Kit for quantitatively detecting CRP (C-reactive protein) by using magnetic particle chemiluminescence method as well as preparation method and detection method of kit

Abstract

The invention provides a kit for quantitatively detecting CRP (C-reactive protein) by using a magnetic particle chemiluminescence method as well as a preparation method and a detection method of the kit. The kit comprises a CRP reagent 1, a CRP reagent 2, a CRP reagent 3, a CRP calibrator and a CRP quality control product, the CRP reagent 1 is prepared from magnetic beads coated with a CRP antibody and a diluent 1; the used CRP antibody is a biotin-labeled CRP antibody, and the used magnetic bead is a magnetic bead coupled with streptavidin; the CRP reagent 2 is a diluent 1; the CRP reagent 3 is prepared from an alkaline phosphatase labeled CRP antibody and a diluent 2; the diluent 1 is a Tris buffer solution containing bovine serum albumin; the diluent 2 is a Tris buffer solution containing bovine serum albumin and a protein stabilizer. The kit provided by the invention can be prepared at a lower cost, can realize high-precision quantitative determination of CRP, and has high sensitivity and good stability.

Description

Technical Field

[0001] This invention relates to the field of CRP kit technology, and in particular to a kit for quantitative detection of CRP using magnetic microparticle chemiluminescence assay, as well as its preparation and detection method. Background Technology

[0002] C-reactive protein (CRP) is an acute-phase reactant used to detect systemic inflammatory processes, assess antibiotic treatment for bacterial infections, detect intrauterine infections associated with preterm amniotic sac rupture, differentiate between active and inactive forms of disease-associated infections, monitor rheumatic diseases for therapeutic purposes and evaluate anti-inflammatory therapy, and identify postoperative complications early. Postoperative monitoring of CRP levels helps identify unexpected complications (persistently high or increasing levels).

[0003] Currently, the main clinical methods for detecting C-reactive protein (CRP) are immunochemical methods, such as immunoturbidimetry, fluorescence immunochromatography, latex immunoassay, chemiluminescence immunoassay, and ELISA. These methods all utilize the reaction between specific CRP antibodies and CRP in the sample, determining the positivity, negativity, or CRP content of the sample based on the resulting turbidity, band color development, agglutination, luminescence, or other immunoreaction results. Immunoturbidimetry, latex immunoturbidimetry, and magnetic particle chemiluminescence immunoassay are commonly used methods in clinical laboratories. However, immunoturbidimetry and latex immunoturbidimetry have limitations such as low sensitivity and poor precision. Magnetic particle chemiluminescence immunoassay, on the other hand, offers high sensitivity, stable results, rapid and easily automated magnetic separation technology, less contamination, and simple operation, making it widely used clinically.

[0004] The magnetic particle chemiluminescence CRP detection kit is used for sample detection. The detection principle is that an antigen-antibody immune reaction occurs, and the enzyme-catalyzed chemiluminescent substrate releases photons. The amount of photons is proportional to the amount of the analyte. This provides a new CRP detection method to solve the problems existing in current detection technologies. Summary of the Invention

[0005] This invention proposes a kit for quantitative detection of CRP using magnetic microparticle chemiluminescence, as well as its preparation and detection method. It can be prepared at a low cost and can achieve highly accurate quantitative determination of CRP, with high sensitivity and good stability.

[0006] The technical solution of the present invention is implemented as follows: a kit for quantitative detection of CRP using magnetic microparticle chemiluminescence method, comprising CRP reagent 1, CRP reagent 2, CRP reagent 3, CRP calibrator and CRP quality control sample;

[0007] The CRP reagent 1 is prepared by magnetic beads coated with CRP antibody and diluent 1; the CRP antibody used is a biotin-labeled CRP antibody, and the magnetic beads used are magnetic beads coupled with streptavidin.

[0008] The CRP reagent 2 is diluent 1;

[0009] The CRP reagent 3 is prepared from alkaline phosphatase-labeled CRP antibody and dilution solution 2;

[0010] Diluent 1 is a Tris buffer containing bovine serum albumin;

[0011] Diluent 2 is a Tris buffer containing bovine serum albumin and protein stabilizers.

[0012] Furthermore, each 1L of dilution 1 contains 10-14g of Tris, 20-35g of NaCl, 15-25g of sucrose, 8-12g of bovine serum albumin, 0.5-1.5mL of ProClin950 and 0.3-0.7mL of Tween-20, with the remainder being purified water.

[0013] Furthermore, each 1L of dilution solution 2 contains 10-14g of Tris, 7-11g of NaCl, 5-15g of D-trehalose, 40-60mL of protein stabilizer, 4-6g of bovine serum albumin, 4-6g of sodium caseinate, 0.05-0.15mL of Triton X100, 0.8-1.2mL of ProClin 300, and 8-12mL of glycerol, with the remainder being purified water.

[0014] Furthermore, the CRP calibrator includes two or more different concentration levels, and the CRP calibrator is prepared from CRP antigen and diluent 3. For example, it includes two different concentration levels, namely 2.5 μg / mL and 50 μg / mL.

[0015] Furthermore, the CRP quality control material includes two or more different concentration levels, and the CRP quality control material is prepared from CRP antigen and diluent 3. For example, it includes two levels (the two quality control levels exist in two different quality control materials, such as quality control material 1 and quality control material 2, where quality control material 1 is a low concentration and quality control material 2 is a high concentration; after testing the two quality control materials, the low and high concentration values ​​are obtained respectively). The concentration of the quality control material is specifically calculated based on the content of CRP antigen; for example, quality control material 1 contains 2.5 μg / mL CRP antigen, and quality control material 2 contains 50 μg / mL CRP antigen.

