortex to mix thoroughly 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., Cat. No.: 47503ES) and place them in a centrifuge tube. Wash twice with 10 mL of binding buffer (PBS, pH 7.4, 0.05% Tween-20, 0.1% BSA), centrifuge, remove the supernatant, add 2.5 mL of binding buffer, and resuspend the beads in binding buffer.

[0033] S3: Add the biotinylated heparin binding protein to the streptavidin immunomagnetic beads described in S2, add the binding buffer to 10 mL, incubate at 37°C for 40 min, and shake gently to avoid magnetic beads sedimentation to form heparin binding protein immune complexes for use. Figure 2 shown.

[0034] The method for preparing polyclonal antibodies from heparin-binding protein immune complexes comprises the following steps:

[0035] Use a syringe to draw up 1 ml of the prepared heparin-binding protein immune complex and inject it subcutaneously into 6-8 week old female Balb / c mice at multiple sites, evenly spaced on the abdomen and one on the back. After the injections, draw up another 1 ml of the immunogen and inject it into the footpad of the mouse. Immunize five mice per group, numbered 1-5, with a total of 2 ml per mouse. This constitutes the first immunization. Two weeks later, perform the second and third immunizations using the same immunogen and immunization method, following the same protocol as the first. Collect serum from mice seven days after the third immunization.

[0036] Example 2

[0037] The method for preparing a heparin-binding protein immune complex comprises 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 thoroughly. Add 420 μL of 0.5 mg / mL heparin-binding protein (Suzhou Nearshore Protein Technology Co., Ltd., Cat. No. DC430) and incubate at room temperature in the dark for 1 hour. Vortex to mix thoroughly every 15 minutes to obtain biotinylated heparin-binding protein.

[0039] S2: 3.15 mL of 10 mg / mL streptavidin immunomagnetic beads (Yisheng Biotechnology (Shanghai) Co., Ltd., Cat. No. 47503ES) were placed in a centrifuge tube. The beads were washed twice with 10 mL of binding buffer (PBS, pH 7.2, 0.03% Tween-20, 0.05% BSA), centrifuged, and the supernatant was removed. 2.1 mL of binding buffer was added and the beads were resuspended in binding buffer.

[0040] S3: Add the biotinylated heparin binding protein to the streptavidin immunomagnetic beads described in S2, add the binding buffer to 10 mL, incubate at 30°C for 30 minutes, and shake gently to avoid magnetic beads sedimentation to form a heparin binding protein immune complex for use. Figure 2 shown.

[0041] The method for preparing polyclonal antibodies from heparin-binding protein immune complexes comprises the following steps:

[0042] Use a syringe to draw up 1 ml of the prepared heparin-binding protein immune complex and inject it subcutaneously into 6-8 week old female Balb / c mice at multiple sites, evenly spaced on the abdomen and one on the back. After the injections, draw up another 1 ml of the immunogen and inject it into the footpad of the mouse. Immunize five mice per group, numbered 1-5, with a total of 2 ml per mouse. This constitutes the first immunization. Two weeks later, perform the second and third immunizations using the same immunogen and immunization method, following the same protocol as the first. Collect serum from mice seven days after the third immunization.

[0043] Example 3

[0044] The method for preparing a heparin-binding protein immune complex comprises 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., Cat. No. DC430) and incubate at room temperature in the dark for 3 h. Vortex to mix 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., Cat. No.: 47503ES) and place them in a centrifuge tube. Wash twice with 10 mL of binding buffer (PBS, pH 7.6, 0.07% Tween-20, 0.15% BSA), centrifuge, remove the supernatant, add 2.8 mL of binding buffer, and resuspend the beads in binding buffer.

[0047] S3: Add the biotinylated heparin binding protein to the streptavidin immunomagnetic beads described in S2, add the binding buffer to 10 mL, incubate at 40°C for 60 min, and shake gently to avoid magnetic bead sedimentation to form a heparin binding protein immune complex for use. Figure 2 shown.

[0048] The method for preparing polyclonal antibodies from heparin-binding protein immune complexes comprises the following steps:

[0049] Use a syringe to draw up 1 ml of the prepared heparin-binding protein immune complex and inject it subcutaneously into 6-8 week old female Balb / c mice at multiple sites, evenly spaced on the abdomen and one on the back. After the injections, draw up another 1 ml of the immunogen and inject it into the footpad of the mouse. Immunize five mice per group, numbered 1-5, with a total of 2 ml per mouse. This constitutes the first immunization. Two weeks later, perform the second and third immunizations using the same immunogen and immunization method, following the same protocol as the first. Collect serum from mice seven days after the third immunization.

