Anti-ovarian antibody chemiluminescence kit and preparation method thereof

Abstract

The invention relates to an anti-ovarian antibody chemiluminiscence kit and a preparation method thereof. The kit comprises amino magnetic beads coated with anti-ovarian antibody recombinant protein, anti-human immune globulin labeled by alkaline phosphatase, an anti-ovarian antibody calibrator and a chemiluminiscence substrate. The method has the beneficial effects that the 7-day acceleration stability of the AOAb coated amino magnetic bead is improved to 95% or above, the signal-to-noise ratio is high, the correlation coefficient is greater than 0.999, the test repeatability CV is less than 3.5%, the reagent correlation and the magnetic bead stability can be better improved, and the test result is ensured to be accurate. By optimizing the amino magnetic bead cross-linking agent, 60 clinical samples are tested and compared with the result of a comparison manufacturer, the total coincidence rate is as high as 98%, and false positive can be effectively reduced. Meanwhile, a detection system is formed by matching with a Guilin superior chemiluminescence immunoassay analyzer IA-260, the testing amount per hour reaches 130 tests, and compared with a traditional colloidal gold immunochromatography method and a traditional enzyme-linked immunosorbent assay method, the detection speed is greatly increased, and the result optimization accuracy is greatly improved.

Description

Technical Field

[0001] This invention relates to the field of chemiluminescence detection technology, and in particular to an anti-ovarian antibody chemiluminescence reagent kit and its preparation method. Background Technology

[0002] In recent years, the involvement of immune factors in the infertility process has been confirmed. Some scholars believe that immune dysregulation is not only an important factor leading to female infertility, but also increases the risk of reproductive tract infections, thus accelerating the infertility process. Therefore, the detection of reproductive immune antibodies plays an important role in the diagnosis and treatment of female infertility. Anti-ovarian antibodies (AOAb) and anticardiolipin antibodies are also common anti-reproductive immune antibodies in clinical practice. AOAb is produced by the body due to increased antigens or excessive immune responses caused by stimuli such as infection or surgery, which can cause damage to ovarian structure and function, resulting in infertility.

[0003] As an autoantibody, AOAb target antigens are mainly cytoplasmic components of oocytes, granulosa cells and luteal cells in the ovary. This antibody is the main cause of ovarian insufficiency, which can lead to follicle atresia before maturation, resulting in irregular menstruation, reduced follicle development quality, and eventually infertility. Therefore, it can be concluded that AOAb positivity plays a certain role in the formation of ovarian insufficiency and can cause infertility caused by immunological factors.

[0004] Currently, the main methods for detecting anti-ovarian antibodies include enzyme-linked immunosorbent assay (ELISA), colloidal gold method, and chemiluminescent immunoassay. Peng Jiexiong et al. (2009) evaluated the clinical application value of an infertility protein chip, finding significant differences in the positive concordance rate for AOAb detection results, indicating that the detection sensitivity needs further improvement. CN107561266A discloses a method for preparing antibody immunomagnetic beads, utilizing carboxyl magnetic beads to coat AOAb, which improves detection sensitivity, but does not mention solutions to the false positive problem.

[0005] Carboxyl magnetic beads work by activating the carboxyl groups on their surface using EDC / NHS, which then bind to the amino groups of the protein, achieving coating. The magnetic beads are primarily linked to the protein molecule via amide bonds (i.e., peptide bonds). Because these bonds are relatively short and rigid, in the complex three-dimensional structure formed by protein folding, they force adjacent amino acid side chain R groups to move closer together, resulting in a significant steric hindrance effect. This steric hindrance directly affects the local conformational flexibility of the protein and may hinder other molecules (such as substrates, ligands, or chemically modified reagents) from approaching the target reaction site.

[0006] Since the active sites of biomolecules typically contain lysine residues and free amino groups, using carboxyl-based magnetic microspheres to couple biomolecules inevitably occupies a portion of these active sites. This can lead to a decrease in biomolecule affinity or conformational changes, thus affecting their biological activity. This effect may be particularly pronounced for certain conformationally sensitive biomolecules (such as enzymes and receptors).

