A high-sensitivity GDF-15 weak-magnetic quantitative detection kit and a preparation method thereof

Abstract

The application discloses a high-sensitivity GDF-15 weak-magnetic quantitative detection kit and a preparation method thereof, relates to the field of in-vitro diagnosis and immunodetection technology, and uses carboxylated superparamagnetic iron oxide microparticles with a particle size of 100-300 nm and a surface carboxyl content of greater than or equal to 80 micromoles per gram as a label carrier. Through fine regulation and control of EDC / NHS activation coupling conditions and blocking processes, the antibody labeling efficiency is increased by more than 30%, the minimum detection limit is reduced to 8 pg / ml while the biological activity of the antibody is maintained, a non-specific binding is effectively inhibited by an optimized chromatography system, a background signal is extremely low, a discrimination degree is not less than 4.0 near a 500 pg / ml clinical critical concentration, a false positive rate and a false negative rate are both controlled to be less than 5%, a linear range covers 0-10000 pg / ml, direct and accurate quantification can be achieved without multiple dilutions of high-value samples, a T / C ratio mode is adopted to eliminate sample matrix effects and operation differences, a batch variation coefficient is less than 10%, and a batch-to-batch variation coefficient is less than 15%.

Description

Technical Field

[0001] This invention relates to the field of in vitro diagnostic immunoassay technology, specifically to a high-sensitivity GDF-15 weak magnetic quantitative detection kit and its preparation method. Background Technology

[0002] Clinical studies have confirmed that GDF-15 is an independent risk stratification marker for cardiovascular diseases. Its concentration is positively correlated with infarct size in patients with acute myocardial infarction, NYHA classification in patients with heart failure, and all-cause mortality. At the same time, it has irreplaceable clinical value in the early screening, efficacy monitoring, and prognostic assessment of malignant tumors such as lung cancer, colorectal cancer, and pancreatic cancer.

[0003] Currently, commonly used clinical methods for GDF-15 detection mainly include: enzyme-linked immunosorbent assay (ELISA), chemiluminescence immunoassay (CIA), and colloidal gold immunochromatography (CIGC). ELISA has a detection limit of approximately 50 pg / ml, a testing cycle of 2-3 hours, is cumbersome, has poor repeatability, and can only perform batch testing, failing to meet the needs of rapid testing in emergency settings. Chemiluminescence immunoassay has high sensitivity (detection limit of approximately 5 pg / ml), but relies on large, fully automated chemiluminescence analyzers, resulting in high testing costs and requiring specialized technicians. Colloidal gold immunochromatography is simple and rapid to operate, but can only provide qualitative or semi-quantitative results, with low sensitivity (detection limit of approximately 1000 pg / ml), failing to meet the requirements for early diagnosis and accurate quantification.

[0004] Weak magnetic quantitative immunochromatography is an emerging point-of-care testing technology that uses magnetic microparticles as markers to achieve quantitative analysis by detecting the magnetic signal intensity of these microparticles in immune complexes. Compared with traditional labeling techniques, the magnetic signal is unaffected by sample color, turbidity, and optical interference, resulting in higher sensitivity and stability. However, existing GDF-15 weak magnetic detection kits generally suffer from low magnetic microparticle activation and coupling efficiency, leading to significant loss of antibody bioactivity and insufficient detection sensitivity. Furthermore, they exhibit severe non-specific binding, high background signal, and poor discrimination of low-concentration samples. Additionally, they have a narrow linear range, require multiple dilutions for high-concentration samples, and introduce additional errors. Summary of the Invention

[0005] The purpose of this invention is to overcome the shortcomings of existing technologies and provide a high-sensitivity GDF-15 weak magnetic quantitative detection kit and its preparation method. Using superparamagnetic microparticles with a particle size of 150-250 nm and a saturation magnetization of ≥50 emu / g as labeling carriers, the kit employs a double-antibody sandwich immunochromatography combined with the principle of weak magnetic quantitative detection. By optimizing the EDC / NHS activation conditions, antibody conjugation parameters, and blocking process, the kit effectively improves the labeling efficiency of magnetic microparticles and the retention rate of antibody bioactivity, while reducing non-specific binding and background signal. This achieves accurate quantitative detection of GDF-15 in the range of 0-10000 pg / ml.

