A RANKL magnetic microparticle chemiluminescence immunoassay kit and detection method thereof

Abstract

The present invention relates to the field of immunoassay, and discloses a RANKL magnetic particle chemiluminescent immunoassay kit and a detection method thereof, wherein the kit comprises a magnetic particle reagent, an AE-labeled RANKL antibody, and a RANKL calibrator, wherein the magnetic particle reagent is a magnetic microsphere suspension coated with the RANKL antibody; the AE-labeled RANKL antibody is obtained by labeling the RANKL antibody with an acridinium ester, and the method comprises the following steps: (1) preparing a magnetic particle reagent and an AE-labeled RANKL antibody; (2) adding a serum sample and the AE-labeled RANKL antibody prepared in step (1) to the magnetic particle reagent prepared in step (1) for incubation; (3) after the incubation is completed, after the magnetic particle reagent is precipitated, the supernatant is removed, the sample is washed, a substrate solution is added, the sample is shaken and mixed, and the relative luminescence intensity is detected. The present invention effectively solves the problem that the RANKL content in the human body cannot be detected quickly, accurately, and in large quantities in the prior art.

Description

Technical Field

[0001] The invention belongs to the field of immunoassay, and more specifically relates to a RANKL magnetic particle chemiluminescence immunoassay kit and a detection method thereof. Background Art

[0002] Osteoprotegerin (OPG) and receptor activator of nuclear factor-KB (RANK) and its ligand (RANKL) are cytokines closely related to bone resorption and reconstruction discovered in recent years. Among them: RANKL (ligand of receptor activator of NF-kB), or osteoclast differentiation factor (ODF): its receptor is RANK (receptor activator of NF-kB) located on the cell membrane of osteoclasts: RANKL's pseudoreceptor osteoprotegerin (OPC). The main content is: Osteoblasts and bone marrow stromal cells express RANKL, which promotes osteoclast differentiation and bone resorption activity after binding to RANK on the surface of osteoclast precursor cells or osteoclasts. Osteoblasts and bone marrow stromal cells secrete and express OPG to competitively bind to RANKL, preventing the binding between RANKL and RANK, and inhibiting osteoclast proliferation and activation. Many hormones and factors in the body affect bone metabolism by affecting RANKL or the expression of RANKL. Activated T lymphocytes and mammary endothelial cells during pregnancy can express RANKL, and play an important role in bone loss in autoimmune diseases such as rheumatoid arthritis, and the development of the mammary gland and fetus during pregnancy. The OPG / RANKL / RANK system closely links bone metabolism, the immune system, and the endocrine system, and opens up new avenues for the treatment of bone-destructive diseases such as osteoporosis, rheumatoid arthritis, and cancer bone metastasis.

[0003] Further research should understand the relationship between the OPF / RANKL / RANK system and other signal transduction pathways, and pay attention to the interaction between the bone, immune and endocrine systems. The existing technology cannot quickly, accurately and in large quantities detect the RANKL content in the human body. Therefore, how to provide a RANKL detection reagent and detection method is an urgent problem to be solved by those skilled in the art. Summary of the invention

[0004] In order to solve the problem that the prior art cannot detect the RANKL content in the human body quickly, accurately and in large quantities, the present invention provides a RANKL magnetic particle chemiluminescence immunoassay kit and a detection method thereof.

[0005] The specific scheme adopted by the present invention is: a magnetic particle chemiluminescent immunoassay kit for RANKL, the kit comprising a magnetic particle reagent, an AE-labeled RANKL antibody, and a RANKL calibrator, the magnetic particle reagent is a magnetic microsphere suspension coated with a RANKL antibody; the AE-labeled RANKL antibody is obtained by labeling the RANKL antibody with an acridinium ester.

[0006] The particle size of the magnetic particles in the magnetic particle reagent is 1-3 μm, and the surface of the magnetic particles is coated with magnetic microspheres with carboxyl active groups. When preparing the magnetic microsphere suspension of RANKL antibody, 1.0-0.5 mg of RANKL antibody is coated per 50 mg of magnetic microspheres. The ratio of RANKL antibody to magnetic beads in the magnetic particle reagent is 1:50-1:100.

[0007] The ratio of RANKL antibody to acridinium ester in the AE-labeled RANKL antibody is 1:1-1:2.

[0008] The ratio of RANKL antibody to magnetic beads in the magnetic particle reagent is 1:50-1:100.

[0009] In another aspect, the present invention provides a magnetic microparticle chemiluminescence immunoassay detection method for RANKL, comprising the following steps:

[0010] (1) preparing magnetic particle reagents and AE-labeled RANKL antibodies;

[0011] (2) adding the serum sample and the AE-labeled RANKL antibody prepared in step (1) to the magnetic particle reagent prepared in step (1) for incubation;

[0012] (3) After the incubation is completed and the magnetic particle reagent is settled, the supernatant is removed, the solution is washed, the substrate solution is added, the solution is shaken and mixed, and the relative luminescence intensity is detected.

[0013] The preparation method of the magnetic particle reagent in step (1) is as follows: dispersing the carboxyl magnetic bead solution under the action of a magnetic particle buffer to obtain a dispersed carboxyl magnetic bead solution; connecting the dispersed carboxyl magnetic bead solution and RANKL antibody to obtain a connection product; placing the connection product in a magnetic field and washing it with a magnetic particle buffer to obtain a magnetic particle reagent connected with the RANKL antibody.

