A monoclonal paired antibody for lipoprotein-associated phospholipase A2 and its application

Abstract

This invention relates to the field of monoclonal antibody technology, and in particular to a monoclonal paired antibody for lipoprotein-associated phospholipase A2 and its applications. The heavy chain sequence of the first antibody is shown in SEQ ID NO:1, and its light chain sequence is shown in SEQ ID NO:2; the heavy chain sequence of the second antibody is shown in SEQ ID NO:3, and its light chain sequence is shown in SEQ ID NO:4. The monoclonal paired antibody for lipoprotein-associated phospholipase A2 of this invention exhibits high specificity and sensitivity, providing accurate and reliable reagent support for relevant detection scenarios. It can be used in multiple detection fields, including biochemical immunoturbidimetric assays, immunochromatographic assays, and ELISA, achieving rapid, efficient, and highly sensitive clinical detection of lipoprotein-associated phospholipase A2 antigen in serum.

Description

Technical Field

[0001] This invention relates to the field of monoclonal antibody technology, specifically to a monoclonal paired antibody against lipoprotein-associated phospholipase A2 and its applications. Background Technology

[0002] Lipoprotein-associated phospholipase A2 (Lp-PLA2) is a specific subtype of the phospholipase A2 superfamily, mainly secreted by macrophages, T lymphocytes, and vascular smooth muscle cells. After secretion, it primarily binds to low-density lipoprotein (LDL) and high-density lipoprotein (HDL) to participate in blood circulation. Lp-PLA2 can specifically hydrolyze phospholipids and platelet-activating factor (PAF) in oxidized lipoproteins, generating pro-inflammatory products such as lysophosphatidylcholine and free fatty acids, thereby promoting the formation and progression of atherosclerotic plaques and serving as an independent risk marker for atherosclerotic cardiovascular disease.

[0003] Lipoprotein-associated phospholipase A2 (Lp-PLA2) is an independent risk marker for atherosclerotic cardiovascular disease, and its quantitative detection in vitro is crucial for risk assessment, early screening, and prognostic monitoring. Immunolabeling is currently the mainstream technique for clinical detection of Lp-PLA2. Its core principle is to utilize the immune binding reaction between Lp-PLA2-specific antibodies and antigens, achieving quantitative detection through marker signal amplification. Therefore, developing a highly specific and high-affinity Lp-PLA2 antibody to achieve highly sensitive detection of Lp-PLA2 antigen levels in serum is of great significance. Summary of the Invention

[0004] The purpose of this invention is to provide a monoclonal paired antibody against lipoprotein-associated phospholipase A2 and its application.

[0005] To achieve the above objectives, the present invention provides the following technical solution:

[0006] A monoclonal paired antibody against lipoprotein-associated phospholipase A2, wherein the heavy chain sequence of the first antibody is shown in SEQ ID NO:1 and the light chain sequence is shown in SEQ ID NO:2; and the heavy chain sequence of the second antibody is shown in SEQ ID NO:3 and the light chain sequence is shown in SEQ ID NO:4.

[0007] The monoclonal paired antibody for lipoprotein-associated phospholipase A2 described in this invention can be used to prepare a latex immunoturbidimetric assay kit for detecting lipoprotein-associated phospholipase A2. The kit includes reagent R1 and reagent R2.

[0008] The reagent R1 is 50mM pH 6.5 MES buffer, Tween 20 1g / L, PEG6000 5g / L, BSA 1w%, Proclin 300 0.08% (v / v);

[0009] The preparation method of the reagent R2 is as follows:

[0010] (1) Coating the first antibody Lp-PLA2mAb01: Take 0.1 mL of PS microspheres, add 2.5 mL of 25 mM pH 6.5 MES buffer and mix, then add 5 μL of 0.1 g / mL EDC solution (prepared and used immediately), mix and stir at room temperature for 15 min; add 0.025 mg of Lp-PLA2mAb01 to the reaction system and stir at room temperature for 1 h; add 10 μL of blocking agent (blocking agent is 1% BSA and 0.2% glycine aqueous solution) to the reaction system and stir for 1 h; centrifuge at 8000 rpm for 15 min, resuspend in 2.5 mL of pH 7.0 PBS solution, and sonicate for 10 min;