[0006] To solve the above-mentioned technical problems, the present invention provides the following technical solution: a high-sensitivity GDF-15 weak magnetic field quantitative detection kit, comprising a detection card and a corporate reference sample; The test card consists of a test strip and a U-shaped buckle. The test strip includes a base plate and a sample pad, a conjugate pad, a nitrocellulose membrane, and an absorbent pad that are sequentially overlapped and pasted on the base plate. The conjugate pad is immobilized with magnetically microparticle-labeled GDF-15 monoclonal antibody and magnetically microparticle-labeled goat anti-rabbit IgG. The nitrocellulose membrane has a detection line and a control line that are separated from each other. The detection line is coated with another monoclonal antibody targeting different antigenic epitopes of GDF-15, and the control line is coated with rabbit IgG. The magnetic microparticles are carboxylated superparamagnetic iron oxide microparticles with a particle size of 100-250 nm, a saturation magnetization of ≥50 emu / g, and a surface carboxyl content of ≥100 μmol / g.

[0007] Furthermore, the enterprise reference is a standard solution prepared with negative serum and a GDF-15 concentration gradient covering 0-10000 pg / ml.

[0008] Furthermore, the enterprise reference materials include low-value enterprise reference materials with a concentration of 1000±50 pg / ml and high-value enterprise reference materials with a concentration of 5000±500 pg / ml.

[0009] Furthermore, the volume ratio of the magnetically microparticle-labeled GDF-15 monoclonal antibody to the magnetically microparticle-labeled goat anti-rabbit IgG is 2:1.

[0010] A method for preparing a high-sensitivity GDF-15 weak magnetic field quantitative detection kit, the method comprising: S1. Preparation of magnetic microparticle-labeled antibodies: magnetic microparticle-labeled GDF-15 monoclonal antibody and magnetic microparticle-labeled goat anti-rabbit IgG were prepared respectively. S2. Preparation of the conjugate pad: The two magnetic microparticle-labeled antibodies were mixed and sprayed onto the conjugate pad using a gold spraying membrane applicator. After drying, the mixture was sealed and stored. S3. Preparation of nitrocellulose membrane: GDF-15 coated antibody and rabbit IgG were diluted to working concentrations and coated onto nitrocellulose membrane using a gold spraying membrane to form detection lines and control lines. After drying, the membrane was sealed and stored. S4. Preparation of sample pads: Soak the sample pads in the treatment solution and then dry them for later use. S5. Assembly of the test card: The sample pad, conjugate pad, nitrocellulose membrane and absorbent pad are overlapped and pasted on the base plate in sequence, cut into test strips, and put into the U-shaped clip to obtain the test card; S6. Kit assembly: Place the test card into an aluminum foil bag containing desiccant and seal it. Then, place the sealed test card into the kit.

[0011] Furthermore, the specific steps of S1 are as follows: Vortex the carboxylated superparamagnetic iron oxide microparticles for 3-10 minutes. Take an appropriate amount of magnetic microparticles into a centrifuge tube, place it on a magnetic rack, and magnetically separate it for 2-5 minutes. Discard the supernatant. Add an appropriate amount of MES buffer (containing 0.02%-0.1% Tween 20) at pH 4.5-5.5, vortex for 20-60 seconds, magnetically separate it, and discard the supernatant. Repeat the washing process 2-4 times. Add an equal volume of freshly prepared 5-15 mg / ml EDC solution and 5-15 mg / ml NHS solution, vortex to mix, and then place on a constant temperature shaker at 20-30℃ and 150-250 rpm for 1-3 hours to activate. After activation, the supernatant is removed by magnetic separation, and the supernatant is washed 1-2 times with an appropriate amount of borate buffer (pH 7.5-8.5). The supernatant is removed by magnetic separation. The antibody to be labeled (GDF-15 monoclonal antibody or goat anti-rabbit IgG) is diluted with borate buffer (pH 7.5-8.5), added to the activated magnetic microparticles, vortexed and mixed, and placed on a constant temperature shaker at 20-30℃ and 100-200 rpm for 2-4 hours, or placed at 2-8℃ for 8-16 hours. After coupling, the supernatant was removed by magnetic separation, and an appropriate amount of blocking buffer (phosphate buffer with pH 7.2-7.6 containing 0.5%-2.0% BSA) was added. After vortexing and mixing, the mixture was placed on a constant temperature shaker and shaken at 20-30℃ and 100-200 rpm for 0.5-2 hours to block unreacted activated carboxyl groups. After sealing, the supernatant is removed by magnetic separation. The supernatant is washed 2-4 times with an appropriate amount of phosphate buffer (pH 7.2-7.6), vortexing for 20-60 seconds each time. The supernatant is then removed by magnetic separation. Finally, the magnetic particles are resuspended in phosphate buffer (pH 7.2-7.6) (containing 0.05%-0.2% BSA and 0.02%-0.1% ProClin300) and stored at 2-10°C in the dark for later use.