[0014] The operation of the dispersion treatment is: placing the carboxyl magnetic bead concentrate in a magnetic field for 1-3 minutes, removing the upper clear liquid after all the carboxyl magnetic beads are precipitated, adding a magnetic particle buffer and oscillating, placing it in a magnetic field for 1-3 minutes, and then removing the upper clear liquid; the oscillation time is 20-30 minutes; the volume ratio of the carboxyl magnetic beads to the magnetic particle buffer is 1:(2-5); and the dispersion treatment is performed 3-5 times.

[0015] The preparation method of the AE-labeled RANKL antibody in step (1) is as follows: preparing a labeling buffer; weighing an appropriate amount of AE solid powder, dissolving it with a 0.1M boric acid buffer, and preparing an AE solution with a concentration of 1.0 mg / mL; mixing the mouse monoclonal anti-RANKL antibody and the AE solution at a mass ratio of 1:(1-2), adding a coating buffer for a linking reaction, and obtaining a linking product of the RANKL antibody and AE; separating and purifying the linking product with a carbonate buffer at a pH of 8 to 9, and obtaining an AE-labeled RANKL antibody.

[0016] The preparation method of the AE-labeled RANKL antibody in step (1) is as follows: preparing a labeling buffer; weighing an appropriate amount of AE solid powder, dissolving it with a 0.1M boric acid buffer, and preparing an AE solution with a concentration of 1.0 mg / mL; mixing the mouse monoclonal anti-RANKL antibody and the AE solution at a mass ratio of 1:(1-2), adding a coating buffer for a linking reaction, and obtaining a linking product of the RANKL antibody and AE; separating and purifying the linking product with a carbonate buffer at a pH of 8 to 9, and obtaining an AE-labeled RANKL antibody.

[0017] The AE marker in the AE-labeled RANKL antibody is a modified acridinium ester.

[0018] The volume ratio of the serum sample, AE-labeled RANKL antibody and magnetic particle reagent in step (2) is 1:2:4.

[0019] After the relative luminescence intensity is detected in step (3), the detection method is calibrated with high and low value two-point calibration products, and a new working curve is generated by fitting for sample measurement; the RANKL quality control product is used as a sample, and the concentration is fitted using the working curve to obtain the corresponding quality control product concentration, and the fitted quality control product concentration is observed to be ±30% of the standard concentration value.

[0020] Compared with the prior art, the present invention has the following beneficial effects:

[0021] The kit of the present invention includes a magnetic particle reagent, an AE-labeled RANKL antibody, and a RANKL calibrator. The magnetic particle reagent is a magnetic microsphere suspension coated with a RANKL antibody; the AE-labeled RANKL antibody is obtained by labeling the RANKL antibody with an acridinium ester. The kit uses magnetic beads as a solid phase, so that the immune response is closer to the liquid phase, the reaction is more sufficient and rapid, and the bound immune complex is easier to separate, reducing nonspecific adsorption. The AE-labeled antibody reagent used in the present invention is a monoclonal antibody mixture, which makes the affinity of the immune response higher, and the production difference of the monoclonal antibody is relatively small, which makes it easier to ensure the stable quality of the product.

[0022] The magnetic microparticle chemiluminescence immunoassay detection method for RANKL described in the present invention relies on the above-mentioned kit to provide the possibility for rapid and accurate RANKL detection. The present invention has low requirements for sample pretreatment, a simple pretreatment process, can quickly and high-throughput detect a large number of samples, and the detection method is convenient and easy, filling the gap in the prior art.

[0023] The kit of the present invention combines chemiluminescence technology with immunomagnetic particles, provides a near-homogeneous reaction system, has higher detection sensitivity, specificity, precision and accuracy, and achieves better performance parameters. The magnetic particle reagent, AE-labeled antibody and calibrator in the kit are the optimal formula under the reaction system, which provides a strong guarantee for the shelf life and detection performance of the kit. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 is the fitted line graph of Example 1;

[0025] Figure 2 is the fitted line graph of Example 2;

[0026] Figure 3 It is the fitted line graph of Example 3;

[0027] Figure 4 Schematic diagram of the modification of acridinium esters. DETAILED DESCRIPTION

[0028] The present invention will be further described in detail below in conjunction with the accompanying drawings. Obviously, it should be understood here that the described embodiments are not all embodiments and are only used to illustrate the present invention rather than to limit the present invention.

[0029] A RANKL magnetic particle chemiluminescent immunoassay kit comprises a magnetic particle reagent, an AE-labeled RANKL antibody, and a RANKL calibrator. The magnetic particle reagent is a magnetic microsphere suspension coated with the RANKL antibody; the AE-labeled RANKL antibody is obtained by labeling the RANKL antibody with an acridinium ester.

[0030] The particle size of the magnetic particles in the magnetic particle reagent is 1-3 μm, the surface of the magnetic particles is coated with magnetic microspheres with carboxyl active groups, the magnetic microsphere suspension of the RANKL antibody is coated with 1.0-0.5 mg of RANKL antibody per 50 mg of magnetic microspheres, and the ratio of RANKL antibody to magnetic beads in the magnetic particle reagent is 1:50-1:100. Improving the coating ratio can improve sensitivity and linear range.

[0031] The ratio of RANKL antibody to acridinium ester in the AE-labeled RANKL antibody is 1:1-1:2. Improving the labeling ratio can effectively improve the low-end sensitivity and widen the linear range.

[0032] The ratio of RANKL antibody to magnetic beads in the magnetic microparticle reagent is 1:50-1:100. Improving the detection ratio can improve sensitivity and linear range while controlling costs.