[0007] Therefore, it is necessary to provide a chemiluminescent immunoassay kit for detecting anti-ovarian antibodies with excellent sensitivity, stability, and specificity, as well as its preparation method. Summary of the Invention

[0008] In summary, to overcome the shortcomings of existing technologies, the technical problem to be solved by this invention is to provide a chemiluminescent reagent kit for anti-ovarian antibodies and its preparation method. Compared with the amide bond linkage of carboxyl magnetic beads, amino magnetic beads, as a carrier, have the advantages of covalently binding the amino groups to the amino groups of proteins through different cross-linking agent arms, and optimizing the spatial region through long cross-linking arms to reduce protein steric hindrance. This allows the target molecule to bind to the protein in the correct manner, effectively reducing false positives. It also features low background luminescence, high signal-to-noise ratio, fewer interfering factors, and good magnetic bead stability. The key technology of this invention is the screening of magnetic beads and cross-linking agents to obtain a chemiluminescent detection kit for anti-ovarian antibodies that improves reagent sensitivity, stability, and reduces false positives.

[0009] The technical solution of the present invention to solve the above-mentioned technical problems is as follows: an anti-ovarian antibody chemiluminescent reagent kit, comprising: amino magnetic beads coated with recombinant anti-ovarian antibody protein, enzyme-labeled antibody working solution, anti-ovarian antibody calibrator and chemiluminescent substrate.

[0010] Based on the above technical solution, the present invention can be further improved as follows:

[0011] Furthermore, the anti-ovarian antibody recombinant protein coated on the amino magnetic beads is coupled to the amino magnetic microparticles via SMCC, PEG-4-SMCC, and PEG-12-SMCC anomalous cross-linking agents.

[0012] Furthermore, the mass ratio of amino magnetic microparticles to anti-ovarian antibody recombinant protein in the amino magnetic beads coated with the anti-ovarian antibody recombinant protein is 1:0.02~1:0.1.

[0013] Furthermore, the chemiluminescent marker labeled with anti-human immunoglobulin in the enzyme-labeled antibody working solution is anti-human immunoglobulin labeled with alkaline phosphatase, and the mass ratio of anti-human immunoglobulin to chemiluminescent marker is 1:1 to 1:4. The chemiluminescent marker is selected from horseradish peroxidase, glucose oxidase, pyruvate kinase or alkaline phosphatase.

[0014] Furthermore, the concentration of the amino group coated with the recombinant anti-ovarian antibody protein is 0.4 mg / ml, the working concentration of the enzyme-labeled antibody working solution is 1 µg / ml, and the concentrations of the anti-ovarian antibody calibrators are solutions of anti-ovarian antibody with concentrations of 1 RU / mL, 10 RU / mL, 50 RU / mL, 100 RU / mL, 500 RU / mL, and 1000 RU / mL, respectively.

[0015] Furthermore, the chemiluminescent substrate is selected from luminol, isoluminol and its derivatives, acridinium ester or adamantane-1,2-dioxane and its derivatives.

[0016] This invention also provides a method for preparing an anti-ovarian antibody chemiluminescent reagent kit, comprising the following steps:

[0017] Step 1: Coat amino magnetic beads with recombinant anti-ovarian antibody protein to obtain amino magnetic beads coated with recombinant anti-ovarian antibody protein;

[0018] Step 2: Label anti-human immunoglobulin with alkaline phosphatase to obtain alkaline phosphatase-labeled anti-human immunoglobulin, and then prepare enzyme-labeled antibody working solution.

[0019] Step 3: Prepare anti-ovarian antibody calibrators;

[0020] Step 4: Prepare a kit by mixing 5 mL of amino magnetic bead working solution coated with recombinant anti-ovarian antibody protein obtained in Step 1, 5 mL of enzyme-labeled antibody working solution obtained in Step 2, 1 mL of anti-ovarian antibody calibrator obtained in Step 3, and 100 mL of chemiluminescent substrate.