[0016] The detection principle of the kit is as follows: The sample to be tested, CRP reagent 1, and reagent 2 are mixed and incubated. C-reactive protein (CRP) in the sample specifically binds to the CRP antibody-biotin-streptomycin-magnetic beads in reagent 2, forming an immune complex. Unbound substances are washed away. Then, CRP reagent 3 is added, and CRP specifically binds to the CRP antibody in alkaline phosphatase. Unbound substances are washed away again. Alkaline phosphatase (ALP) catalyzes the substrate to emit light, and the relative luminescence intensity (RLU) is measured. Within a certain range, RLU is positively correlated with the CRP concentration in the sample. The CRP content in the sample can be calculated from the working curve using RLU.

[0017] Furthermore, each 1L of dilution solution 3 contains 10-14g of Tris, 7-11g of NaCl, 15-25g of D-trehalose, 8-12g of sodium caseinate, 0.01-0.03g of 4-aminoantipyrine, 0.05-0.15mL of Triton X 100, 0.8-1.2mL of ProClin 300, 8-12mL of glycerol, and 0.4-0.6g of CaCl2, with the remainder being purified water.

[0018] A method for preparing a kit for the quantitative detection of CRP using magnetic microparticle chemiluminescence includes the following steps:

[0019] Step 1: Prepare CRP reagent 1: Wash 0.02 mL of magnetic bead stock solution with dilution buffer 1 at room temperature to obtain washed magnetic beads. Then add 0.1 mL of dilution buffer 1 to the magnetic beads and mix well. Add 0.2 mg of biotin-labeled CRP antibody and mix well. React for 30 min. Then add 0.01 mL of blocking buffer and mix well. React for 15 min.

[0020] After sealing, the supernatant was discarded by magnetic separation, and then 0.5 mL of diluent 1 was added and the mixture was suspended and mixed. Then 0.04 mg of blocking agent was added and mixed to complete the coating of the magnetic bead suspension mother liquor. Finally, 0.1 mL of diluent 1 was added to obtain CRP reagent 1.

[0021] Step 2: Use diluent 1 as CRP reagent 2;

[0022] Step 3: Prepare CRP reagent 3: Mix 800 mL of dilution buffer 2 and 0.5 mg of alkaline phosphatase-labeled CRP antibody, and bring the volume to 1 L with purified water to obtain CRP reagent 3.

[0023] Step 4: Prepare CRP calibrators: Use diluent 3 to prepare CRP antigen at two or more different concentration points;

[0024] Step 5: Prepare CRP quality control samples: Use diluent 3 to prepare CRP antigen at two or more different concentration points.

[0025] Further, in steps 1 and 2, the preparation method of diluent 1 is as follows: Add 800 mL of purified water, 10-14 g of Tris and 20-35 g of NaCl, stir until completely dissolved, adjust the pH value to 8.0±0.1, then add 15-25 g of sucrose, 8-12 g of bovine serum albumin, 0.5-1.5 mL of ProClin950 and 0.3-0.7 mL of Tween-20, stir until completely dissolved, and bring the volume to 1 L with purified water;

[0026] In step 3, the preparation method of diluent 2 is as follows: Add 800mL of purified water, 10-14g of Tris and 7-11g of NaCl, stir until completely dissolved, adjust the pH value to 8.0±0.1, then add 5-15g of D-trehalose, 40-60mL of protein stabilizer, 4-6g of bovine serum albumin, 4-6g of sodium caseinate, 0.05-0.15mL of Triton X 100, 0.8-1.2mL of ProClin 300 and 8-12mL of glycerol, stir until completely dissolved, and bring the volume to 1L with purified water.

[0027] In steps 4 and 5, the preparation method of diluent 3 is as follows: Stir 800 mL of purified water, 10-14 g of Tris and 7-11 g of NaCl until completely dissolved, adjust the pH value to 7.4±0.1, then add 15-25 g of D-trehalose, 8-12 g of sodium caseinate, 0.01-0.03 g of 4-aminoantipyrine, 0.05-0.15 mL of Triton X100, 0.8-1.2 mL of ProClin 300, 8-12 mL of glycerol and 0.4-0.6 g of CaCl2, stir until completely dissolved, and bring the volume to 1 L with purified water.

[0028] A detection method for a kit using magnetic microparticle chemiluminescence immunoassay to quantitatively detect CRP includes the following steps:

[0029] (1) After mixing CRP calibrator, CRP reagent 1 and CRP reagent 2, incubate at 37°C for 5 min, perform magnetic separation, remove the supernatant, add cleaning solution to wash, repeat the washing multiple times to obtain intermediate A;

[0030] (2) Add CRP reagent 3 to intermediate A, mix well, incubate at 37°C for 5 min, perform magnetic separation, and remove the supernatant; then add washing solution to wash, repeat washing several times to obtain intermediate B;

[0031] (3) Add the substrate solution for the fully automated immunoassay system to intermediate B, mix well, and use a fully automated chemiluminescence immunoassay analyzer to detect the luminescence intensity to obtain the working curve after CRP calibration.

[0032] (4) Replace the CRP calibrator with the CRP quality control sample, repeat steps (1)-(3), obtain the luminescence value of the CRP quality control sample, fit the luminescence value with the working curve obtained in step (3), and obtain the concentration value of the CRP quality control sample.

[0033] (5) Replace the CRP calibrator with the sample to be tested, and repeat steps (1)-(3) to obtain the concentration value of the sample to be tested.

[0034] Compared with the prior art, the present invention has the following advantages:

[0035] 1. High sensitivity; the detection limit of this kit is no higher than 0.1 μg / mL.

[0036] 2. The components have high activity. The stability of reagent 3 is increased by adding a protein stabilizer. Accelerated treatment is carried out at 37°C. After 14 days, the signal retention rate is greater than 85%.

[0037] 3. It has good specificity, with cross-reactivity rates of less than 0.1% with both procalcitonin (PCT) and heparin-binding protein (HBP).

[0038] 4. It has good stability and can be stored at 37℃ for more than 7 days. Unopened reagent kits can be stored at 2℃~8℃ in a sealed container protected from light for 15 months.