[0033] On the other hand, the present invention provides a magnetic particle chemiluminescent immunoassay detection method for RANKL. Chemiluminescent immunoassay technology is a highly sensitive micro-assay technology. All antigenic substances (including haptens) can be measured by chemiluminescent immunoassay technology. It has the following characteristics: ① High sensitivity, with a limit of 10-17-10-19M / L, much higher than RIA and EIA, comparable to TRFIA but cheaper than TRFIA. ② Strong specificity and good repeatability. ③ Wide measurement range, up to 7 orders of magnitude. ④ Good reagent stability, no pollution and a validity period of 6-12 months. ⑤ Simple operation and easy automation. Chemiluminescent immunoassay is divided into chemiluminescent enzyme immunoassay and flash chemiluminescent immunoassay. Among them, chemiluminescent enzyme immunoassay is a type of enzyme immunoassay, but the substrate used in the last step of the enzymatic reaction is a luminescent agent, and the light from the luminescent reaction is measured on a specific instrument. Commonly used labeling enzymes include horseradish peroxidase (HP) and alkaline phosphatase (AP), while flash chemiluminescent immunoassay uses acridinium esters to label antigens or antibodies.

[0034] Magnetic particles refer to colloidal composite materials formed by the combination of magnetic nanoparticles and inorganic or organic molecules, which can be uniformly dispersed in a certain base liquid and have high stability. Due to the magnetic responsiveness, low cost, low energy consumption and no pollution of magnetic particles, people can fix enzymes, antibodies, oligonucleotides and other bioactive substances on the surface of magnetic particles or through the functional groups on the surface of magnetic particles (such as amino, carboxyl, thiol and ethylene oxide, etc.), which can be further used in enzyme immobilization, targeted drug carriers, cell sorting, immunoassay, separation and purification of proteins and nucleic acids, and hybridization detection.

[0035] Traditional immunological tests mostly use ELISA plates as solid phase carriers. Suspended magnetic particles as carriers have a higher specific surface area and can react with samples more fully. In addition, the flexible application of the external magnetic field has the advantages of higher sensitivity, faster detection speed and better repeatability than ELISA plate carriers. It has been widely used in biological and medical testing and other fields. It includes the following steps:

[0036] (1) preparing magnetic particle reagents and AE-labeled RANKL antibodies;

[0037] (2) adding the serum sample and the AE-labeled RANKL antibody prepared in step (1) to the magnetic particle reagent prepared in step (1) for incubation;

[0038] (3) After the incubation is completed and the magnetic particle reagent is settled, the supernatant is removed, the solution is washed, the substrate solution is added, the solution is shaken and mixed, and the relative luminescence intensity is detected.

[0039] The preparation method of the magnetic particle reagent in step (1) is as follows: dispersing the carboxyl magnetic bead solution under the action of a magnetic particle buffer to obtain a dispersed carboxyl magnetic bead solution; connecting the dispersed carboxyl magnetic bead solution and RANKL antibody to obtain a connection product; placing the connection product in a magnetic field and washing it with a magnetic particle buffer to obtain a magnetic particle reagent connected with the RANKL antibody.

[0040] The operation of the dispersion treatment is: placing the carboxyl magnetic bead concentrate in a magnetic field for 1-3 minutes, removing the upper clear liquid after all the carboxyl magnetic beads are precipitated, adding a magnetic particle buffer and oscillating, placing it in a magnetic field for 1-3 minutes, and then removing the upper clear liquid; the oscillation time is 20-30 minutes; the volume ratio of the carboxyl magnetic beads to the magnetic particle buffer is 1:(2-5); and the dispersion treatment is performed 3-5 times.

[0041] The preparation method of the AE-labeled RANKL antibody in step (1) is as follows: preparing a labeling buffer; weighing an appropriate amount of AE solid powder, dissolving it with a 0.1M boric acid buffer, and preparing an AE solution with a concentration of 1.0 mg / mL; mixing the mouse monoclonal anti-RANKL antibody and the AE solution at a mass ratio of 1:(1-2), adding a coating buffer for a linking reaction, and obtaining a linking product of the RANKL antibody and AE; separating and purifying the linking product with a carbonate buffer at a pH of 8 to 9, and obtaining an AE-labeled RANKL antibody.

[0042] The preparation method of the AE-labeled RANKL antibody in step (1) is as follows: preparing a labeling buffer; weighing an appropriate amount of AE solid powder, dissolving it with a 0.1M boric acid buffer, and preparing an AE solution with a concentration of 1.0 mg / mL; mixing the mouse monoclonal anti-RANKL antibody and the AE solution at a mass ratio of 1:(1-2), adding a coating buffer for a linking reaction, and obtaining a linking product of the RANKL antibody and AE; separating and purifying the linking product with a carbonate buffer at a pH of 8 to 9, and obtaining an AE-labeled RANKL antibody.

[0043] The AE marker in the AE-labeled RANKL antibody is a modified acridinium ester.

[0044] The volume ratio of the serum sample, AE-labeled RANKL antibody and magnetic particle reagent in step (2) is 1:2:4.

[0045] After the relative luminescence intensity is detected in step (3), the detection method is calibrated with high and low value two-point calibration products, and a new working curve is generated by fitting for sample measurement; the RANKL quality control product is used as a sample, and the concentration is fitted using the working curve to obtain the corresponding quality control product concentration, and the fitted quality control product concentration is observed to be ±30% of the standard concentration value.

[0046] The labeling buffer is a PB buffer with a pH of 7.0-7.5, and each 1L of the buffer contains 20-25g of casein sodium salt, 10-15g of SDS, 10-15g of BSA and 0.5-1mL of Proclin-300. The first addition of an appropriate amount of BSA to the labeling buffer in the present invention can effectively improve the labeling efficiency, reduce antibody loss, and reduce acridinium ester waste; the addition of SDS can reduce the detection luminescence value of low-value samples, avoid false positives, and improve correlation and sensitivity.