[0011] (1) Coating the second antibody Lp-PLA2mAb02: Take 0.05 mL of PS microspheres, add 2.5 mL of 25 mM pH 6.5 MES buffer and mix, then add 5 μL of 0.1 g / mL EDC solution (prepared and used immediately), mix and stir at room temperature for 15 min; add 0.025 mg of Lp-PLA2mAb02 to the reaction system and stir at room temperature for 1 h; add 10 μL of blocking agent (blocking agent is 1% BSA and 0.2% glycine aqueous solution) to the reaction system and stir for 1 h; centrifuge at 8000 rpm for 15 min, resuspend in 2.5 mL of pH 7.0 PBS solution, and sonicate for 10 min;

[0012] (3) Mix the two microsphere antibody-labeled complexes and store at 2-8℃ for later use.

[0013] The monoclonal paired antibody for lipoprotein-associated phospholipase A2 described in this invention can be used to prepare an immunochromatographic assay kit for detecting lipoprotein-associated phospholipase A2. The preparation method is as follows:

[0014] (1) Labeling antibody: Take 0.05 mg of time-resolved fluorescent microspheres into a centrifuge tube, add 1 mL of 25 mM pH 6.5 MES buffer, and vortex to mix; add 5 μL of 100 mg / mL EDC and 5 μL of 100 mg / mL NHS respectively, vortex to mix, and incubate at room temperature in the dark for 30 min; centrifuge the activated microspheres at 15000 rpm for 20 min, remove the supernatant, resuspend the microspheres with 1 mL of 25 mM pH 6.5 MES buffer, and repeat 3 times; add 0.05 mg of Lp-PLA2mAb 01, vortex to mix, and incubate at room temperature in the dark for 2 h; add 100 μL of 10% (w / v) BSA, and incubate at room temperature for 30 min; centrifuge at 15000 rpm for 15 min, remove the supernatant, resuspend the microspheres with 1 mL of 50 mM pH 7.4 PBS, repeat 3 times, and store at 2-8℃ for later use.

[0015] (2) Preparation of nitrocellulose membrane: The second antibody Lp-PLA2mAb02 was coated on the nitrocellulose membrane at a coating amount of 1 μL / cm as the detection line, and the goat anti-mouse IgG antibody at a concentration of 1 mg / mL was coated on the nitrocellulose membrane at a coating amount of 1 μL / cm as the quality control line. The membrane was dried overnight at 45°C.

[0016] (3) Preparation of conjugate pad: Time-resolved fluorescent microsphere-labeled antibody complex was sprayed onto glass cellulose membrane at a coating amount of 6 μL / cm and dried overnight at 45°C to prepare conjugate pad.

[0017] (4) Test strip assembly: attach the coated nitrocellulose membrane to the support base plate, attach the conjugate pad to one end of the marking line, place the sample pad (glass cellulose membrane) on the conjugate pad, and attach the absorbent pad to one end of the control line.

[0018] The monoclonal paired antibody for lipoprotein-associated phospholipase A2 described in this invention can also be used to prepare an ELISA kit for detecting lipoprotein-associated phospholipase A2.

[0019] Compared with the prior art, the beneficial effects of the present invention are:

[0020] (1) The monoclonal paired antibody of lipoprotein-associated phospholipase A2 of the present invention has high specificity and sensitivity, and can provide accurate and reliable reagent support for relevant detection scenarios.

[0021] (2) The monoclonal paired antibody of lipoprotein-associated phospholipase A2 of the present invention can be used in multiple detection fields such as biochemical immunoturbidimetric detection, immunochromatographic detection and ELISA, realizing rapid, efficient and high-sensitivity clinical detection of lipoprotein-associated phospholipase A2 antigen in serum. Attached Figure Description

[0022] Figure 1 Linearity of the Lp-PLA2 detection kit by biochemical immunoturbidimetric assay.

[0023] Figure 2 The linear relationship is shown for the Lp-PLA2 detection kit using fluorescence immunochromatography. Detailed Implementation

[0024] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0025] Example 1: Preparation of monoclonal paired antibodies against lipoprotein-associated phospholipase A2

[0026] (1) Immunized animals

[0027] The immunization regimen is selected based on the characteristics of the antigen. For soluble antigens with weak immunogenicity, adjuvants are generally added. Commonly used adjuvants are Freund's complete adjuvant and Freund's incomplete adjuvant. The antigen and adjuvant should be mixed in equal volumes and ground into a water-in-oil emulsion.