[0021] Based on the above technical solution, the present invention can be further improved as follows:

[0022] Furthermore, the preparation method of the anti-ovarian antibody chemiluminescent reagent kit is characterized in that step 1 specifically comprises:

[0023] Step 1.1, prepare the following buffer solutions:

[0024] Phosphate buffer: composed of 18 mmol / L disodium hydrogen phosphate, 2 mmol / L sodium dihydrogen phosphate, and 150 mM sodium chloride, with pH adjusted to 7.4;

[0025] Blocking solution: prepared by adding 0.5% bovine serum albumin and 0.5% Tween 20 to phosphate buffer;

[0026] Preservative solution: prepared based on phosphate buffer, with the addition of 0.5% bovine serum albumin, 0.5% Tween 20 and 1% Proclin-300;

[0027] Step 1.2, Magnetic bead pretreatment: Take amino magnetic beads with a particle size of 1μm~5μm, mix them with a vortex mixer, place them in an EP tube according to the required amount, add phosphate buffer to make the final concentration of magnetic beads 10mg / mL, mix with a vortex mixer, remove the supernatant by magnetic separation, wash again with phosphate buffer, and finally add phosphate buffer to maintain the final concentration of magnetic beads 10mg / mL to obtain a magnetic bead suspension for later use.

[0028] Step 1.3, magnetic bead activation reaction: Prepare an aqueous solution of 2-IT and add it to the magnetic bead suspension to make the final concentration of 2-IT 1 mg / ml. After vortexing and mixing, suspend at room temperature in the dark for 1 h.

[0029] Step 1.4, Antibody activation reaction: Prepare a DMSO solution of PEG-12-SMCC and add it to the recombinant anti-ovarian antibody protein solution to make the final concentration of PEG-12-SMCC 0.5 mg / mL and the concentration of anti-ovarian antibody 2 mg / mL. After vortexing and mixing, suspend at room temperature in the dark for 1 h. The activated anti-ovarian antibody is then desalted using a desalting column.

[0030] Step 1.5, Magnetic bead antibody conjugation reaction: After the magnetic beads are activated, the supernatant is removed by magnetic separation. First, phosphate buffer is added to make the final concentration of magnetic beads 10 mg / mL. The mixture is vortexed and mixed. Then, the activated and desalted anti-ovarian antibody is added at a mass ratio of 1:0.05 between magnetic beads and antibody. The mixture is vortexed and mixed again. The mixture is then suspended at room temperature in the dark for 2 hours or incubated at 4 degrees for 10 hours.

[0031] Step 1.6, magnetic bead blocking: After the coupling reaction is completed, the supernatant is removed by magnetic separation, and blocking solution is added to make the final concentration of magnetic beads 10 mg / mL. The mixture is then suspended at room temperature for 1-2 hours.

[0032] Step 1.7, Cleaning and Preservation: After blocking, add phosphate buffer and wash three times to obtain amino magnetic beads coated with recombinant anti-ovarian antibody protein;

[0033] Step 1.8, Prepare magnetic bead working solution: Prepare a 0.4 mg / ml solution of amino magnetic beads coated with recombinant anti-ovarian antibody protein obtained in step 1.7 using preservation solution.

[0034] Furthermore, step 2 specifically involves:

[0035] Step 2.1, Activation of anti-human immunoglobulin: Prepare a glutaraldehyde aqueous solution with a final concentration of 20%, add anti-human immunoglobulin to make the final concentration of anti-human immunoglobulin 2 mg / mL, vortex to mix, and then let it stand at room temperature in the dark for 30 min to activate.

[0036] Step 2.2, Enzyme-labeled conjugation and desalting: Alkaline phosphatase was added to the activated anti-human immunoglobulin solution at a mass ratio of 1:2. After mixing, the mixture was reacted at room temperature in the dark for 2 hours. After the reaction, desalting was performed to obtain alkaline phosphatase-labeled anti-human immunoglobulin. An equal volume of glycerol was added, and the mixture was stored at -20°C for later use.