[0039] 5. This invention employs a two-step sandwich reaction mode (the test sample, calibrator, and quality control contain CRP antigen, and reagents 1 and 3 both contain CRP antibody; the reaction process after adding the test sample, calibrator, quality control, and reagents to the reaction vessel is a double-antibody sandwich method). Utilizing the principle of combining magnetic microparticle chemiluminescence detection technology with magnetic microparticle immunoassay technology, it quantitatively detects the C-reactive protein content in human serum and plasma samples, significantly improving the sensitivity, linear range, accuracy, and precision of the detection. Furthermore, it has low requirements for sample pretreatment and can rapidly and efficiently detect large batches of samples, facilitating clinical reagent application. This invention provides a more accurate, precise, convenient, rapid, and simple method for clinical detection of C-reactive protein in human serum and plasma.

[0040] 6. The detection method described herein, when used in conjunction with a fully automated chemiluminescence immunoassay analyzer, achieves fully automated operation, is easy to operate, and provides objective results. It has significant value for clinical diagnosis and has broad application prospects.

[0041] 7. This invention's kit uses magnetic microparticle chemiluminescence immunoassay for the quantitative detection of C-reactive protein (CRP). Reagent 1 consists of biotin-labeled CRP antibody coated with streptomycin-conjugated magnetic beads and diluent 1; reagent 3 consists of alkaline phosphatase-labeled CRP antibody and diluent 2. This ensures accurate detection while increasing the linear detection range, achieving an upper detection limit of 100 μg / ml, and maintaining linearity within the detection range. This invention is specifically designed for CRP, improving the upper detection limit. Two control concentrations are provided based on CRP content, ensuring excellent detection performance throughout the linear range. The magnetic microparticle chemiluminescence immunoassay method offers advantages such as less contamination, higher sensitivity, simpler operation, higher automation, and lower cost.

[0042] 8. The preparation method of this invention selects specific components and specific reagents in specific amounts, realizing high-precision and rapid detection of CRP. In CRP reagent 1, the CRP antibody used is a biotin-labeled CRP antibody, and the magnetic beads used are magnetic beads coupled with streptavidin. The biotin-streptavidin reaction is an in vitro biological technique. Biotin and streptavidin have a strong affinity and a very fast binding speed. The magnetic bead coating of the antibody only needs to react at room temperature for 30 minutes. Traditional plate luminescence requires the coating reaction to be carried out at 2℃~8℃, and the entire coating process takes about 2~3 days. Detailed Implementation

[0043] The technical solutions in the embodiments of the present invention will be clearly and completely described below. 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 of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0044] In the specific embodiments of this invention, the protein stabilizer used is protein stabilizer 5 from Shenzhen Kemisen Biotechnology Co., Ltd.; the blocking solution, cleaning solution, and substrate solution for the fully automated immunoassay system were all purchased from Sichuan Xieguang Biotechnology Co., Ltd. The registration number of the cleaning solution is: Chuanrong Medical Device Registration No. 20190083, and the registration number of the substrate solution for the fully automated immunoassay system is: Chuanrong Medical Device Registration No. 20190085.

[0045] Example 1

[0046] A kit for the quantitative detection of human C-reactive protein (CRP) using magnetic microparticle chemiluminescence assay, the kit comprising CRP reagent 1, CRP reagent 2, CRP reagent 3, CRP calibrator, and CRP quality control.

[0047] The preparation method of the reagent kit includes the following steps:

[0048] Step 1

[0049] Step 1: Prepare diluent 1:

[0050] Add 800 mL of purified water, 12 g of Tris and 27 g of NaCl to a 1 L container and stir until completely dissolved. Adjust the pH to 8.0 ± 0.1. Then add 20 g of sucrose, 10 g of bovine serum albumin, 1 mL of ProClin950 and 0.5 mL of Tween-20 and stir until completely dissolved. Make up the volume to 1 L with purified water to obtain dilution 1. Store the obtained dilution 1 at 2℃~8℃ for later use.

[0051] Preparation of CRP reagent 1:

[0052] At room temperature, remove the magnetic beads and mix well; take 0.05 mL of dilution buffer 1, add 0.02 mL of the original magnetic bead solution, and mix well; place on a magnetic separator and react for 15 min, then discard the supernatant. Repeat 3 times to complete the washing; add 0.1 mL of dilution buffer 1 and mix well; then add 0.2 mg of biotin-labeled CRP antibody and mix well; place on a mixer and react for 30 min; add 0.01 mL of blocking buffer and mix well; place on a mixer and react for 15 min; after blocking, place on a magnetic separator at room temperature. After 15 minutes, discard the supernatant; add 0.5 mL of diluent 1, suspend and mix well; add 0.02 mg of inhibitor 1 (abbreviation of inhibitor BRO1), 0.01 mg of inhibitor 12, and 0.01 mg of inhibitor 13 (inhibitors 1, 12, and 13 were all purchased from Shenzhen Kemisen Biotechnology Co., Ltd.), and mix well; the magnetic microsphere suspension mother liquor coating is complete; add 0.1 mL of diluent 1 to obtain the CRP reagent 1, and store the obtained CRP reagent 1 at 2℃~8℃ for later use;

[0053] Step 2: Prepare CRP reagent 2, using the same method as diluent 1;

[0054] Step 3: Prepare diluent 2:

[0055] Add 800 mL of purified water, 12 g of Tris, and 9 g of NaCl to a 1 L container and stir until completely dissolved. Adjust the pH to 8.0 ± 0.1. Then add 10 g of D-trehalose, 50 mL of protein stabilizer, 5 g of bovine serum albumin, 5 g of sodium caseinate, 0.1 mL of Triton X 100, 1 mL of ProClin 300, and 10 mL of glycerol. Stir until completely dissolved and bring the volume to 1 L with purified water to obtain dilution 2. Store the obtained dilution 2 at 2℃~8℃ for later use.

[0056] Preparation of CRP reagent 3:

[0057] Add 800 mL of diluent 2 to a 1 L container, then add 0.5 mg of alkaline phosphatase-labeled CRP antibody; stir until completely mixed, and bring the volume up to 1 L with purified water to obtain the CRP reagent 3. Store the obtained CRP reagent 3 at 2℃~8℃ for later use.