[0028] The initial immunization was performed by subcutaneous injection of 50 μg antigen with Freund's complete adjuvant at multiple sites; the second immunization was performed 3 weeks later (same dose of antigen with Freund's incomplete adjuvant); the third immunization was performed by intraperitoneal injection without adjuvant, and the titer was tested 7 days later; the final booster immunization was performed (50 μg antigen intraperitoneal injection), and spleen cells were harvested for fusion 3 days later.

[0029] (2) Cell fusion

[0030] Myeloma cell suspension: Select a good myeloma cell line, isolate myeloma cells from tumors growing in vivo, and prepare a cell suspension;

[0031] Spleen cell suspension: Mice that received a booster 3 days prior underwent orbital bloodletting, and the serum was separated and frozen for later use. The mice were euthanized, and the cells were immersed in 75% ethanol for 3-5 minutes. The spleen was aseptically removed and washed in a petri dish containing 5 mL of incomplete culture medium. The surrounding connective tissue was removed, and the spleen was transferred to a steel mesh in another petri dish containing 5 mL of incomplete culture medium. It was first cut into 3-5 small pieces with scissors, then ground with a syringe core. The spleen cell suspension was transferred to a 50 mL centrifuge tube, 50 mL of incomplete culture medium was added, and the tube was centrifuged at 1000 rpm for 5 minutes. The supernatant was discarded, and the cells were washed and centrifuged again using the same method. The precipitated cells were then resuspended in 10 mL of incomplete culture medium, and the viable cell count was recorded. 0.5-2 × 10⁶ cells were obtained from one mouse. 8 One spleen cell.

[0032] Feeder Cells: After euthanizing, disinfecting, and fixing the mice, use sterile scissors to lift the abdominal skin from the posterior abdomen to expose the peritoneum. Disinfect the peritoneum with an alcohol swab. Inject 10 mL of incomplete culture medium into the peritoneal cavity using a syringe, taking care to avoid perforation of the intestine. Hold the syringe firmly with your right hand, leaving the needle in the peritoneal cavity, and gently massage the abdomen with an alcohol swab in your left hand for 1 minute. Then aspirate the injected culture medium. Centrifuge at 1000 rpm for 10 minutes and discard the supernatant. Resuspend the pelleted cells in 5 mL of HAT medium. Based on the cell count, add more HAT medium to bring the cell concentration to 2 × 10⁶ cells / mL. 5 / mL, for later use.

[0033] Cell fusion: Mix the prepared myeloma cells and mouse spleen cells at a ratio of 1:10, add 20 mL of PRMI-1640 solution; centrifuge at 1000 r / min for 10 min, discard the supernatant, and aspirate as much supernatant as possible; gently tap the bottom of the centrifuge tube with your finger to disperse the precipitated cells, and place the centrifuge tube in a 37℃ water bath; slowly add 1 mL of 50% PEG pre-warmed at 37℃ into the centrifuge tube over about 45 seconds, stirring gently while adding, and let stand at 37℃ for 5 min; add 20 mL of incomplete culture medium (pre-warmed at 37℃) over 5 min, adding 1 mL in the first minute, 2 mL in the second minute, 5 mL in the third minute, and 6 mL each in the fourth and fifth minutes, while gently rotating the centrifuge tube. The medium should be added along the tube wall, not directly onto the precipitated cells, to prevent dislodging the newly fused cells. Then add PRMI-1640 solution to 50 mL to terminate the PEG effect; centrifuge at 800 r / min for 10 min, discard the supernatant, gently suspend the precipitated cells in the required volume of HAT culture medium, and seed 0.10-0.15 mL per well in a 96-well culture plate. After seeding, place the culture plate in a 37℃ 5% CO2 incubator for culture.

[0034] (3) Selective culture

[0035] Five days after inoculating 96-well plates, replace half of the medium with HAT medium. After 7-10 days, replace the HAT medium with HT medium. After the 14th day, ordinary complete medium can be used.

[0036] (4) Screening and cloning of hybridoma-positive clones

[0037] Coating: The antigen was coated with Na2CO3-NaHCO3 buffer at pH 9.6. 20 μg of antigen was added to each microplate, and the coating volume per well was 50 μL. Coating was carried out at 37°C for 2 h.