[0050] Comparative Example 1

[0051] A method for preparing polyclonal antibodies by combining heparin-binding protein with Freund's adjuvant comprises the following steps:

[0052] Transfer 500 μl of a PBS solution containing heparin-binding protein (250 μg total) to a 1.5 ml EP tube. Add an equal volume of Freund's adjuvant to the EP tube. Use a 2 ml syringe to draw the mixture from the EP tube for emulsification. Repeat the pipetting and vortexing for approximately 30-50 times until a stable emulsion is obtained (a drop of the emulsion will form a sphere when dropped into water without dispersing). This will be used as the immunogen. Use a syringe to draw 1 ml of the prepared immunogen and inject it subcutaneously into 6-8 week old B / c female mice at multiple sites, selecting four evenly spaced sites on the abdomen and one site on the back. After the injections are complete, draw another 1 ml of the immunogen and inject it into the footpad of the mouse. Immunize five mice per group, numbered 1-5, for a total of 2 ml per mouse. This constitutes the first immunization. Two weeks later, perform the second and third immunizations using the same immunogen and immunization method, respectively, following the same protocol as the first. Collect mouse serum seven days after the third immunization.

[0053] Comparative Example 2

[0054] The method for preparing polyclonal antibodies by combining heparin-binding protein with a water-soluble adjuvant comprises the following steps:

[0055] Take 950ul of PBS solution containing heparin binding protein (total amount 250μg) to a 1.5ml EP tube, take another 50ul of water-soluble adjuvant and add it to the EP tube, vortex mix, and use as an immunogen. Use a syringe to draw 1ml of the prepared immunogen and inject it subcutaneously at multiple points in 6-8 week old Balb / c female mice. Select 4 points evenly on the abdomen and 1 point on the back. After the injection is completed, draw 1ml of immunogen again for injection into the mouse foot pad. Each group immunizes 5 mice and numbers them 1-5. Each mouse is immunized with a total of 2ml. This is the first immunization. Thereafter, the second and third immunizations are carried out with the same immunogen and the same immunization method at intervals of 2 weeks. The immunization method is the same as the first immunization. Collect mouse serum on the seventh day after the third immunization.

[0056] Comparative Example 3

[0057] The method for preparing polyclonal antibodies by coupling magnetic beads with anti-GST tag antibodies and then capturing HBP-GST fusion protein comprises the following steps:

[0058] The amino acid sequence of the heparin-binding protein shown in SEQ ID NO: 1 was optimized to obtain the gene encoding the heparin-binding protein (HBP). This sequence was then inserted into the multiple cloning site of the pGEX-4T-1 vector. The plasmid was transformed into Escherichia coli BL21 and fermented in shake flasks using LB medium. Prokaryotic expression was induced with IPTG, and the expression product was purified to obtain the GST-HBP fusion protein. Commercially available carboxyl-coated magnetic beads were washed with MES buffer (pH 6.0) to remove the preservative from the storage solution. The beads were separated using a magnetic stand, and the supernatant discarded. The beads were resuspended in a buffer containing EDC / NHS and incubated at room temperature for 30 minutes to activate the carboxyl groups to form an NHS ester intermediate. Anti-GST tag antibody was prepared in PBS (pH 7.4) at a ratio of 10 μg antibody / mg beads. 5 mL of activated magnetic beads at a concentration of 5 mg / mL were mixed with the antibody and incubated at room temperature for 1 hour with gentle shaking to prevent beads from settling. Wash the beads twice with 10 mL of binding buffer and resuspend them in 2.5 mL of binding buffer. Add 250 μL of 1 mg / mL GST-HBP fusion protein to the bead complex and bring the volume up to 5 mL with binding buffer. Incubate at 37°C for 40 minutes with gentle shaking to prevent beads from settling to form a heparin-binding protein immune complex for later use. Use a syringe to draw up 1 mL of the prepared immunogen and inject it subcutaneously into 6-8 week-old Balb / c female mice at four evenly spaced sites on the abdomen and one on the back. After each injection, a fresh 1 mL of immunogen should be injected into the footpads of the mice. Five mice per group were immunized, numbered 1-5, with a total of 2 mL injected per mouse. This constituted the first immunization. Two weeks later, the second and third immunizations were performed using the same immunogen and immunization protocol, respectively, following the same protocol as the first. Serum was collected seven days after the third immunization.

[0059] Experimental Example 1

[0060] The method for detecting the titer of serum heparin-binding protein antibodies comprises the following steps:

[0061] (1) Design the number of ELISA plates to be coated according to the experimental needs and mark them on the strips.

[0062] (2) Dilute the heparin-binding protein to a concentration of 0.5 μg / ml using PBS coating solution, mix well, and add to the enzyme-labeled plate, 100 μl per well, and store in a refrigerator at 4°C overnight.

[0063] (3) After coating, discard the coating solution, wash the plate once, add 200 μl of blocking solution (1% casein) to each well, incubate in a 37°C incubator for 2 h, remove the ELISA plate, discard the inner solution, and pat dry for later use.