[0058] Step 4: Prepare diluent 3:

[0059] Add 800 mL of purified water, 12 g of Tris and 9 g of NaCl to a 1 L container and stir until completely dissolved. Adjust the pH to 7.4 ± 0.1. Then add 20 g of D-trehalose, 10 g of sodium caseinate, 0.02 g of 4-aminoantipyrine, 0.1 mL of Triton X 100, 1 mL of ProClin 300, 10 mL of glycerol and 0.555 g of CaCl2 and stir until completely dissolved. Make up the volume to 1 L with purified water to obtain dilution 3. Store the obtained dilution 3 at 2℃~8℃ for later use.

[0060] Preparation of CRP calibrators:

[0061] The C-reactive protein (CRP) antigen was prepared into two concentrations of 2.5 μg / mL and 50 μg / mL using the above dilution solution 3.

[0062] Step 4: Prepare CRP quality control samples:

[0063] The C-reactive protein (CRP) antigen was prepared into two concentrations of 2.5 μg / mL and 50 μg / mL using the above dilution solution 3.

[0064] Example 2

[0065] A kit for the quantitative detection of human C-reactive protein (CRP) using magnetic microparticle chemiluminescence assay, the kit comprising CRP reagent 1, CRP reagent 2, CRP reagent 3, CRP calibrator, and CRP quality control.

[0066] The preparation method of the reagent kit includes the following steps:

[0067] Step 1: Prepare diluent 1:

[0068] Add 800 mL of purified water, 10 g of Tris and 20 g of NaCl to a 1 L container and stir until completely dissolved. Adjust the pH to 8.0 ± 0.1. Then add 15 g of sucrose, 8 g of bovine serum albumin, 0.5 mL of ProClin950 and 0.3 mL of Tween-20 and stir until completely dissolved. Make up the volume to 1 L with purified water to obtain dilution 1. Store the obtained dilution 1 at 2℃~8℃ for later use.

[0069] Preparation of CRP reagent 1:

[0070] At room temperature, remove the magnetic beads and mix well; take 0.05 mL of dilution buffer 1, add 0.02 mL of the original magnetic bead solution, and mix well; place on a magnetic separator and react for 15 min, then discard the supernatant, repeat 3 times to complete the washing; add 0.1 mL of dilution buffer 1 and mix well; then add 0.2 mg of biotin-labeled CRP antibody and mix well; place on a mixer and react for 30 min; add 0.01 mL of blocking buffer and mix well; place on a mixer and react for 15 min; after blocking, at room temperature, place on a magnetic separator and react for 15 min, then discard the supernatant; add 0.5 mL of dilution buffer 1 and suspend and mix well; add 0.02 mg of blocking agent 1, 0.01 mg of blocking agent 12, and 0.01 mg of blocking agent 13, and mix well; the magnetic microsphere suspension mother liquor coating is complete; add 0.1 mL of dilution buffer 1 to obtain the CRP reagent 1, and store the obtained CRP reagent 1 at 2℃~8℃ for later use;

[0071] Step 2: Prepare CRP reagent 2, using the same method as diluent 1;

[0072] Step 3: Prepare diluent 2:

[0073] Add 800 mL of purified water, 10 g of Tris and 7 g of NaCl to a 1 L container and stir until completely dissolved. Adjust the pH to 8.0 ± 0.1. Then add 5 g of D-trehalose, 40 mL of protein stabilizer, 4 g of bovine serum albumin, 4 g of sodium caseinate, 0.05 mL of Triton X 100, 0.8 mL of ProClin 300 and 8 mL of glycerol and stir until completely dissolved. Make up the volume to 1 L with purified water to obtain dilution 2. Store the obtained dilution 2 at 2℃~8℃ for later use.

[0074] Preparation of CRP reagent 3:

[0075] Add 800 mL of diluent 2 to a 1 L container, then add 0.5 mg of alkaline phosphatase-labeled CRP antibody; stir until completely mixed, and bring the volume up to 1 L with purified water to obtain the CRP reagent 3. Store the obtained CRP reagent 3 at 2℃~8℃ for later use.

[0076] Step 4: Prepare diluent 3:

[0077] Add 800 mL of purified water, 10 g of Tris, and 7 g of NaCl to a 1 L container and stir until completely dissolved. Adjust the pH to 7.4 ± 0.1. Then add 15 g of D-trehalose, 8 g of sodium caseinate, 0.01 g of 4-aminoantipyrine, 0.05 mL of Triton X 100, 0.8 mL of ProClin 300, 8 mL of glycerol, and 0.4 g of CaCl2. Stir until completely dissolved and bring the volume up to 1 L with purified water to obtain dilution 3. Store the obtained dilution 3 at 2℃~8℃ for later use.

[0078] Preparation of CRP calibrators:

[0079] The C-reactive protein (CRP) antigen was prepared into two concentrations of 2.5 μg / mL and 50 μg / mL using the above dilution solution 3.

[0080] Step 4: Prepare CRP quality control samples:

[0081] The C-reactive protein (CRP) antigen was prepared into two concentrations of 2.5 μg / mL and 50 μg / mL using the above dilution solution 3.

[0082] Example 3

[0083] A kit for the quantitative detection of human C-reactive protein (CRP) using magnetic microparticle chemiluminescence assay, the kit comprising CRP reagent 1, CRP reagent 2, CRP reagent 3, CRP calibrator, and CRP quality control.

[0084] The preparation method of the reagent kit includes the following steps:

[0085] Step 1: Prepare diluent 1:

[0086] Add 800 mL of purified water, 14 g of Tris and 35 g of NaCl to a 1 L container and stir until completely dissolved. Adjust the pH to 8.0 ± 0.1. Then add 25 g of sucrose, 12 g of bovine serum albumin, 1.5 mL of ProClin950 and 0.7 mL of Tween-20 and stir until completely dissolved. Make up the volume to 1 L with purified water to obtain dilution 1. Store the obtained dilution 1 at 2℃~8℃ for later use.