[0038] Blocking: Discard the antigen, blot dry the liquid in the wells of the ELISA plate, and block with 1% BSA at 37°C for 2 hours.

[0039] Primary antibody: After blocking, discard the liquid in the well, pat dry, add 100 μL of the supernatant of the cells to be tested to each well, and incubate at 37°C for 1 h.

[0040] Secondary antibody: After incubation with primary antibody, discard the liquid in the wells, blot dry, and wash three times with PBST buffer (pH 7.4) for 5 min each time. Dilute the secondary antibody according to the instructions, add 100 μL to each well, and incubate at 37°C for 1 h.

[0041] Color development: After incubation with the secondary antibody, discard the liquid in the wells, pat dry, add the colorimetric reagent, and stop the reaction with the stop solution when the color is the deepest. Read the OD value of each well with a microplate reader, select the positive wells, expand the culture and freeze them.

[0042] (5) Purification of monoclonal antibodies

[0043] Affinity purification was used to purify monoclonal paired antibodies against lipoprotein-associated phospholipase A2. Staphylococcal A protein was cross-linked with a carrier, and an affinity chromatography column was prepared to bind the antibodies before elution. The antibodies were then concentrated to 5 mg / mL. The heavy chain sequence of the purified monoclonal paired antibody, Lp-PLA2mAb01, is shown in SEQ ID NO:1, and the light chain sequence is shown in SEQ ID NO:2. The heavy chain sequence of the second antibody, Lp-PLA2mAb02, is shown in SEQ ID NO:3, and the light chain sequence is shown in SEQ ID NO:4.

[0044] Example 2: An Lp-PLA2 detection kit (latex immunoturbidimetric assay) was prepared using the Lp-PLA2 monoclonal paired antibody from Example 1.

[0045] Reagent R1: 50mM pH6.5 MES buffer, Tween20 1g / L, PEG6000 5g / L, BSA 1w%, Proclin300 0.08% (v / v).

[0046] Preparation of reagent R2:

[0047] (1) Coating the first antibody Lp-PLA2mAb01: Take 0.1 mL of PS microspheres (Nanjing Liding Medical Technology Co., Ltd., catalog number: LD102021), add 2.5 mL of 25 mM pH 6.5 MES buffer, mix, add 5 μL of 0.1 g / mL EDC solution (prepared fresh for use), mix and stir at room temperature for 15 min; add 0.025 mg of Lp-PLA2mAb01 prepared in Example 1 to the reaction system, stir at room temperature for 1 h; add 10 μL of blocking agent (blocking agent is 1% BSA and 0.2% glycine aqueous solution) to the reaction system, stir for 1 h; centrifuge at 8000 rpm for 15 min, resuspend in 2.5 mL of pH 7.0 PBS solution, and sonicate for 10 min.

[0048] (2) Coating the second antibody Lp-PLA2mAb02: Take 0.05 mL of PS microspheres (Nanjing Liding Medical Technology Co., Ltd., catalog number: LD102025), add 2.5 mL of 25 mM pH 6.5 MES buffer, mix, add 5 μL of 0.1 g / mL EDC solution (prepared fresh for use), mix and stir at room temperature for 15 min; add 0.025 mg of Lp-PLA2mAb02 prepared in Example 1 to the reaction system, stir at room temperature for 1 h; add 10 μL of blocking agent (blocking agent is 1% BSA and 0.2% glycine aqueous solution) to the reaction system, stir for 1 h; centrifuge at 8000 rpm for 15 min, resuspend in 2.5 mL of pH 7.0 PBS solution, and sonicate for 10 min.

[0049] (3) Mix the two microsphere antibody-labeled complexes and store at 2-8℃ for later use.

[0050] Testing instrument: Hitachi 7180 fully automated biochemical analyzer.

[0051] Parameters: Sample volume 12μL, reagent R1 240μL, reagent R2 60μL, fitting method Spline, main wavelength 546nm, secondary wavelength none.

[0052] Example 3: Lp-PLA2 detection kit (fluorescent immunochromatography) was prepared using the Lp-PLA2 monoclonal paired antibody from Example 1.