[0047] The kit of the present invention combines chemiluminescence technology with immunomagnetic particles, provides a near-homogeneous reaction system, has higher detection sensitivity, specificity, precision and accuracy, and achieves better performance parameters. The magnetic particle reagent, AE-labeled antibody and calibrator in the kit are the optimal formula under the reaction system, which provides a strong guarantee for the shelf life and detection performance of the kit.

[0048] The cleaning solution for the fully automatic chemiluminescence detection system of the present invention is a buffer solution containing Tris, NaCl and a surfactant, etc.; the substrate solution (for the fully automatic chemiluminescence detection system) is divided into an acid solution and an alkaline solution. The acid solution is a boric acid buffer solution containing urea peroxide; and the alkaline solution is a buffer solution containing sodium hydroxide.

[0049] Operation requirements of the fully automatic chemiluminescence immunoassay system:

[0050] 1. Preparation before the test: Turn on the instrument before the test and perform power-on maintenance; follow the system operation instructions to load the reaction tube (cup), add or replace the supporting reagents, clean the waste liquid and waste tube, and fill the liquid line.

[0051] 2. Loading of reagents and samples: Before loading the reagents, place the reagents to be used on the mixing device and mix them thoroughly. Visually check that the reagent solution components should be clear, without foreign matter, sediment and floccules. The magnetic particle reagent should be a uniform suspension without obvious agglutination. According to the system operation instructions, scan the reagent barcode to complete the loading of magnetic particle reagents and antibody reagents. After mixing, move the calibrator to the instrument's dedicated reaction cup and load it into the instrument's sample position. After scanning, the plasma sample obtained by centrifugation is directly loaded into the instrument's sample position.

[0052] 3. Detection steps: To achieve the best detection performance, follow the relevant requirements of the detection kit (magnetic microparticle chemiluminescence method) provided by the present invention and the operation manual of the fully automatic chemiluminescence analyzer. The detection reaction process and related parameters have been pre-defined in the instrument operation software.

[0053] 4. Standard curve calibration: Scan the main curve card, read the standard curve data, and the instrument background automatically fits the standard curve. Note: The standard curve is calibrated by the high and low value two-point calibration products, and the instrument background automatically fits to generate a new working curve for sample measurement.

[0054] 5. Calibration: By measuring the high and low calibrators, each calibration point on the pre-defined master curve will be adjusted (recalibrated) to a new, instrument-specific measurement level, i.e. the working curve.

[0055] Example 1

[0056] The invention provides a magnetic particle chemiluminescent immunoassay kit for nuclear factor KB receptor activator (RANKL). The magnetic particle chemiluminescent immunoassay kit for nuclear factor KB receptor activator comprises a magnetic particle reagent, an AE-labeled RANKL antibody, and a RANKL calibrator; the magnetic particle reagent is a magnetic microsphere suspension coated with the RANKL antibody; the AE-labeled RANKL antibody is labeled with an acridinium ester.

[0057] The concentration of the magnetic microsphere suspension coated with RANKL antibody is prepared at a ratio of 1:100; the AE-labeled RANKL antibody is labeled at a ratio of 1:2 to the marker acridinium ester.

[0058] The labeling buffer is a PB buffer with a pH of 7.0, and each 1L of the buffer contains 20g of casein sodium salt, 10g of SDS, 10g of BSA and 0.5mL of Proclin-300.

[0059] The concentration of the RANKL calibrator is 50pg / mL and 500pg / mL;

[0060] In the present invention, the cleaning solution (for the fully automatic chemiluminescence detection system) is a buffer solution containing Tris, NaCl and a surfactant, etc.; the substrate solution (for the fully automatic chemiluminescence detection system) is divided into an acid solution and an alkali solution. The acid solution is a boric acid buffer solution containing urea peroxide; and the alkali solution is a buffer solution containing sodium hydroxide.

[0061] The preparation method of the magnetic particle reagent comprises the following steps:

[0062] (1) dispersing the carboxyl magnetic bead solution under the action of a magnetic particle buffer to obtain a dispersed carboxyl magnetic bead solution;

[0063] (2) connecting the dispersed carboxyl magnetic bead solution and the RANKL antibody to obtain a connection product;

[0064] (3) placing the ligated product in a magnetic field and washing it with a magnetic particle buffer to obtain a magnetic particle reagent ligated with RANKL antibody.

[0065] In the above step (1), the carboxyl magnetic bead concentrate is placed in a magnetic field for 1 min, the supernatant is removed after all the carboxyl magnetic beads are completely precipitated, the magnetic particle buffer is added and oscillated, and the concentrate is placed in a magnetic field for 1 min, and the supernatant is removed; the oscillation time is 20 min; the volume ratio of the carboxyl magnetic beads to the magnetic particle buffer is 1:2; and the dispersion treatment is performed 3 times.

[0066] In the above step (2), the mass ratio of RANKL antibody to dispersed carboxyl magnetic bead solution is 1:100; the concentration of dispersed carboxyl magnetic bead solution is 10 mg / mL; the temperature of the connection reaction is room temperature, and the time of the connection reaction is 16 h.

[0067] In the above step (2), the connection product is placed in the magnetic field for 5 minutes; then washed; and a magnetic particle solution connected with RANKL antibody is obtained.

[0068] The RANKL calibrator is a Tris buffer solution containing RANKL antigen, 0.5% by mass BSA, and 0.05% by mass prolin300.