[0012] Furthermore, the specific steps of S2 are as follows: The prepared magnetic microparticle-labeled GDF-15 monoclonal antibody and magnetic microparticle-labeled goat anti-rabbit IgG were mixed at a volume ratio of 2:1, and the conjugate pad diluent was added to adjust the total protein concentration to 0.2-1.0 mg / ml. The mixed antibody solution was evenly sprayed onto the conjugate pad using a gold spraying membrane applicator, with a spraying amount of 0.5-4 μl / cm. After spraying, place the bonding pad in a constant temperature drying oven at 35-40℃ for 2-6 hours. After removing it, immediately put it into an aluminum foil bag, add desiccant, seal it, and store it at room temperature for later use.

[0013] Furthermore, the specific steps of S3 are as follows: Dilute the GDF-15 coated monoclonal antibody to 0.3-3.0 mg / ml with coating working solution (phosphate buffer, pH 7.2-7.6), and dilute the rabbit IgG to 0.1-1.5 mg / ml. Place the diluted antibody solution in a high-speed centrifuge and centrifuge at 10,000-15,000 rpm for 5-15 minutes to remove insoluble precipitates and collect the supernatant for later use. The GDF-15-coated monoclonal antibody was coated onto the NC membrane using a gold spraying membrane to form a detection line (T line), and rabbit IgG was coated onto the NC membrane to form a control line (C line). The distance between the T line and the C line was 4-6 mm, and the coating amount was 0.3-2.0 μl / cm. Place the coated NC film in a constant temperature drying oven at 35-40℃ for 8-24 hours. After removing it, immediately pack it into an aluminum foil bag, add desiccant, seal it, and store it at room temperature for later use.

[0014] Furthermore, the specific steps of S4 are as follows: Prepare the sample pad treatment solution: phosphate buffer solution with pH 7.2-7.6, containing 0.2%-1.0% BSA, 0.05%-0.2% Tween 20, 0.02%-0.1% ProClin 300, and 0.8%-1.0% sodium chloride; Completely immerse the sample pad in the treatment solution and soak at room temperature for 20-60 minutes, squeezing it 1-3 times during the process to remove air bubbles. Remove the sample pad, squeeze out excess liquid, and dry it in a constant temperature drying oven at 35-40℃ for 8-24 hours. After drying, put it in an aluminum foil bag, seal it, and store it at room temperature for later use.

[0015] Furthermore, the specific steps of S5 are as follows: The prepared NC film is pasted in the middle of the PVC base plate, and an absorbent pad is pasted on one end of the NC film. The overlap width between the absorbent pad and the NC film is 1-3mm. A bonding pad is attached to the other end of the NC membrane, with an overlap width of 1-2mm between the bonding pad and the NC membrane. Attach the sample pad to the other end of the conjugate pad, with an overlap width of 1-3mm between the sample pad and the conjugate pad; Use a cutting machine to cut the assembled large board into test strips 3-5mm wide; Insert the test strip into the U-shaped plastic clip, press them together to obtain the test card, put it into an aluminum foil bag, add desiccant and seal for storage.

[0016] Compared with existing technologies, this high-sensitivity GDF-15 weak magnetic field quantitative detection kit and its preparation method have the following advantages: This invention uses carboxylated superparamagnetic iron oxide microparticles with a particle size of 100-250 nm and a surface carboxyl content ≥100 μmol / g as a labeling carrier. By finely controlling the EDC / NHS activation coupling conditions and blocking process, the antibody labeling efficiency is improved by more than 30% compared to the conventional one-step activation method. While maintaining the antibody's biological activity, the limit of detection is reduced to 8 pg / ml. The optimized chromatography system effectively inhibits non-specific binding, with extremely low background signal. The discrimination is not less than 4.0 near the clinical critical concentration of 500 pg / ml, and the false positive rate and false negative rate are both controlled below 5%. The linear range covers 0-10000 pg / ml, and accurate quantification can be performed directly without multiple dilutions of high-value samples. The T / C ratio mode eliminates sample matrix effects and operational differences, with an intra-batch coefficient of variation of <10% and an inter-batch coefficient of variation of <15%.