[0064] (4) The mouse sera of Examples 1-3 and Comparative Examples 1-3 were diluted 1:100 for the first well, and the remaining wells were serially diluted 5-fold in 11 gradients, with 100ul per well, and incubated in a 37°C incubator for 40 minutes.

[0065] (5) Remove the ELISA plate, discard the internal solution, wash the plate 4 times, add 100ul 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, and incubate at 37℃ in a constant temperature box for 40min.

[0066] (6) Remove the ELISA plate, discard the internal solution, wash the plate 4 times, add 100 μl of TMB colorimetric solution to each well, and let it stand at room temperature for 5 minutes.

[0067] (7) Add 50 μl of stop solution (dilute sulfuric acid) to each well to terminate the reaction, and immediately read the reading on a microplate reader at 450 nm. The dilution corresponding to the well with an OD value greater than 2.1 times the set negative control OD value is determined as the titer of the mouse serum heparin binding protein antibody.

[0068] The results of the serum heparin binding protein antibody titer test in the examples and comparative examples are shown in Tables 1-6 below.

[0069] Table 1: Example 1 Serum heparin binding protein antibody titer detection results

[0070]

[0071] Table 2: Example 2 Serum heparin binding protein antibody titer detection results

[0072]

[0073]

[0074] Table 3: Example 3 Serum heparin binding protein antibody titer detection results

[0075]

[0076] Table 4: Comparative Example 1 Serum titer test results

[0077]

[0078] Table 5: Comparative Example 2 Serum Titer Test Results

[0079]

[0080]

[0081] Table 6: Comparative Example 3 Serum Titer Test Results

[0082]

[0083] As shown in Table 1-6, in Example 1-3, heparin-binding protein was biotinylated and then bound to streptavidin immunomagnetic beads to form a heparin-binding protein immune complex to immunize experimental animals. The titers of the prepared mouse serum heparin-binding protein antibodies were all 1:1562500. In Comparative Example 1 (heparin-binding protein was mixed with equal volumes of Freund's adjuvant and emulsified), the serum titers of 5 mice were all 1:62500. In Comparative Example 2 (heparin-binding protein was mixed with a water-soluble adjuvant), except for the serum titer of mouse No. 3 at 1:12500, the serum titers of the other 4 mice were all 1:62500. In Comparative Example 3, magnetic beads were coupled to anti-GST tag 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. The method can be used to obtain high-titer heparin-binding protein antibodies, provide a method for producing heparin-binding protein monoclonal antibodies using hybridoma cells, and provide high-quality raw materials for the development of clinical diagnostic reagents.

[0084] Experimental Example 2

[0085] The method for detecting nonspecific antibody titer comprises the following steps:

[0086] (1) Design the number of ELISA plates to be coated according to the experimental needs and mark them on the strips.

[0087] (2) Dilute the GST antibody / GST protein to a concentration of 0.5 μg / ml using PBS coating solution, mix well, and add to the enzyme-labeled plate, 100 μl per well, and store in a refrigerator at 4°C overnight.

[0088] (3) After coating, discard the coating solution, wash the plate once, add 200 μl of blocking solution (1% casein) to each well, incubate in a 37°C incubator for 2 h, remove the ELISA plate, discard the inner solution, and pat dry for later use.

[0089] (4) The serum of the mouse in comparative example 3 was diluted 1:100 for the first well, and the remaining wells were serially diluted 5 times in 11 gradients, with 100ul per well, and incubated in a 37°C incubator for 40 minutes.

[0090] (5) Remove the ELISA plate, discard the internal solution, wash the plate 4 times, add 100ul 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, and incubate at 37℃ in a constant temperature box for 40min.

[0091] (6) Remove the ELISA plate, discard the internal solution, wash the plate 4 times, add 100 μl of TMB colorimetric solution to each well, and let it stand at room temperature for 5 minutes.

[0092] (7) Add 50 μl of stop solution (dilute sulfuric acid) to each well to terminate the reaction, and immediately read the plate at 450 nm on a microplate reader. The dilution corresponding to the well with an OD value greater than 2.1 times the set negative control OD value is determined as the titer of the nonspecific antibody in the mouse serum.

[0093] The results of the non-specific antibody titer test in the serum of Comparative Example 3 are shown in Tables 7-8 below:

[0094] Table 7: Antibody titer test results of serum GST antibodies in comparative example 3

[0095]

[0096] Table 8: Comparative Example 3 serum GST antibody titer test results

[0097]

[0098] As shown in Tables 7-8, the antibody titer of GST antibody in Comparative Example 3 is 1:1562500, and the antibody titer of GST is 1:312500, indicating that the method of Comparative Example 3, in addition to producing HBP antibodies, also produces GST antibodies and antibodies to GST antibodies, which to a certain extent affects the titer and specificity of HBP antibodies in serum.