[0069] The preparation method of the AE-labeled RANKL antibody comprises the following steps:

[0070] (1) Prepare coating buffer: pH 7.0 PB buffer, each 1 L of buffer contains 20 g casein sodium salt, 10 g SDS, 10 g BSA and 0.5 mL Proclin-300;

[0071] (2) Weigh an appropriate amount of AE solid powder and dissolve it in 0.1 M boric acid buffer to prepare an AE solution with a concentration of 1.0 mg / mL;

[0072] (3) mixing the mouse monoclonal anti-RANKL antibody and the AE solution at a mass ratio of 1:2, adding a coating buffer to carry out a linking reaction, and obtaining a linking product of the RANKL antibody and the AE;

[0073] (4) The ligation product was separated and purified by a PB-10 desalting column using a carbonate buffer solution at pH 8 to obtain an AE-labeled RANKL antibody.

[0074] The magnetic microparticle chemiluminescence immunoassay detection method for RANKL is as follows: the serum sample and the prepared AE-labeled RANKL antibody are added to the prepared magnetic microparticle reagent for incubation; after the incubation is completed and the magnetic microparticle reagent is precipitated, the supernatant is removed, the mixture is washed, the substrate solution is added, the mixture is shaken and mixed, and the relative luminescence intensity is detected.

[0075] Example 2

[0076] The specific scheme adopted by the present invention is as follows: the present invention provides a magnetic particle chemiluminescent immunoassay kit for nuclear factor KB receptor activating factor (RANKL), wherein the magnetic particle chemiluminescent immunoassay kit for nuclear factor KB receptor activating factor comprises a magnetic particle reagent, an AE-labeled RANKL antibody, and a RANKL calibrator; the magnetic particle reagent is a magnetic microsphere suspension coated with a RANKL antibody; the AE-labeled RANKL antibody is labeled with an acridinium ester.

[0077] The concentration of the magnetic microsphere suspension coated with RANKL antibody is prepared at a ratio of 1:80; the AE-labeled RANKL antibody is labeled at a ratio of 1:1.5 to the marker acridinium ester.

[0078] The labeling buffer is a PB buffer with a pH of 7.5, and each 1L of the buffer contains 25g of casein sodium salt, 15g of SDS, 15g of BSA and 1.0mL of Proclin-300.

[0079] The concentration of the RANKL calibrator is 50pg / mL and 500pg / mL;

[0080] In the present invention, the cleaning solution (for the fully automatic chemiluminescence detection system) is a buffer solution containing Tris, NaCl and a surfactant, etc.; the substrate solution (for the fully automatic chemiluminescence detection system) is divided into an acid solution and an alkali solution. The acid solution is a boric acid buffer solution containing urea peroxide; and the alkali solution is a buffer solution containing sodium hydroxide.

[0081] The preparation method of the magnetic particle reagent comprises the following steps:

[0082] (4) dispersing the carboxyl magnetic bead solution under the action of a magnetic particle buffer to obtain a dispersed carboxyl magnetic bead solution;

[0083] (5) connecting the dispersed carboxyl magnetic bead solution and the RANKL antibody to obtain a connection product;

[0084] (6) The ligated product is placed in a magnetic field and washed with a magnetic particle buffer to obtain a magnetic particle reagent linked to the RANKL antibody.

[0085] In the above step (1), the carboxyl magnetic bead concentrate is placed in a magnetic field for 3 minutes, the supernatant is removed after all the carboxyl magnetic beads are completely precipitated, the magnetic particle buffer is added and shaken, and the concentrate is placed in a magnetic field for 3 minutes, and then the supernatant is removed; the shaking time is 30 minutes; the volume ratio of the carboxyl magnetic beads to the magnetic particle buffer is 1:5; and the dispersion treatment is performed 5 times.

[0086] In the above step (2), the mass ratio of RANKL antibody to dispersed carboxyl magnetic bead solution is 1:80; the concentration of dispersed carboxyl magnetic bead solution is 10 mg / mL; the temperature of the connection reaction is room temperature, and the time of the connection reaction is 20 h.

[0087] In the above step (2), the connection product is placed in the magnetic field for 5 minutes; then washed; and a magnetic particle solution connected with RANKL antibody is obtained.

[0088] The RANKL calibrator is a Tris buffer solution containing RANKL antigen, 0.5% by mass BSA, and 0.05% by mass prolin300.

[0089] The preparation method of the AE-labeled RANKL antibody comprises the following steps:

[0090] (5) Prepare coating buffer: pH 7.0 PB buffer, each 1 L of buffer contains 25 g casein sodium salt, 10 g SDS, 10 g BSA and 0.5 mL Proclin-300;

[0091] (6) Weigh an appropriate amount of AE solid powder and dissolve it in 0.1 M boric acid buffer to prepare an AE solution with a concentration of 1.0 mg / mL;

[0092] (7) mixing the mouse monoclonal anti-RANKL antibody and the AE solution at a mass ratio of 1:1.5, adding a coating buffer to carry out a linking reaction, and obtaining a linking product of the RANKL antibody and the AE;

[0093] (8) The ligation product was separated and purified by a PB-10 desalting column using a carbonate buffer at pH 9 to obtain an AE-labeled RANKL antibody.

[0094] The magnetic microparticle chemiluminescence immunoassay detection method for RANKL is as follows: the serum sample and the prepared AE-labeled RANKL antibody are added to the prepared magnetic microparticle reagent for incubation; after the incubation is completed and the magnetic microparticle reagent is precipitated, the supernatant is removed, the mixture is washed, the substrate solution is added, the mixture is shaken and mixed, and the relative luminescence intensity is detected.