[0053] (1) Antibody labeling: Take 0.05 mg of time-resolved fluorescent microspheres (Nanjing Liding Medical Technology Co., Ltd., catalog number: LD302017) into a centrifuge tube, add 1 mL of 25 mM pH 6.5 MES buffer, and vortex to mix; add 5 μL of 100 mg / mL EDC and 5 μL of 100 mg / mL NHS respectively, vortex to mix, and incubate at room temperature in the dark for 30 min; centrifuge the activated microspheres at 15000 rpm for 20 min, remove the supernatant, and resuspend the microspheres with 1 mL of 25 mM pH 6.5 MES buffer, repeat 3 times; add 0.05 mg of Lp-PLA2mAb 01 from Example 1, vortex to mix, and incubate at room temperature in the dark for 2 h; add 100 μL of 10% (w / v) BSA, and incubate at room temperature for 30 min; centrifuge at 15000 rpm for 15 min, remove the supernatant, and resuspend the microspheres with 1 mL of 50 mM pH 7.4 MES buffer. Resuspend the microspheres in PBS, repeat 3 times, and store at 2-8℃ for later use.

[0054] (2) Preparation of nitrocellulose membrane: The second antibody Lp-PLA2mAb02 was coated on the nitrocellulose membrane at a coating amount of 1 μL / cm as the detection line, and the goat anti-mouse IgG antibody at a concentration of 1 mg / mL was coated on the nitrocellulose membrane at a coating amount of 1 μL / cm as the quality control line. The membrane was dried overnight at 45°C.

[0055] (3) Preparation of conjugate pad: Time-resolved fluorescent microsphere-labeled antibody complex was sprayed onto glass cellulose membrane at a coating amount of 6 μL / cm and dried overnight at 45°C to prepare conjugate pad.

[0056] (4) Test strip assembly: attach the coated nitrocellulose membrane to the support base plate, attach the conjugate pad to one end of the marking line, place the sample pad (glass cellulose membrane) on the conjugate pad, and attach the absorbent pad to one end of the control line.

[0057] Testing instrument: Lambert AFS1520 fluorescence immunoassay analyzer.

[0058] To highlight the beneficial effects of the present invention, the following comparative experiments are provided.

[0059] Comparative Example 1: An Lp-PLA2 detection kit (latex immunoturbidimetric assay) was prepared using Lp-PLA2 monoclonal antibody from Medix Biochemica China.

[0060] Reagent R1: 50mM pH 6.5 MES buffer, Tween 20 1g / L, PEG6000 5g / L, BSA 1w%, Proclin 300 0.08% (v / v).

[0061] Preparation of reagent R2:

[0062] (1) Coating the first antibody Lp-PLA2mAb01: Take 0.1 mL of PS microspheres (Nanjing Liding Medical Technology Co., Ltd., catalog number: LD102021), add 2.5 mL of 25 mM pH 6.5 MES buffer, mix, add 5 μL of 0.1 g / mL EDC solution (prepared and used immediately), mix and stir at room temperature for 15 min; add 0.025 mg of Lp-PLA2mAb (HM820, Medix Biochemica China) to the reaction system, stir at room temperature for 1 h; add 10 μL of blocking agent (blocking agent is 1% BSA and 0.2% glycine aqueous solution) to the reaction system, stir for 1 h; centrifuge at 8000 rpm for 15 min, resuspend in 2.5 mL of pH 7.0 PBS solution, and sonicate for 10 min.

[0063] (2) Coating the second antibody Lp-PLA2mAb02: Take 0.05 mL of PS microspheres (Nanjing Liding Medical Technology Co., Ltd., catalog number: LD102025), add 2.5 mL of 25 mM pH 6.5 MES buffer, mix, add 5 μL of 0.1 g / mL EDC solution (prepared and used immediately), mix and stir at room temperature for 15 min; add 0.025 mg of Lp-PLA2mAb02 (HM821, Medix Biochemica China) to the reaction system, stir at room temperature for 1 h; add 10 μL of blocking agent (blocking agent is 1% BSA and 0.2% glycine aqueous solution) to the reaction system, stir for 1 h; centrifuge at 8000 rpm for 15 min, resuspend in 2.5 mL of pH 7.0 PBS solution, and sonicate for 10 min.

[0064] (3) Mix the two microsphere antibody-labeled complexes and store at 2-8℃ for later use.

[0065] Testing instrument: Hitachi 7180 fully automated biochemical analyzer.

[0066] Parameters: Sample volume 12μL, reagent R1 240μL, reagent R2 60μL, fitting method Spline, main wavelength 546nm, secondary wavelength none.