[0017] Other advantages, objectives and features of the invention will be set forth in part in the description which follows, and in part will be apparent to those skilled in the art from the following examination or study, or may be learned from the practice of the invention. Attached Figure Description

[0018] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are merely some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without any creative effort.

[0019] Figure 1 A flowchart illustrating the preparation method of a high-sensitivity GDF-15 weak magnetic field quantitative detection kit; Figure 2 This is a flowchart of step S1 in the preparation method of a high-sensitivity GDF-15 weak magnetic quantitative detection kit. Detailed Implementation

[0020] To further illustrate the technical means and effects of the present invention in achieving its intended purpose, the following detailed description of the specific implementation methods, structures, features, and effects of the present invention, in conjunction with the accompanying drawings and preferred embodiments, is provided below.

[0021] Example 1 In this embodiment, magnetic microparticle-labeled GDF-15 monoclonal antibody and magnetic microparticle-labeled goat anti-rabbit IgG were prepared respectively.

[0022] The magnetic microparticle-labeled antibody preparation method described in this invention was used as the experimental group: Carboxylated superparamagnetic iron oxide microparticles: particle size 200 nm, saturation magnetization 60 emu / g, surface carboxyl content 120 μmol / g; like Figure 2 As shown, take 10 mg of magnetic microparticles, vortex mix for 5 minutes, transfer to a 2 ml centrifuge tube, magnetically separate for 3 minutes, and discard the supernatant; add 1 ml of 20 mM MEST buffer (20 mM MES, pH 5.0, containing 0.05% Tween 20), vortex mix for 30 seconds, magnetically separate to remove the supernatant, and repeat the washing 3 times. Add 500 μl of freshly prepared 10 mg / ml EDC solution and 500 μl of prepared 10 mg / ml NHS solution, vortex to mix, and place on a constant temperature shaker at 25°C and 180 rpm for 2 hours to activate. After activation, the supernatant was removed by magnetic separation, and the mixture was washed once with 1 ml of pH 8.0 20 mM borate buffer. The supernatant was removed by magnetic separation. 500 μg of GDF-15 monoclonal antibody or goat anti-rabbit IgG (diluted to 1 ml with pH 8.0 borate buffer) was added respectively. The mixture was vortexed and placed on a constant temperature shaker at 25°C and 150 rpm for 3 hours for coupling. After coupling, the supernatant was removed by magnetic separation, and 1 ml of blocking buffer (20 mM pH 7.4 PBS containing 1.0% BSA) was added. The mixture was then blocked at 25°C and 150 rpm for 1 hour. After blocking, the supernatant was removed by magnetic separation, washed three times with 20mM pH7.4 PBS buffer, and finally resuspended in 1ml of storage buffer (20mM pH7.4 PBS containing 0.1% BSA and 0.05% ProClin 300). Store at 2-8℃ protected from light for later use.

[0023] A control group was set up using existing conventional magnetic microparticle labeling technology. The specific steps are as follows: Pretreatment of magnetic particles: Same as the experimental group; Activation of magnetic microparticles: Add 1 ml of 5 mg / ml EDC solution, vortex to mix, and then activate at 25°C and 180 rpm for 1 hour. Antibody conjugation: Add 200 μg GDF-15 monoclonal antibody or goat anti-rabbit IgG, and conjugate at 25°C and 150 rpm for 2 hours. Sealing and preservation: Same as the experimental group procedure.

[0024] The content of free antibody in the supernatant after labeling was determined by the BCA method, and the antibody labeling efficiency was calculated. The immunological activity of the labeled antibody was determined by indirect ELISA. The results are shown in Table 1. Table 1. Comparison of antibody labeling efficiency and activity under different labeling processes The results show that the present invention significantly improves antibody labeling efficiency and biological activity by using an EDC / NHS dual activation system and optimizing activation time and antibody conjugation amount.

[0025] Example 2 In this embodiment, the magnetic microparticle-labeled antibody prepared in Example 1 was used to prepare a complete GDF-15 weak magnetic quantitative detection kit according to the optimal process parameters.