[0087] Preparation of CRP reagent 1:

[0088] At room temperature, remove the magnetic beads and mix well; take 0.05 mL of dilution buffer 1, add 0.02 mL of the original magnetic bead solution, and mix well; place on a magnetic separator and react for 15 min, then discard the supernatant, repeat 3 times to complete the washing; add 0.1 mL of dilution buffer 1 and mix well; then add 0.2 mg of biotin-labeled CRP antibody and mix well; place on a mixer and react for 30 min; add 0.01 mL of blocking buffer and mix well; place on a mixer and react for 15 min; after blocking, at room temperature, place on a magnetic separator and react for 15 min, then discard the supernatant; add 0.5 mL of dilution buffer 1 and suspend and mix well; add 0.02 mg of blocking agent 1, 0.01 mg of blocking agent 12, and 0.01 mg of blocking agent 13, and mix well; the magnetic microsphere suspension mother liquor coating is complete; add 0.1 mL of dilution buffer 1 to obtain the CRP reagent 1, and store the obtained CRP reagent 1 at 2℃~8℃ for later use;

[0089] Step 2: Prepare CRP reagent 2, using the same method as diluent 1;

[0090] Step 3: Prepare diluent 2:

[0091] Add 800 mL of purified water, 14 g of Tris, and 11 g of NaCl to a 1 L container and stir until completely dissolved. Adjust the pH to 8.0 ± 0.1. Then add 15 g of D-trehalose, 60 mL of protein stabilizer, 6 g of bovine serum albumin, 6 g of sodium caseinate, 0.15 mL of Triton X 100, 1.2 mL of ProClin 300, and 12 mL of glycerol. Stir until completely dissolved and bring the volume to 1 L with purified water to obtain dilution 2. Store the obtained dilution 2 at 2℃~8℃ for later use.

[0092] Preparation of CRP reagent 3:

[0093] Add 800 mL of diluent 2 to a 1 L container, then add 0.5 mg of alkaline phosphatase-labeled CRP antibody; stir until completely mixed, and bring the volume up to 1 L with purified water to obtain the CRP reagent 3. Store the obtained CRP reagent 3 at 2℃~8℃ for later use.

[0094] Step 4: Prepare diluent 3:

[0095] Add 800 mL of purified water, 14 g of Tris, and 11 g of NaCl to a 1 L container and stir until completely dissolved. Adjust the pH to 7.4 ± 0.1. Then add 25 g of D-trehalose, 12 g of sodium caseinate, 0.03 g of 4-aminoantipyrine, 0.15 mL of Triton X 100, 1.2 mL of ProClin 300, 12 mL of glycerol, and 0.6 g of CaCl2. Stir until completely dissolved and bring the volume up to 1 L with purified water to obtain dilution 3. Store the obtained dilution 3 at 2℃~8℃ for later use.

[0096] Preparation of CRP calibrators:

[0097] The C-reactive protein (CRP) antigen was prepared into two concentrations of 2.5 μg / mL and 50 μg / mL using the above dilution solution 3.

[0098] Step 4: Prepare CRP quality control samples:

[0099] The C-reactive protein (CRP) antigen was prepared into two concentrations of 2.5 μg / mL and 50 μg / mL using the above dilution solution 3.

[0100] Comparative Example 1

[0101] A kit for the quantitative detection of human C-reactive protein (CRP) using magnetic microparticle chemiluminescence assay, the kit comprising CRP reagent 1, CRP reagent 2, CRP reagent 3, CRP calibrator, and CRP quality control.

[0102] The preparation method of the reagent kit includes the following steps:

[0103] Step 1: Prepare diluent 1:

[0104] Add 800 mL of purified water, 12 g of Tris and 27 g of NaCl to a 1 L container and stir until completely dissolved. Adjust the pH to 8.0 ± 0.1. Then add 20 g of sucrose, 10 g of bovine serum albumin, 1 mL of ProClin950 and 0.5 mL of Tween-20 and stir until completely dissolved. Make up the volume to 1 L with purified water to obtain dilution 1. Store the obtained dilution 1 at 2℃~8℃ for later use.

[0105] Preparation of CRP reagent 1:

[0106] At room temperature, remove the magnetic beads and mix well; take 0.05 mL of dilution buffer 1, add 0.02 mL of the original magnetic bead solution, and mix well; place on a magnetic separator and react for 15 min, then discard the supernatant, repeat 3 times to complete the washing; add 0.1 mL of dilution buffer 1 and mix well; then add 0.2 mg of biotin-labeled CRP antibody and mix well; place on a mixer and react for 30 min; add 0.01 mL of blocking buffer and mix well; place on a mixer and react for 15 min; after blocking, at room temperature, place on a magnetic separator and react for 15 min, then discard the supernatant; add 0.5 mL of dilution buffer 1 and suspend and mix well; add 0.02 mg of blocking agent 1, 0.01 mg of blocking agent 12, and 0.01 mg of blocking agent 13, and mix well; the magnetic microsphere suspension mother liquor coating is complete; add 0.1 mL of dilution buffer 1 to obtain the CRP reagent 1, and store the obtained CRP reagent 1 at 2℃~8℃ for later use;

[0107] Step 2: Prepare CRP reagent 2, using the same method as diluent 1;

[0108] Step 3: Prepare diluent 2:

[0109] Add 800 mL of purified water, 12 g of Tris and 9 g of NaCl to a 1 L container and stir until completely dissolved. Adjust the pH to 8.0 ± 0.1. Then add 10 g of D-trehalose, 5 g of bovine serum albumin, 5 g of sodium caseinate, 0.1 mL of Triton X100, 1 mL of ProClin 300 and 10 mL of glycerol and stir until completely dissolved. Make up the volume to 1 L with purified water to obtain dilution 2. Store the obtained dilution 2 at 2℃~8℃ for later use.