[0067] Comparative Example 2: An Lp-PLA2 detection kit (fluorescent immunochromatography) was prepared using Lp-PLA2 monoclonal antibody from Medix Biochemica China.

[0068] (1) Antibody labeling: Take 0.05 mg of time-resolved fluorescent microspheres (Nanjing Liding Medical Technology Co., Ltd., catalog number: LD302017) into a centrifuge tube, add 1 mL of 25 mM pH 6.5 MES buffer, and vortex to mix; add 5 μL of 100 mg / mL EDC and 5 μL of 100 mg / mL NHS respectively, vortex to mix, and incubate at room temperature in the dark for 30 min; centrifuge the activated microspheres at 15000 rpm for 20 min, remove the supernatant, and resuspend the microspheres with 1 mL of 25 mM pH 6.5 MES buffer, repeat 3 times; add 0.05 mg of Lp-PLA2mAb (HM820, Medix Biochemica China), vortex to mix, and incubate at room temperature in the dark for 2 h; add 100 μL of 10% (w / v) BSA, incubate at room temperature for 30 min; centrifuge at 15000 rpm for 15 min, remove the supernatant, and resuspend the microspheres with 1 mL of 25 mM pH 6.5 MES buffer. Resuspend the microspheres in 50mM pH 7.4 PBS, repeat 3 times, and store at 2-8℃ for later use.

[0069] (2) Preparation of nitrocellulose membrane: The second antibody Lp-PLA2mAb (HM821, Medix Biochemica China) was applied to the nitrocellulose membrane at a coating amount of 1 μL / cm as the detection line, and the goat anti-mouse IgG antibody at a concentration of 1 mg / mL was applied to the nitrocellulose membrane at a coating amount of 1 μL / cm as the quality control line. The membrane was dried overnight at 45°C.

[0070] (3) Preparation of conjugate pad: Time-resolved fluorescent microsphere-labeled antibody complex was sprayed onto glass cellulose membrane at a coating amount of 6 μL / cm and dried overnight at 45°C to prepare conjugate pad.

[0071] (4) Test strip assembly: attach the coated nitrocellulose membrane to the support base plate, attach the conjugate pad to one end of the marking line, place the sample pad (glass cellulose membrane) on the conjugate pad, and attach the absorbent pad to one end of the control line.

[0072] Testing instrument: Lambert AFS1520 fluorescence immunoassay analyzer.

[0073] Table 1 shows the precision detection of Example 2 and Comparative Example 1 at two concentration levels. The results show that, in terms of concentration consistency, the concentration CV of Example 2 at both levels is less than 5%, which is significantly better than that of Comparative Example 1. Furthermore, in terms of signal response performance, the signal values ​​of Example 2 are higher than those of Comparative Example 1 at the same concentration level. Therefore, Example 2 has significant technical advantages in core indicators such as concentration consistency, signal response intensity, and detection repeatability, and its detection system has better overall performance than Comparative Example 1.

[0074] Table 1. Lp-PLA2 Detection Kit (Latex Immunoturbidimetric Assay) for Clinical Sample Testing

[0075]

[0076] Note: a: Unit ng / mL; b: Absorbance value.

[0077] Figure 1 To illustrate the linear relationship of the Lp-PLA2 detection kit using biochemical immunoturbidimetric assay, the coefficient of determination R for linear fitting in Example 2 is given. 2 The R value reached 0.9997, compared to R in Comparative Example 1. 2 The value is 0.9967, indicating that the linear range of Example 2 is better than that of Comparative Example 1.

[0078] Figure 2 To illustrate the linear relationship of the Lp-PLA2 detection kit using fluorescence immunochromatography, the coefficient of determination R for linear fitting in Example 3 is given. 2 The R-value is 0.9722, which is the value of Comparative Example 2. 2 The correlation coefficient was 0.9698, indicating that both examples exhibited a basic linear correlation, but Example 3 showed better linearity than Comparative Example 2.

[0079] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make some modifications or alterations to the above-disclosed technical content to create equivalent embodiments without departing from the scope of the present invention. Any simple modifications, equivalent changes and alterations made to the above embodiments based on the technical essence of the present invention without departing from the scope of the present invention shall still fall within the scope of the present invention.