[0095] Example 3

[0096] The specific scheme adopted by the present invention is as follows: the present invention provides a magnetic particle chemiluminescent immunoassay kit for nuclear factor KB receptor activating factor (RANKL), wherein the magnetic particle chemiluminescent immunoassay kit for nuclear factor KB receptor activating factor comprises a magnetic particle reagent, an AE-labeled RANKL antibody, and a RANKL calibrator; the magnetic particle reagent is a magnetic microsphere suspension coated with a RANKL antibody; the AE-labeled RANKL antibody is labeled with an acridinium ester.

[0097] The AE marker in the AE-labeled RANKL antibody is a modified acridinium ester.

[0098] Modify the acridinium ester and add a certain proportion of condensing agent EDC to form an active intermediate NHS ester. NHS ester is a reactive group formed by EDC-activated carboxylic acid molecules. NHS ester reacts with the primary amino group of the labeled antibody. This reaction process is a bonding reaction between carboxyl and amino groups, which can produce a stable amide bond. This process is called covalent coupling. Then react with the primary amino group of the labeled antibody. Under alkaline conditions, the leaving group is replaced, and the antibody and the acridinium ester form a stable amide bond. Covalent coupling is more advanced than physical adsorption, mainly in that it overcomes the two disadvantages of physical adsorption: the antibody covalently coupled to the solid surface (1) has less steric hindrance, and the arrangement and orientation of the antibody are determined to a certain extent; (2) the binding is more stable and not easy to detach from the solution. After the reaction is completed, under alkaline conditions, the leaving group is replaced, and the excess acridinium salt is removed through a desalting column.

[0099] The concentration of the magnetic microsphere suspension coated with RANKL antibody is prepared at a ratio of 1:80; the AE-labeled RANKL antibody is labeled at a ratio of 1:1.5 to the marker acridinium ester.

[0100] The labeling buffer is a PB buffer with a pH of 7.5, and each 1L of the buffer contains 25g of casein sodium salt, 15g of SDS, 15g of BSA and 1.0mL of Proclin-300.

[0101] The concentration of the RANKL calibrator is 50pg / mL and 500pg / mL;

[0102] In the present invention, the cleaning solution (for the fully automatic chemiluminescence detection system) is a buffer solution containing Tris, NaCl and a surfactant, etc.; the substrate solution (for the fully automatic chemiluminescence detection system) is divided into an acid solution and an alkali solution. The acid solution is a boric acid buffer solution containing urea peroxide; and the alkali solution is a buffer solution containing sodium hydroxide.

[0103] The preparation method of the magnetic particle reagent comprises the following steps:

[0104] (7) dispersing the carboxyl magnetic bead solution under the action of a magnetic particle buffer to obtain a dispersed carboxyl magnetic bead solution;

[0105] (8) subjecting the dispersed carboxyl magnetic bead solution and the RANKL antibody to a ligation reaction to obtain a ligation product;

[0106] (9) The ligated product is placed in a magnetic field and washed with a magnetic particle buffer to obtain a magnetic particle reagent linked to the RANKL antibody.

[0107] In the above step (1), the carboxyl magnetic bead concentrate is placed in a magnetic field for 3 minutes, the supernatant is removed after all the carboxyl magnetic beads are completely precipitated, the magnetic particle buffer is added and shaken, and the concentrate is placed in a magnetic field for 3 minutes, and then the supernatant is removed; the shaking time is 30 minutes; the volume ratio of the carboxyl magnetic beads to the magnetic particle buffer is 1:5; and the dispersion treatment is performed 5 times.

[0108] In the above step (2), the mass ratio of RANKL antibody to dispersed carboxyl magnetic bead solution is 1:80; the concentration of dispersed carboxyl magnetic bead solution is 10 mg / mL; the temperature of the connection reaction is room temperature, and the time of the connection reaction is 20 h.

[0109] In the above step (2), the connection product is placed in the magnetic field for 5 minutes; then washed; and a magnetic particle solution connected with RANKL antibody is obtained.

[0110] The RANKL calibrator is a Tris buffer solution containing RANKL antigen, 0.5% by mass BSA, and 0.05% by mass prolin300.

[0111] The preparation method of the AE-labeled RANKL antibody comprises the following steps:

[0112] (9) Prepare coating buffer: pH 7.0 PB buffer, each 1 L of buffer contains 25 g casein sodium salt, 10 g SDS, 10 g BSA and 0.5 mL Proclin-300;

[0113] (10) Weigh an appropriate amount of AE solid powder and dissolve it in 0.1 M boric acid buffer to prepare an AE solution with a concentration of 1.0 mg / mL;

[0114] (11) mixing the mouse monoclonal anti-RANKL antibody and the AE solution at a mass ratio of 1:1.5, adding a coating buffer to carry out a linking reaction, and obtaining a linking product of the RANKL antibody and the AE;

[0115] (12) The ligation product was separated and purified by a PB-10 desalting column using a carbonate buffer at pH 9 to obtain an AE-labeled RANKL antibody.

[0116] The magnetic microparticle chemiluminescence immunoassay detection method for RANKL is as follows: the serum sample and the prepared AE-labeled RANKL antibody are added to the prepared magnetic microparticle reagent for incubation; after the incubation is completed and the magnetic microparticle reagent is precipitated, the supernatant is removed, the mixture is washed, the substrate solution is added, the mixture is shaken and mixed, and the relative luminescence intensity is detected.

[0117] The performance indicators of the magnetic microparticle chemiluminescent immunoassay kit for RANKL in Examples 1-3 of the present invention were compared and tested:

[0118] 1. Linear

[0119] The linear range is (10-1000) pg / mL, and the correlation coefficient is r≥0.9900.