[0110] Preparation of CRP reagent 3:

[0111] Add 800 mL of diluent 2 to a 1 L container, then add 0.5 mg of alkaline phosphatase-labeled CRP antibody; stir until completely mixed, and bring the volume up to 1 L with purified water to obtain the CRP reagent 3. Store the obtained CRP reagent 3 at 2℃~8℃ for later use.

[0112] Step 4: Prepare diluent 3:

[0113] Add 800 mL of purified water, 12 g of Tris and 9 g of NaCl to a 1 L container and stir until completely dissolved. Adjust the pH to 7.4 ± 0.1. Then add 20 g of D-trehalose, 10 g of sodium caseinate, 0.02 g of 4-aminoantipyrine, 0.1 mL of Triton X 100, 1 mL of ProClin 300, 10 mL of glycerol and 0.555 g of CaCl2 and stir until completely dissolved. Make up the volume to 1 L with purified water to obtain dilution 3. Store the obtained dilution 3 at 2℃~8℃ for later use.

[0114] Preparation of CRP calibrators:

[0115] The C-reactive protein (CRP) antigen was prepared into two concentrations of 2.5 μg / mL and 50 μg / mL using the above dilution solution 3.

[0116] Step 4: Prepare CRP quality control samples:

[0117] The C-reactive protein (CRP) antigen was prepared into two concentrations of 2.5 μg / mL and 50 μg / mL using the above dilution solution 3.

[0118] Experiment 1

[0119] Precision comparison test: After 6 months of storage, the reagents prepared using the formulations of Examples 1-3 and Comparative Example 1 were tested on low-value samples (target value 2.5 μg / mL) and high-value samples (target value 50 μg / mL). Each sample was tested 20 times, and the mean, standard deviation, and coefficient of variation were calculated. The results are shown in Tables 1-1 and 1-2.

[0120] Table 1-1 Precision Test Data for Low-Value Samples

[0121] Example 1 Example 2 Example 3 Comparative Example 1 average value 2.48 2.47 2.51 2.37 Standard deviation 0.052 0.075 0.088 0.099 coefficient of variation 2.09％ 3.02％ 3.49％ 4.17％

[0122] Table 1-2 Precision Test Data for High-Value Samples

[0123] Example 1 Example 2 Example 3 Comparative Example 1 average value 50.10 49.08 49.83 44.03 Standard deviation 1.461 1.856 1.891 1.933 coefficient of variation 2.92％ 3.78％ 3.79％ 4.39％

[0124] As shown by the coefficients of variation in Tables 1-1 and 1-2, compared with Comparative Example 1, the reagents prepared according to the formulations in Examples 1-3 have detection values ​​closer to the target values, with smaller standard deviations and coefficients of variation, indicating higher intra-batch precision. This demonstrates that the addition of a protein stabilizer to reagent 3 of the present invention optimizes the reaction system and greatly improves the intra-batch precision of the reagent.

[0125] Experiment 2

[0126] Accuracy comparison test: After 6 months of storage, the reagents prepared using the formulations of Examples 1-3 and Comparative Example 1 were tested on low-value samples (target value 2.5 μg / mL) and high-value samples (target value 50 μg / mL). Each sample was tested three times, and the relative deviation between the measured value and the labeled value was calculated. The results are shown in Tables 2-1 and 2-2.

[0127] Table 2-1 Accuracy Test Data for Low-Value Samples

[0128] Example 1 Example 2 Example 3 Comparative Example 1 Relative deviation 1 0.40％ 0.80％ -1.20％ -5.20％ Relative deviation 2 0.42％ -0.84％ 1.16％ 3.85％ Relative deviation 3 -0.51％ 0.93％ 1.34％ 4.72％

[0129] Table 2-2 Accuracy Test Data for High-Value Samples

[0130] Example 1 Example 2 Example 3 Comparative Example 1 Relative deviation 1 0.73％ -1.12％ 1.54％ 3.96％ Relative deviation 2 -0.80％ 0.97％ 1.73％ -4.31％ Relative deviation 3 0.94％ 1.04％ 2.00％ -4.54％

[0131] As can be seen from the relative deviations in Tables 2-1 and 2-2, compared with Comparative Example 1, the reagents prepared according to the formulations in Examples 1-3 all showed smaller relative deviations and higher accuracy. This indicates that the addition of a protein stabilizer to reagent 3 of the present invention optimized the reaction system and greatly improved the accuracy of the reagent.

[0132] Experiment 3

[0133] Sensitivity comparison test: After 6 months of storage, the reagents prepared using the formulations of Examples 1-3 and Comparative Example 1 were used to detect 10 concentration samples diluted from the linear high-value sample from low to high concentration. The detection results were compared with the theoretical concentration. The results are shown in Table 3.

[0134] Table 3 Sensitivity Test Data

[0135] Theoretical concentration Example 1 Example 2 Example 3 Comparative Example 1 0.1 μg / mL 0.10 0.09 0.11 0.04 0.25 μg / mL 0.24 0.26 0.26 0.18 0.5 μg / mL 0.52 0.49 0.53 0.37 1μg / mL 1.03 1.12 1.08 0.86 2.5 μg / mL 2.54 2.51 2.49 2.33 5μg / mL 5.02 5.13 5.07 4.50 10 μg / mL 10.17 10.39 11.05 8.74 25μg / mL 25.30 26.12 24.38 23.16 50 μg / mL 50.08 49.37 48.61 44.07 100 μg / mL 100.34 98.74 97.85 85.36

[0136] As shown in Table 3, when the sample concentration was as low as 0.1 μg / mL, the detection value of Comparative Example 1 was 0.04 μg / mL, while the reagents prepared in Examples 1-3 could still detect the accurate value of the sample. Furthermore, compared to Comparative Example 1, the reagents prepared in Examples 1-3 showed higher accuracy in detecting low-value samples (0-1 μg / mL) close to the lower limit of linearity. Additionally, when the sample concentration was at the upper limit of linearity (100 μg / mL), the detection value of Comparative Example 1 was 85.36 μg / mL, which was significantly lower than expected, while the reagents prepared in Examples 1-3 could still detect the accurate value of the sample. This indicates that the reagents prepared in Examples 1-3 have higher analytical sensitivity, accuracy, and a wider linear range. This demonstrates that the addition of a protein stabilizer to Reagent 3 of the present invention optimizes the reaction system and greatly improves the analytical sensitivity, accuracy, and linear range of the reagent.