Claims

1. A monoclonal pair of antibodies to lipoprotein-associated phospholipase A2, characterized in that: The heavy chain sequence of the first antibody is shown as SEQ ID NO: 1, and the light chain sequence is shown as SEQ ID NO: 2; the heavy chain sequence of the second antibody is shown as SEQ ID NO: 3, and the light chain sequence is shown as SEQ ID NO:

4.

2. The use of the lipoprotein-associated phospholipase A2 monoclonal paired antibody of claim 1 in the preparation of a latex immunoturbidimetric detection kit for detecting lipoprotein-associated phospholipase A2.

3. Use according to claim 2, characterized in that: The kit comprises reagent R1 and reagent R2; The reagent R1 is 50 mM pH 6.5 MES buffer, Tween 20 1 g / L, PEG6000 5 g / L, BSA 1 w%, Proclin 300 0.08% v / v; The preparation method of the reagent R2 is: (1) coating the first antibody Lp-PLA2 mAb01: take 0.1 mL PS microspheres, add 2.5 mL 25 mM pH 6.5 MES buffer, then add 5 μL 0.1 g / mL EDC solution, mix and stir at room temperature for 15 min; add 0.025 mg Lp-PLA2 mAb01 to the reaction system, stir at room temperature for 1 h; add 10 μL blocking agent to the reaction system, stir for 1 h; centrifuge at 8000 rpm for 15 min, resuspend with 2.5 mL pH 7.0 PBS solution, and ultrasonic for 10 min; (2) coating the second antibody Lp-PLA2 mAb02: take 0.05 mL PS microspheres, add 2.5 mL 25 mM pH 6.5 MES buffer, then add 5 μL 0.1 g / mL EDC solution, mix and stir at room temperature for 15 min; add 0.025 mg Lp-PLA2 mAb02 to the reaction system, stir at room temperature for 1 h; add 10 μL blocking agent to the reaction system, stir for 1 h; centrifuge at 8000 rpm for 15 min, resuspend with 2.5 mL pH 7.0 PBS solution, and ultrasonic for 10 min; (3) mix the two microsphere antibody labeled complexes, and store at 2-8℃ for standby.

4. The use of the lipoprotein-associated phospholipase A2 monoclonal paired antibody of claim 1 in the preparation of an immunochromatographic detection kit for detecting lipoprotein-associated phospholipase A2.

5. Use according to claim 4, characterized in that, The preparation method of the kit is: (1) Labeling antibody: Take 0.05 mg time-resolved fluorescent microspheres in a centrifuge tube, add 1 mL 25 mM pH 6.5 MES buffer, vortex mix; add 5 μL 100 mg / mL EDC and 5 μL 100 mg / mL NHS, vortex mix, incubate at room temperature for 30 min in the dark; centrifuge the activated microspheres at 15000 rpm for 20 min, remove the supernatant, resuspend the microspheres in 1 mL 25 mM pH 6.5 MES buffer, repeat 3 times; add 0.05 mg Lp-PLA2 mAb01, vortex mix, incubate at room temperature for 2 h in the dark; add 100 μL 10% w / v BSA, incubate at room temperature for 30 min; centrifuge at 15000 rpm for 15 min, remove the supernatant, resuspend the microspheres in 1 mL 50 mM pH 7.4 PBS, repeat 3 times, store at 2-8°C for standby; (2) Preparation of nitrocellulose membrane: the second antibody Lp-PLA2 mAb02 is coated on the nitrocellulose membrane as a detection line at a coating amount of 1 μL / cm, and the goat anti-mouse IgG antibody with a concentration of 1 mg / mL is coated on the nitrocellulose membrane as a quality control line at a coating amount of 1 μL / cm, and dried at 45°C overnight; (3) Preparation of binding pad: the time-resolved fluorescent microsphere-labeled antibody complex is sprayed on the glass fiber membrane at a coating amount of 6 μL / cm, and dried at 45°C overnight to prepare a binding pad; (4) Assembly of test strip: the coated nitrocellulose membrane is attached to the support base plate, the binding pad is attached to one end of the marker line, the sample pad is attached to the binding pad, and the quality control line is attached to the water absorption pad.

6. Use of the lipoprotein-associated phospholipase A2 monoclonal paired antibody of claim 1 in the preparation of an ELISA detection kit for detecting lipoprotein-associated phospholipase A2.

Images