[0026] like Figure 1 As shown, the magnetic microparticle-labeled GDF-15 monoclonal antibody and the magnetic microparticle-labeled goat anti-rabbit IgG prepared in Example 1 were mixed at a volume ratio of 2:1, and the conjugate pad dilution buffer (20mM pH7.4 PBS, containing 0.5% BSA, 5% sucrose, and 0.05% ProClin300) was added to adjust the total protein concentration to 0.5 mg / ml. The mixed antibody solution was uniformly sprayed onto the glass fiber conjugate pad using a gold spraying film scrubber, with a spraying amount of 2 μl / cm. After spraying, place the bonding pad in a 37°C constant temperature drying oven for 3 hours. After removing it, immediately put it into an aluminum foil bag, add desiccant, seal it, and store it at room temperature for later use.

[0027] The GDF-15 coated monoclonal antibody was diluted to 1.0 mg / ml using the coating working solution (20 mM pH 7.4 PBS buffer), and the rabbit IgG was diluted to 0.5 mg / ml. Place the diluted antibody solution in a high-speed centrifuge and centrifuge at 12,000 rpm for 10 minutes to remove insoluble precipitate. Collect the supernatant for later use. GDF-15-coated monoclonal antibody was coated onto the NC membrane using a gold spraying membrane scribing device to form a detection line (T line), and rabbit IgG was coated onto the NC membrane to form a control line (C line). The distance between the T line and the C line was 5 mm, and the coating amount for both was 1 μl / cm. The coated NC film was placed in a 37°C constant temperature drying oven and dried for 18 hours. After removal, it was immediately placed in an aluminum foil bag, desiccant was added and it was sealed and stored at room temperature for later use.

[0028] Prepare the sample pad treatment solution: 20mM pH7.4 PBS buffer, containing 0.5% BSA, 0.1% Tween 20, 0.05% ProClin 300, and 0.9% sodium chloride; Completely immerse the glass fiber sample pad in the treatment solution and soak at room temperature for 30 minutes, gently squeezing it twice during the process to remove air bubbles. Remove the sample pad, squeeze out excess liquid with a wringer, and dry it in a 37°C constant temperature drying oven for 18 hours. After drying, put it in an aluminum foil bag, seal it, and store it at room temperature for later use.

[0029] The prepared NC film is pasted in the middle of the PVC base plate, and an absorbent pad is pasted on the upper end of the NC film. The overlap width between the absorbent pad and the NC film is 2mm. A bonding pad is attached to the lower end of the NC membrane, with an overlap width of 1mm between the bonding pad and the NC membrane. The sample pad is attached to the lower end of the conjugate pad, with an overlap width of 2mm between the sample pad and the conjugate pad. Use a cutting machine to cut the assembled large plate into 4mm wide test strips; Insert the test strip into the U-shaped plastic clip, press them together to obtain the test card, put it into an aluminum foil bag, add desiccant and seal for storage.

[0030] Example 3 This embodiment performs a comprehensive performance verification of the reagent kit prepared in Example 2.

[0031] Linear range verification: Experimental objective: To verify the linearity of the kit within the specified concentration range and to determine the effective detection interval.

[0032] Experimental materials: the reagent kit prepared in Example 2, GDF-15 series calibrators (0, 500, 1000, 2500, 5000, 10000 pg / ml), and a magnetic quantitative immunoassay analyzer.

[0033] Operating steps: The enterprise reference sample was brought to room temperature (25±2℃) and equilibrated for 30 minutes. Take 80 μl of each concentration calibrator and add it to the sample well of the test card respectively. React at room temperature for 15 minutes. Insert the test card into the magnetic quantitative immunoassay analyzer, which will automatically read the magnetic signal intensity of the T line and C line and calculate the T / C ratio. Each concentration calibrator was tested twice, and the results were recorded.

[0034] Data processing: Using the theoretical concentration of GDF-15 as the abscissa (Y) and the average T / C ratio as the ordinate (X), linear regression analysis was performed using the least squares method to calculate the regression equation and the correlation coefficient R².

[0035] Judgment criteria: A correlation coefficient R² ≥ 0.990 indicates a good linear relationship.

[0036] Experimental results: Table 2. Linearity range test results of GDF-15 series calibrators The linear regression equation is: y=0.00022x+0.038, with a correlation coefficient R²=0.998, which meets the judgment criteria, indicating that the kit has a good linear relationship in the range of 0-10000pg / ml.

[0037] Minimum detection limit verification: Experimental objective: To determine the lowest GDF-15 concentration that the kit can reliably detect.

[0038] Experimental materials: the kit prepared in Example 2, zero-concentration calibrator (0 pg / ml), and magnetic quantitative immunoassay analyzer.