[0137] Inspection of the kit described in this invention:

[0138] (1) Appearance: All components should be complete and intact, with no liquid leakage; packaging labels should be clear, easy to identify, and secure;

[0139] (2) Limit of detection: The limit of detection of the kit is not higher than 0.1 μg / mL;

[0140] (3) Linearity: The correlation coefficient (r) is not less than 0.9900 in the range of 0.1 to 100 μg / mL when the line is fitted by the least squares method.

[0141] (4) Accuracy: High-value and low-value reference materials are used as samples for testing. The test is repeated 3 times. The relative deviation between the measurement result and the labeled concentration is calculated. Relative deviation = (measurement result - labeled concentration) / labeled concentration × 100%. The result should meet the requirement that the relative deviation is within ±10.0%.

[0142] (5) Precision: High-value and low-value reference samples were used as samples for testing. The test was repeated 10 times, and the mean and standard deviation of the 10 test results were calculated. Repeatability (CV) = standard deviation / mean × 100%; 3 batches of products were used to conduct 10 tests, and the mean of the 10 measurement results was calculated. i and the average value of 30 measurements Batch variation The results should meet the following requirements: repeatability coefficient of variation (CV) not greater than 10.0%; inter-batch relative range not greater than 15.0%.

[0143] (6) Specificity: Cross-reactivity meets the requirements in the table below:

[0144] Testing cross-reactants concentration Interference Procalcitonin (PCT) 100ng / mL <0.1％ Heparin-binding protein (HBP) 100 μg / mL <0.1％

[0145] (7) Stability: After being placed at 37℃ for 7 days, the measured values ​​should meet all the above requirements;

[0146] Example 2

[0147] A detection method for a kit for quantitative detection of CRP using magnetic microparticle chemiluminescence immunoassay, the detection method of the kit comprising the following steps:

[0148] Step 1: Place the CRP calibrator in the test position of the fully automated chemiluminescence immunoassay analyzer. The instrument scans the QR code of the main calibration curve. After calibration with the calibrator, a working curve is obtained, which can then be used to test samples.

[0149] Step 2: Place the CRP quality control sample at the test position of the analyzer to obtain the luminescence value of the quality control sample output by the fully automated chemiluminescence immunoassay analyzer and the concentration value of the CRP quality control sample obtained by fitting the working curve obtained in Step 1.

[0150] Step 3: Place the sample to be tested at the test position of the analyzer to obtain the concentration value of the sample output by the fully automated chemiluminescence immunoassay analyzer.

[0151] The detection method using the magnetic microparticle chemiluminescence reagent kit for quantitative detection of CRP described above includes fully automated detection steps in steps 1, 2, and 3 using a fully automated chemiluminescence immunoassay analyzer.

[0152] Step 1) For step 1, add 5 μL of CRP calibrator to the reaction vessel; for step 2, add 5 μL of CRP quality control to the reaction vessel; for step 3, add 5 μL of the sample to be tested to the reaction vessel.

[0153] Step 2) Add 50 μL of CRP reagent 1 and 100 μL of CRP reagent 2 to the reaction vessel described in Step 1), mix well, incubate at 37°C for 5 min, perform magnetic separation, and remove the supernatant;

[0154] Step 3) Add 500 μL of cleaning solution to the reaction vessel, mix well, perform magnetic separation, and remove the supernatant;

[0155] Step 4) Repeat step 3) twice;

[0156] Step 5) Add 100 μL of CRP reagent 3 to the reaction vessel described in step 3), mix well, incubate at 37°C for 5 min, perform magnetic separation, and remove the supernatant;

[0157] Step 6) Add 500 μL of cleaning solution to the reaction vessel, mix well, perform magnetic separation, and remove the supernatant;

[0158] Step 7) Repeat step 6) twice;

[0159] Step 8) Add 200 μL of substrate solution for the fully automated immunoassay system to the reaction vessel, mix well, and detect the luminescence intensity.

[0160] Methodological evaluation results of the reagent kit of this invention:

[0161] Detection range: 0.1–100 μg / mL. For samples with a concentration greater than 100 μg / mL, they should be diluted before measurement.

[0162] Limit of detection: 0.1 μg / mL;

[0163] Precision: less than 8%;

[0164] Accuracy: Relative deviation within ±10.0%;

[0165] Specificity: Cross-reactivity with both PCT and HBP is less than 0.1%;

[0166] Stability: The reagent components in the kit showed good stability after being placed at 37°C for 7 days.

[0167] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A kit for the quantitative detection of CRP using magnetic microparticle chemiluminescence immunoassay, characterized in that: This includes CRP reagent 1, CRP reagent 2, CRP reagent 3, CRP calibrators, and CRP quality control products; The CRP reagent 1 is prepared by magnetic beads coated with CRP antibody and diluent 1; the CRP antibody used is a biotin-labeled CRP antibody, and the magnetic beads used are magnetic beads coupled with streptavidin. The CRP reagent 2 is diluent 1; The CRP reagent 3 is prepared from alkaline phosphatase-labeled CRP antibody and dilution solution 2; Diluent 1 is a Tris buffer containing bovine serum albumin; Diluent 2 is a Tris buffer containing bovine serum albumin and protein stabilizers.

2. The kit for quantitative detection of CRP using magnetic microparticle chemiluminescence immunoassay according to claim 1, characterized in that: Each 1L of dilution 1 contains 10-14g of Tris, 20-35g of NaCl, 15-25g of sucrose, 8-12g of bovine serum albumin, 0.5-1.5mL of ProClin950 and 0.3-0.7mL of Tween-20, with the remainder being purified water.