[0120] Here’s how to do it:

[0121] Serum or serum substitute close to the upper limit of the linear range is diluted into five concentrations with sample diluent at a certain ratio. The five concentration samples before and after dilution are measured three times using a chemiluminescence analyzer, and the average value is calculated. A four-parameter fitting curve is used based on the average value and the dilution ratio, and the linear correlation coefficient r (obtained by the X / Y axis linear fitting of the column of 1-1 / 100 and the RLU average value) is calculated. The result should be ≥0.9900. The specific values ​​of the test are shown in the following table:

[0122]

[0123] According to the above table, the luminescence value of Example 1 is too high when detecting low-value samples. It is speculated that the false positive phenomenon is caused by incorrect antibody coating ratio. Therefore, the ratio of antibody coating and labeling is reduced in Example 2, but the result is still not ideal, and the ratio is further reduced in Example 3. It can be obtained that the curve of Example 3 rises steadily and has a good trend in the linear range, which best meets the requirements.

[0124] The measured linear correlation coefficients (r) of the reaction curves of Examples 1-3 are 0.9905, 0.9960, and 0.9992, respectively, all of which are >0.9900, which are significantly better than the prior art. Among them, Example 3 has the best effect.

[0125] 2. Accuracy (recovery rate)

[0126] The recovery rates in Examples 1-3 of the present invention are in the range of 85 to 115%.

[0127] Here’s how to do it:

[0128] Add sample A of known RANKL concentration to sample B with the same matrix as the sample to be tested. The volume of A added should not exceed 10% of the total volume (A+B). Repeat the measurement three times and calculate according to formula (1):

[0129] Recovery rate

[0130] Where:

[0131] R: Recovery rate

[0132] V: volume of sample A liquid;

[0133] V0 is the volume of sample B solution;

[0134] C: the detection concentration after adding sample B solution into A solution;

[0135] C0: concentration of sample B solution;

[0136] Cs: concentration of sample A solution.

[0137] Data case (V = 0.1mL, V0 = 0.9mL, C0 = 0pg / mL, Cs = 800pg / mL, where V and V0 are in a 1:9 relationship according to the above method, and C0 and Cs are the concentrations of the high concentration point and the low concentration point in the calibration product, respectively): The test results are shown in the following table:

[0138]

[0139] The accuracy (recovery rate) of Examples 1-3 measured was 86.4%, 97%, and 100.17%, respectively, all within the range of 85-115%, meeting the requirements. Among them, Example 3 was the best.

[0140] 3. Minimum detection limit (sensitivity)

[0141] The minimum detection limit is ≤10pg / mL.

[0142] Here’s how to do it:

[0143] Use zero-concentration calibrator or sample diluent as the sample to be tested, and repeat the measurement 20 times. Obtain the RLU value (relative luminescence value) of the 20 measurement results, calculate its mean value (M) and standard deviation (SD), and obtain M+2SD. Perform two-point regression fitting based on the concentration-RLU value results between the zero-concentration calibrator and the adjacent calibrator to obtain a linear equation. Substitute the RLU value of M+2SD into the above equation to obtain the corresponding concentration value, which is the minimum detection limit, as shown in the following table:

[0144]

[0145] Since the linearity of the scheme of Example 1.2 does not meet the requirements, only Example 3 is further tested and verified. The average luminescence value of the RANKL sample dilution measured in Example 3 is M=476.8, SD=101.59. The value of (M+2SD) is substituted into this reaction curve, and its sensitivity is: 0.1pg / mL (<10pg / mL), which meets the requirements.

[0146] 4. Repeatability

[0147] The coefficient of variation CV≤8%. The operation method is as follows:

[0148] Quality control product 1 (10 pg / mL) and quality control product 2 (1000 ng / mL) were taken as the test samples, and the RANKL content in the test samples was measured 10 times using a chemiluminescence analyzer. The coefficient of variation (CV) was calculated according to formula (2).

[0149]

[0150] Where: —Average of 10 test results

[0151] SD—Standard deviation of 10 test results

[0152] CV—Coefficient of Variation

[0153] Data examples:

[0154] Example 1 data:

[0155]

[0156] Example 2 data:

[0157]

[0158] Example 3 data:

[0159]

[0160] Analysis: Examples 1-3 tested the quality control products 1.2 ten times each, and calculated the CV. The quality control product 1SD measured by the scheme of Example 1 was: 0.7, CV: 6.46%; the quality control product 2SD was: 17.33, CV: 1.70%; the quality control product 1SD measured by the scheme of Example 2 was: 0.53, CV: 5.02%; the quality control product 2SD was: 7.83, CV: 0.77%; the quality control product 1SD measured by the scheme of Example 3 was: 0.20, CV: 0.44%; the quality control product 2SD was: 13.45, CV: 0.40%. All CVs were <8%.

[0161] Conclusion: All CVs were <8%. Comparison of SD revealed that the scheme in Example 3 had more stable repeatability, smaller deviation, and met the requirements.

[0162] 5. Stability

[0163] The reagent kit was placed in a 37°C thermostat for accelerated destructive experiments, and the acceleration was performed for 1 day, 5 days, and 7 days, respectively, to observe the degree of decrease in the signal value of the reagent kit. According to the Arrhenius reaction equation, 7 days of acceleration at 37°C is equivalent to one year of storage at 4°C. If the signal value decreases by less than 20%, the reagent stability is considered to be extremely excellent, and if the signal value decreases by less than 50%, the reagent stability is considered to be qualified.