[0039] Operating steps: The enterprise reference sample was brought to room temperature (25±2℃) and equilibrated for 30 minutes. Take 80 μl of zero-concentration calibrator and add it to the sample well of the test card. React at room temperature for 15 minutes. The T / C ratio was read using a magnetic quantitative immunoassay analyzer. Repeat the test on 20 zero-concentration calibrators and record all test results.

[0040] Data processing: Calculate the average value of 20 test results. and standard deviation (SD), in ( The concentration corresponding to +3SD was used as the lowest detection limit and substituted into the linear regression equation to calculate the specific value.

[0041] Judgment criteria: Limit of detection ≤ 10 pg / ml.

[0042] Experimental results: The average T / C value of 20 zero-concentration calibrator tests was calculated. =0.0456, standard deviation SD=0.0019, then +3SD=0.0513, substituting into the linear regression equation, the limit of detection is 8pg / ml, which meets the judgment criteria.

[0043] Precision verification: Experimental objective: To verify the repeatability and batch-to-batch consistency of the test results obtained by the kit.

[0044] Experimental materials: three different batches of reagent kits prepared in Example 2, low-value corporate reference material (500 pg / ml), medium-value corporate reference material (1000 pg / ml), high-value corporate reference material (5000 pg / ml), and a magnetic quantitative immunoassay analyzer.

[0045] Operating steps: Intra-batch precision: Take the same batch of reagent kits and test the low, medium and high value enterprise references respectively. Each enterprise reference is tested 10 times and the T / C ratio is recorded. Inter-batch precision: Take 3 different batches of reagent kits and test the above 3 concentrations of the enterprise reference material. Repeat the test 3 times for each batch and each enterprise reference material, and record the T / C ratio.

[0046] Data processing: Calculate the mean and standard deviation of the within-batch and between-batch test results respectively, and calculate the coefficient of variation according to the formula. : ; Judgment criteria: Intra-batch coefficient of variation <8%, inter-batch coefficient of variation <10%.

[0047] The experimental results are shown in Table 3: Table 3. Results of intra- and inter-batch precision assays for the reagent kits. The results showed that the intra-batch coefficient of variation of the kit was <8%, and the inter-batch coefficient of variation was <10%, which met the requirements for clinical testing.

[0048] Stability verification: Experimental objective: To verify the stability of the kit under accelerated degradation conditions and predict its shelf life.

[0049] Experimental materials: the reagent kit prepared in Example 2, low-value, medium-value, and high-value enterprise reference materials, magnetic quantitative immunoassay analyzer, and 37°C incubator.

[0050] Operating steps: Unopened reagent kits were placed in a 37°C incubator to accelerate degradation; The kits were removed on day 0 (initial value), day 3, and day 7, and after being brought to room temperature, the three concentrations of the enterprise reference products were tested. Each enterprise reference product was tested three times, and the average T / C ratio was recorded.

[0051] Data processing: Using the test results on day 0 as a baseline, calculate the rate of change between the test results on day 3 and day 7: Rate of change (%) = (Test value - Initial value) / Initial value × 100% Judgment criterion: After 7 days of accelerated degradation, the absolute value of the change rate of the enterprise reference test results at each concentration is <10%.

[0052] The experimental results are shown in Table 4: Table 4. Results of accelerated degradation stability test of the reagent kit at 37℃ The results showed that after 7 days of accelerated degradation, the change rate of the T / C value of the enterprise reference at each concentration was <10%, indicating that the kit has good stability and can be stably stored at 2-8℃ for 18 months.

[0053] 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 high-sensitivity GDF-15 weak magnetic field quantitative detection kit, characterized in that, Includes test cards and enterprise reference materials (not part of the reagent kit components); The test card consists of a test strip and a U-shaped buckle. The test strip includes a base plate and a sample pad, a conjugate pad, a nitrocellulose membrane, and an absorbent pad that are sequentially overlapped and pasted on the base plate. The conjugate pad is immobilized with magnetically microparticle-labeled GDF-15 monoclonal antibody and magnetically microparticle-labeled goat anti-rabbit IgG. The nitrocellulose membrane has a detection line and a control line that are separated from each other. The detection line is coated with another monoclonal antibody targeting different antigenic epitopes of GDF-15, and the control line is coated with rabbit IgG. The magnetic microparticles are carboxylated superparamagnetic iron oxide microparticles with a particle size of 150-250 nm, a saturation magnetization of ≥50 emu / g, and a surface carboxyl content of ≥100 μmol / g.