[0099] Experimental Example 3

[0100] The method for detecting nonspecific antibody titer comprises the following steps:

[0101] (1) Design the number of ELISA plates to be coated according to the experimental needs and mark them on the strips.

[0102] (2) Dilute the SA protein to a concentration of 0.5 μg / ml using PBS coating solution, mix well, and add it to the enzyme-labeled plate, 100 μl per well, and store in a refrigerator at 4°C overnight.

[0103] (3) After coating, discard the coating solution, wash the plate once, add 200 μl of blocking solution (1% casein) to each well, incubate in a 37°C incubator for 2 h, remove the ELISA plate, discard the inner solution, and pat dry for later use.

[0104] (4) The mouse serum from Example 1 was diluted 1:100 for the first well, and the remaining wells were serially diluted 5-fold in 11 gradients, with 100 μl per well, and incubated in a 37°C incubator for 40 min.

[0105] (5) Remove the ELISA plate, discard the internal solution, wash the plate 4 times, add 100ul 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, and incubate at 37℃ in a constant temperature box for 40min.

[0106] (6) Remove the ELISA plate, discard the internal solution, wash the plate 4 times, add 100 μl of TMB colorimetric solution to each well, and let it stand at room temperature for 5 minutes.

[0107] (7) Add 50 μl of stop solution (dilute sulfuric acid) to each well to terminate the reaction, and immediately read the plate at 450 nm on a microplate reader. The dilution corresponding to the well with an OD value greater than 2.1 times the set negative control OD value is determined as the titer of the nonspecific antibody in the mouse serum.

[0108] The results of the non-specific antibody titer test in the serum of Example 1 are shown in Table 9 below.

[0109] Table 9: Example 1 Serum GST antibody titer detection results

[0110]

[0111] As shown in Table 9, the antibody titer of the SA antibody produced in Example 1 was 1:500. The heparin-binding protein was biotinylated and then bound to streptavidin immunomagnetic beads to form a heparin-binding protein immune complex. The antibody obtained by immunizing animals with this complex produced a low amount of nonspecific antibodies, which was much lower than the method of using magnetic beads to couple anti-GST tag antibodies and then capture HBP-GST fusion protein.

[0112] The method of biotinylating heparin-binding protein and then binding it to streptavidin immunomagnetic beads can produce higher-titer antibodies compared to Freund's adjuvant and water-soluble adjuvants. Compared to the method of using magnetic beads to couple anti-GST-tagged antibodies and then capture HBP-GST fusion proteins, it can produce higher-titer HBP antibodies and lower nonspecific antibodies. The antibodies prepared using the method used 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 proteins and providing high-quality raw materials for the development of detection reagents.

[0113] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the technical principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for preparing a heparin-binding protein immune complex, characterized in that: The following steps are involved: S1: After biotin is dissolved, it is added to the coating buffer, and heparin-binding protein is added. Incubate 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: Add biotinylated heparin-binding protein to streptavidin immunomagnetic beads, add binding buffer, and incubate to form heparin-binding protein immune complexes.

2. The method for preparing the heparin-binding protein immune complex according to claim 1, characterized in that: In S1, the biotin is dissolved in dimethyl sulfoxide to obtain a concentration of 3-7 mg / mL of biotin, and the volume ratio of the biotin to the coating buffer is 1:(80-120).

3. The method for preparing the heparin-binding protein immune complex according to claim 1, characterized in that: In S1, the concentration of the heparin-binding protein is 0.5-1.5 mg / mL, the molar ratio of the heparin-binding protein to biotin is 1:(5-15); and the incubation time at room temperature in the dark is 1-3 h.

4. The method for preparing the heparin-binding protein immune complex according to claim 1, characterized in that: In S1, the coating buffer is 0.1 M potassium phosphate buffer with a pH of 7.3-7.

7.

5. The method for preparing the heparin-binding protein immune complex according to claim 1, characterized in that: The ratio of binding buffer and streptavidin immunomagnetic beads added to S2 is 1 mL: (15-25 mg).

6. The method for preparing the heparin-binding protein immune complex according to claim 1, characterized in that: In S2, the binding buffer comprises: 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%.

7. The method for preparing the heparin-binding protein immune complex according to claim 1, characterized in that: In S3, the mass ratio of the streptavidin immunomagnetic beads to the biotinylated heparin binding protein is (50-150):

1.

8. The method for preparing the heparin-binding protein immune complex according to claim 1, characterized in that: In S3, the incubation temperature is 30-40° C. and the incubation time is 30-60 min.

9. A heparin-binding protein immune complex, characterized in that The method is obtained by the preparation method according to any one of claims 1 to 8.

10. Use of the heparin-binding protein immune complex according to claim 9 in animal immunization.

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