3. The kit for quantitative detection of CRP using magnetic microparticle chemiluminescence as described in claim 1, characterized in that: Each 1L of dilution buffer 2 contains 10-14g of Tris, 7-11g of NaCl, 5-15g of D-trehalose, 40-60mL of protein stabilizer, 4-6g of bovine serum albumin, 4-6g of sodium caseinate, 0.05-0.15mL of Triton X 100, 0.8-1.2mL of ProClin 300, and 8-12mL of glycerol, with the remainder being purified water.

4. The kit for quantitative detection of CRP using magnetic microparticle chemiluminescence immunoassay according to claim 1, characterized in that: The CRP calibrator includes two or more different concentration levels, and the CRP calibrator is prepared from CRP antigen and diluent 3.

5. A kit for quantitative detection of CRP using magnetic microparticle chemiluminescence as described in claim 1, characterized in that: The CRP quality control material includes two or more different concentration levels, and the CRP quality control material is prepared by CRP antigen and diluent 3.

6. A kit for quantitative detection of CRP using magnetic microparticle chemiluminescence immunoassay according to claim 4 or 5, characterized in that: Each 1L of dilution solution 3 contains 10-14g Tris, 7-11g NaCl, 15-25g D-trehalose, 8-12g sodium caseinate, 0.01-0.03g 4-aminoantipyrine, 0.05-0.15mL Triton X 100, 0.8-1.2mL ProClin 300, 8-12mL glycerol, and 0.4-0.6g CaCl2, with the remainder being purified water.

7. A method for preparing a kit for quantitative detection of CRP using magnetic microparticle chemiluminescence as described in any one of claims 1-6, characterized in that, Includes the following steps: Step 1: Prepare CRP reagent 1: Wash 0.02 mL of magnetic bead stock solution with dilution buffer 1 at room temperature to obtain washed magnetic beads. Then add 0.1 mL of dilution buffer 1 to the magnetic beads and mix well. Add 0.2 mg of biotin-labeled CRP antibody and mix well. React for 30 min. Then add 0.01 mL of blocking buffer and mix well. React for 15 min. After sealing, the supernatant was discarded by magnetic separation, and then 0.5 mL of diluent 1 was added and the mixture was suspended and mixed. Then 0.04 mg of blocking agent was added and mixed to complete the coating of the magnetic bead suspension mother liquor. Finally, 0.1 mL of diluent 1 was added to obtain CRP reagent 1. Step 2: Use diluent 1 as CRP reagent 2; Step 3: Prepare CRP reagent 3: Mix 800 mL of dilution buffer 2 and 0.5 mg of alkaline phosphatase-labeled CRP antibody, and bring the volume to 1 L with purified water to obtain CRP reagent 3. Step 4: Prepare CRP calibrators: Use diluent 3 to prepare CRP antigen at two or more different concentration points; Step 5: Prepare CRP quality control samples: Use diluent 3 to prepare CRP antigen at two or more different concentration points.

8. A kit for quantitative detection of CRP using magnetic microparticle chemiluminescence immunoassay according to claim 7, characterized in that: In steps 1 and 2, the preparation method of diluent 1 is as follows: Add 800mL of purified water, 10-14g of Tris and 20-35g of NaCl, stir until completely dissolved, adjust the pH value to 8.0±0.1, then add 15-25g of sucrose, 8-12g of bovine serum albumin, 0.5-1.5mL of ProClin950 and 0.3-0.7mL of Tween-20, stir until completely dissolved, and bring the volume to 1L with purified water; In step 3, the preparation method of diluent 2 is as follows: Add 800mL of purified water, 10-14g of Tris and 7-11g of NaCl, stir until completely dissolved, adjust the pH value to 8.0±0.1, then add 5-15g of D-trehalose, 40-60mL of protein stabilizer, 4-6g of bovine serum albumin, 4-6g of sodium caseinate, 0.05-0.15mL of Triton X 100, 0.8-1.2mL of ProClin 300 and 8-12mL of glycerol, stir until completely dissolved, and bring the volume to 1L with purified water. In steps 4 and 5, the preparation method of diluent 3 is as follows: Stir 800 mL of purified water, 10-14 g of Tris and 7-11 g of NaCl until completely dissolved, adjust the pH value to 7.4±0.1, then add 15-25 g of D-trehalose, 8-12 g of sodium caseinate, 0.01-0.03 g of 4-aminoantipyrine, 0.05-0.15 mL of Triton X100, 0.8-1.2 mL of ProClin 300, 8-12 mL of glycerol and 0.4-0.6 g of CaCl2, stir until completely dissolved, and bring the volume to 1 L with purified water.

9. A detection method for a kit for quantitative detection of CRP using magnetic microparticle chemiluminescence as described in any one of claims 1-6, comprising the following steps: (1) After mixing CRP calibrator, CRP reagent 1 and CRP reagent 2, incubate at 37°C for 5 min, perform magnetic separation, remove the supernatant, add cleaning solution to wash, repeat the washing multiple times to obtain intermediate A; (2) Add CRP reagent 3 to intermediate A, mix well, incubate at 37°C for 5 min, perform magnetic separation, and remove the supernatant; then add washing solution to wash, repeat washing several times to obtain intermediate B; (3) Add the substrate solution for the fully automated immunoassay system to intermediate B, mix well, and use a fully automated chemiluminescence immunoassay analyzer to detect the luminescence intensity to obtain the working curve after CRP calibration. (4) Replace the CRP calibrator with the CRP quality control sample, repeat steps (1)-(3), obtain the luminescence value of the CRP quality control sample, fit the luminescence value with the working curve obtained in step (3), and obtain the concentration value of the CRP quality control sample. (5) Replace the CRP calibrator with the sample to be tested, and repeat steps (1)-(3) to obtain the concentration value of the sample to be tested.