2. The high-sensitivity GDF-15 weak magnetic field quantitative detection kit according to claim 1, characterized in that, The enterprise reference materials include low-value enterprise reference materials with a concentration of 1000±50 pg / ml and high-value enterprise reference materials with a concentration of 5000±500 pg / ml.

3. The high-sensitivity GDF-15 weak magnetic field quantitative detection kit according to claim 1, characterized in that, The volume ratio of the magnetically microparticle-labeled GDF-15 monoclonal antibody to the magnetically microparticle-labeled goat anti-rabbit IgG is 2:

1.

4. A method for preparing a high-sensitivity GDF-15 weak magnetic field quantitative detection kit as described in any one of claims 1-3, characterized in that, Includes the following steps: S1. Preparation of magnetic microparticle-labeled antibodies: magnetic microparticle-labeled GDF-15 monoclonal antibody and magnetic microparticle-labeled goat anti-rabbit IgG were prepared respectively. S2. Preparation of the conjugate pad: The two magnetic microparticle-labeled antibodies were mixed and sprayed onto the conjugate pad using a gold spraying membrane applicator. After drying, the mixture was sealed and stored. S3. Preparation of nitrocellulose membrane: GDF-15 coated antibody and rabbit IgG were diluted to working concentrations and coated onto nitrocellulose membrane using a gold spraying membrane to form detection lines and control lines. After drying, the membrane was sealed and stored. S4. Preparation of sample pads: Soak the sample pads in the treatment solution and then dry them for later use. S5. Assembly of the test card: The sample pad, conjugate pad, nitrocellulose membrane and absorbent pad are overlapped and pasted on the base plate in sequence, cut into test strips, and put into the U-shaped clip to obtain the test card; S6. Kit assembly: Place the test card into an aluminum foil bag containing desiccant and seal it. Then, place the sealed test card into the kit.

5. The preparation method of a high-sensitivity GDF-15 weak magnetic field quantitative detection kit according to claim 4, characterized in that, In step S1, the preparation of the magnetic microparticle-labeled antibody specifically includes: After mixing the magnetic microparticles for 3-10 minutes, transfer them to a centrifuge tube, magnetically separate them for 2-5 minutes, remove the supernatant, and wash them 2-4 times with MES buffer at pH 4.5-5.

5. Add equal volumes of 5-15 mg / ml EDC solution and 5-15 mg / ml NHS solution to disperse the magnetic particles, and activate by shaking at 20-30℃ and 150-250 rpm for 1-3 hours. Remove the supernatant by magnetic attraction, disperse the antibody to be labeled in borate buffer at pH 7.5-8.5, add the activated magnetic microparticles, and couple by shaking at 20-30℃ and 100-200 rpm for 2-4 hours, or by standing at 2-8℃ for 8-16 hours. Remove the supernatant with a magnetic aspirator, add phosphate buffer (pH 7.2-7.6) containing 0.5%-2.0% bovine serum albumin, and incubate at 20-30°C and 100-200 rpm for 0.5-2 hours. Remove the supernatant using a magnetic aspirator, wash 2-4 times with phosphate buffer solution at pH 7.2-7.6, redisperse, and store at 2-10°C protected from light for later use.

6. The preparation method of a high-sensitivity GDF-15 weak magnetic field quantitative detection kit according to claim 4, characterized in that, The total protein concentration of the magnetic microparticle-labeled antibody mixture in S2 is 0.2-1.0 mg / ml, the spraying amount is 0.5-4 μl / cm, and it is dried at 35-40℃ for 2-6 hours.

7. The preparation method of the high-sensitivity GDF-15 weak magnetic field quantitative detection kit according to claim 4, characterized in that, In S3, the working concentration of GDF-15 coated antibody is 0.3-3.0 mg / ml, the working concentration of rabbit IgG is 0.1-1.5 mg / ml, the coating amount is 0.3-2.0 μl / cm, and it is dried at 35-40℃ for 8-24 hours.

8. The preparation method of a high-sensitivity GDF-15 weak magnetic field quantitative detection kit according to claim 4, characterized in that, In step S4, the sample pad is soaked in the treatment solution and then dried for later use. The soaking time is 20-60 minutes, and the pad is dried at 35-40℃ for 8-24 hours.

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