[0037] Step 2.3, prepare enzyme-labeled dilution solution: the components and ratios are 50mM Tris, 9g / L sodium chloride, 10g / L glucose, 10g / L BSA, 0.01mM zinc ions, 10mM magnesium ions, 0.5% TW20, 1% proclin-300, and adjust the pH to 7.4;

[0038] Step 2.4, Prepare enzyme-labeled antibody working solution: Prepare a 1µg / ml solution of alkaline phosphatase-labeled anti-human immunoglobulin obtained in step 2.2 using the enzyme-labeled diluent prepared in step 2.3.

[0039] Furthermore, step 3 specifically involves:

[0040] Step 3.1: Prepare a calibrator dilution solution with pH 6.8 using the following raw materials: 50 mmol / L PBS, 150 mM sodium chloride, 10 g / L bovine serum albumin, 0.1% Tween 20, 0.1 g / L glucose, 10 g / L PEG4000, and 1% proclin-300.

[0041] Step 3.2: Prepare solutions of anti-ovarian antibody with concentrations of 1 RU / mL, 10 RU / mL, 50 RU / mL, 100 RU / mL, 500 RU / mL and 1000 RU / mL using the calibrator diluent prepared in Step 3.1, thereby obtaining anti-ovarian antibody calibrators.

[0042] The beneficial effects of this invention are: it provides an amino magnetic bead coupling scheme that improves the 7-day accelerated stability of AOAb-coated amino magnetic beads to over 95%, with a high signal-to-noise ratio, a correlation coefficient greater than 0.999, and a test repeatability CV < 3.5%, effectively improving reagent correlation and magnetic bead stability, and ensuring accurate test results. Through optimization of the amino magnetic bead crosslinking agent, the overall concordance rate of 60 clinical samples compared with manufacturer results reached 98%, effectively reducing false positives. Simultaneously, when combined with the Guilin Ulite IA-260 chemiluminescence immunoassay analyzer, the detection system can perform up to 130 tests per hour, significantly improving testing speed and optimizing result accuracy compared to traditional colloidal gold immunochromatography and enzyme-linked immunosorbent assay (ELISA). Detailed Implementation

[0043] The principles and features of the present invention are described below with reference to specific examples. The examples are only used to explain the present invention and are not intended to limit the scope of the present invention.

[0044] Example 1: This example yields an anti-ovarian antibody chemiluminescent detection kit 1

[0045] A method for preparing a chemiluminescent detection kit for anti-ovarian antibodies includes the following steps:

[0046] Step 1, preparing amino magnetic beads coated with recombinant anti-ovarian antibody protein, specifically includes the following steps:

[0047] Step 1.1, prepare the following buffer solution:

[0048] Phosphate buffer: 18 mmol / L disodium hydrogen phosphate, 2 mmol / L sodium dihydrogen phosphate, 150 mM sodium chloride, pH 7.4;

[0049] Blocking buffer: phosphate buffer containing 0.5% bovine serum albumin and 0.5% Tween 20;

[0050] Preservative solution: Phosphate buffer containing 0.5% bovine serum albumin, 0.5% Tween 20, and 1% Proclin-300.

[0051] Step 1.2: Mix 2μm amino magnetic beads using a vortex mixer. The required amount of magnetic beads is taken and placed in an EP tube. Add phosphate buffer to achieve a final concentration of 10 mg / mL. Mix again using a vortex mixer. After magnetic separation to remove the supernatant, wash once more with phosphate buffer. Finally, add phosphate buffer to achieve a final concentration of 10 mg / mL to obtain a magnetic bead suspension for later use.

[0052] Step 1.3, magnetic bead activation reaction: Prepare 2-IT aqueous solution, add 2-IT to the magnetic bead suspension to make the final concentration of 2-IT 1mg / ml, vortex and mix again, and suspend at room temperature in the dark for 1h.

[0053] Step 1.4, Antibody Activation Reaction: Prepare a DMSO solution of PEG-12-SMCC. Add PEG-12-SMCC to the recombinant anti-ovarian antibody protein solution to achieve a final concentration of 0.5 mg / mL for PEG-12-SMCC and 2 mg / mL for the anti-ovarian antibody. Vortex to mix thoroughly and suspend in the dark at room temperature for 1 hour. Desalt the activated anti-ovarian antibody using a desalting column.