[0164] Data examples:

[0165] The data of Example 1 are as follows:

[0166]

[0167]

[0168] Example 2 data are as follows:

[0169]

[0170]

[0171] The data of Example 3 are as follows:

[0172]

[0173]

[0174] Analysis: Acceleration at 37°C for 7 days is equivalent to storage at 4°C for one year. Through the accelerated test, the stability of the kit can be quickly seen. After baking at 37°C for one day, the test values ​​of some samples increased. The reason is that after the accelerated destructive experiment, some unstable antibodies fell off and combined with the samples. With the initial progress of the accelerated experiment, the overall trend is more stable and balanced, but after a certain period of time, the theoretical balance will be broken, the antibody activity will decrease, and the signal value of the test sample will decrease. At this time, continue to observe whether the decrease meets the requirements.

[0175] Conclusion: It can be found from the data in the table that the decrease in the sample signal value detected by the scheme in Example 3 is less than 20%. The present invention obtains a kit with more stable performance by modifying acridinium ester, which has a good improvement effect.

[0176] In summary, the nuclear factor KB receptor activating factor magnetic microparticle chemiluminescence immunoassay kit disclosed in the present invention has higher detection sensitivity, specificity, precision and accuracy, and achieves better performance parameters. The magnetic microparticle reagent, AE labeled antibody and calibrator in the kit are the optimal formula under the reaction system, which provides a strong guarantee for the shelf life and detection performance of the kit.

[0177] The above drawings and explanations are only a specific implementation of the present invention, but the specific protection scope of the present invention is not limited to the above explanations. Any simple replacement or change within the technical idea disclosed in the present invention and the technical solution of the present invention should be within the protection scope of the present invention.

Claims

1. A method for preparing a magnetic microparticle chemiluminescent immunoassay kit for RANKL, characterized in that: The kit comprises a magnetic particle reagent, an AE-labeled RANKL antibody, and a RANKL calibrator. The magnetic particle reagent is a magnetic microsphere suspension coated with a RANKL antibody. The AE-labeled RANKL antibody is obtained by labeling the RANKL antibody with an acridinium ester. The particle size of the magnetic particles in the magnetic particle reagent is 1-3 μm, and the surface of the magnetic particles is coated with magnetic microspheres with carboxyl active groups; The preparation method comprises the following steps: preparing magnetic particle reagents, AE-labeled RANKL antibody RANKL calibrator; The preparation method of the AE-labeled RANKL antibody comprises: preparing a labeling buffer; weighing an appropriate amount of AE solid powder, dissolving it with a 0.1M boric acid buffer, and preparing an AE solution with a concentration of 1.0 mg / mL; mixing the mouse monoclonal anti-RANKL antibody and the AE solution at a mass ratio of 1:1.5, adding the labeling buffer for a linking reaction, and obtaining a linking product of the RANKL antibody and AE; separating and purifying the linking product with a carbonate buffer at a pH of 8 to 9, and obtaining an AE-labeled RANKL antibody; wherein the AE marker in the AE-labeled RANKL antibody is a modified acridinium ester; The acridinium ester is modified by adding a condensing agent EDC to form an active intermediate NHS ester, and the NHS ester reacts with the primary amino group of the labeled antibody; The labeling buffer is a PB buffer at pH 7.5, and each 1L of the buffer contains 25g of casein sodium salt, 15g of SDS, 15g of BSA and 1.0mL of Proclin-300; The preparation method of the magnetic particle reagent is as follows: dispersing a carboxyl magnetic bead solution under the action of a magnetic particle buffer to obtain a dispersed carboxyl magnetic bead solution; connecting the dispersed carboxyl magnetic bead solution and a RANKL antibody to obtain a connection product; placing the connection product in a magnetic field, and washing it with a magnetic particle buffer to obtain a magnetic particle reagent connected with the RANKL antibody, wherein the mass ratio of the RANKL antibody to the dispersed carboxyl magnetic bead solution is 1:80; and the concentration of the dispersed carboxyl magnetic bead solution is 10 mg / mL; The RANKL calibrator is a Tris buffer solution containing RANKL antigen, 0.5% BSA by weight, and 0.05% prolin300 by weight.

2. The method for preparing a RANKL magnetic microparticle chemiluminescence immunoassay kit according to claim 1, characterized in that: The operation of the dispersion treatment is as follows: placing the carboxyl magnetic bead concentrate in a magnetic field for 1-3 minutes, removing the upper clear liquid after all the carboxyl magnetic beads are precipitated, adding a magnetic particle buffer and oscillating, placing it in a magnetic field for 1-3 minutes, and then removing the upper clear liquid; the oscillation time is 20-30 minutes; the volume ratio of the carboxyl magnetic beads to the magnetic particle buffer is 1: (2-5); and the dispersion treatment is performed 3-5 times.

3. A method for detecting RANKL using the magnetic microparticle chemiluminescence immunoassay kit for RANKL according to any one of claims 1 to 2, characterized in that: (1) adding the serum sample and the prepared AE-labeled RANKL antibody to the prepared magnetic particle reagent for incubation; (2) After the incubation is completed and the magnetic particle reagent has settled, remove the supernatant, wash, add the substrate solution, shake and mix, and detect the relative luminescence intensity.

4. The method for detecting RANKL using the magnetic microparticle chemiluminescence immunoassay kit for RANKL according to claim 3, characterized in that: The volume ratio of the serum sample, AE-labeled RANKL antibody and magnetic particle reagent in step (1) is 1:2:

4.

5. The method for detecting RANKL using the magnetic microparticle chemiluminescence immunoassay kit for RANKL according to claim 4, characterized in that: The detection method, after detecting the relative luminescence intensity in step (2), performs calibration with high and low value two-point calibrants, and generates a new working curve by fitting for sample measurement; the RANKL quality control product is used as a sample, and the concentration is fitted using the working curve to obtain the corresponding quality control product concentration, and the fitted quality control product concentration is observed to be ±30% of the standard concentration value.

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