[0054] Step 1.5, Magnetic bead antibody conjugation reaction: After the magnetic beads are activated, the supernatant is removed by magnetic separation. First, add phosphate buffer to make the final concentration of the magnetic beads 10 mg / mL, vortex to mix, then add the activated antibody, with a mass ratio of magnetic beads to antibody of 1:0.05, vortex again to mix, and suspend at room temperature in the dark for 2 hours.

[0055] Step 1.6, Magnetic bead blocking: After the coupling reaction is completed, the supernatant is removed by magnetic separation and blocking solution is added. The final concentration of magnetic beads is 10 mg / mL, and the mixture is suspended at room temperature for 2 hours.

[0056] Step 1.7, Cleaning and Preservation: After sealing, add phosphate buffer and wash three times to obtain amino magnetic beads coated with recombinant anti-ovarian antibody protein.

[0057] Step 1.8, Prepare magnetic bead working solution: Prepare a 0.4 mg / ml solution of amino magnetic beads coated with recombinant anti-ovarian antibody protein obtained in step 1.7 using preservation solution.

[0058] Step 2, preparing alkaline phosphatase-labeled anti-human immunoglobulin, specifically includes the following steps:

[0059] Step 2.1: Prepare an aqueous solution of glutaraldehyde with a final concentration of 20%. Add anti-human immunoglobulin to the prepared aqueous solution of glutaraldehyde to achieve a final concentration of 2 mg / mL. Vortex to mix thoroughly and allow to stand at room temperature in the dark for 30 min to activate.

[0060] Step 2.2: Then add alkaline phosphatase at a mass ratio of 1:2 and mix well. React at room temperature in the dark for 2 hours. After the reaction is complete, desalt to obtain alkaline phosphatase-labeled anti-human immunoglobulin. Add an equal volume of glycerol and store at -20°C for later use.

[0061] Step 2.3: The enzyme-labeled dilution solution consists of 50 mM Tris, 9 g / L sodium chloride, 10 g / L glucose, 10 g / L BSA, 0.01 mM zinc ions, 10 mM magnesium ions, 0.5% TW20, and 1% proclin-300, with a pH of 7.4.

[0062] Step 2.4: Prepare an enzyme-labeled antibody working solution by diluting alkaline phosphatase-labeled anti-human immunoglobulin with enzyme-labeled diluent to a concentration of 1 µg / ml.

[0063] Step 3, prepare the anti-ovarian antibody calibrator, which includes the following steps:

[0064] Step 3.1, prepare calibrator dilution solution: 50 mmol / L PBS, 150 mM sodium chloride, 10 g / L bovine serum albumin, 0.1% Tween 20, 0.1 g / L glucose, 10 g / L PEG4000, 1% proclin-300, pH 6.8;

[0065] Step 3.2: Prepare anti-ovarian antibody calibrators by diluting the anti-ovarian antibody with calibrator diluent to concentrations of 1RU / mL, 10RU / mL, 50RU / mL, 100RU / mL, 500RU / mL and 1000RU / mL.

[0066] Step 4: Prepare kit 1 by mixing 5 mL of amino magnetic bead working solution coated with recombinant anti-ovarian antibody protein obtained in step 1, 5 mL of enzyme-labeled antibody working solution obtained in step 2, 1 mL of anti-ovarian antibody calibrator obtained in step 3, and 100 mL of chemiluminescent substrate.

[0067] Example 2: The magnetic bead crosslinking agent used was SMCC, and the remaining steps were the same as in Example 1. This example yielded the anti-ovarian antibody chemiluminescence detection kit 2.

[0068] Example 3: The magnetic bead cross-linking agent used was PEG-4-SMCC, and the remaining steps were the same as in Example 1. This example yielded the anti-ovarian antibody chemiluminescence detection kit 3.

[0069] Example 4:

[0070] The magnetic beads conjugated with recombinant anti-ovarian antibody protein are carboxyl magnetic beads, and their preparation method is as follows:

[0071] Step 1, prepare the following buffer solution

[0072] Coupling buffer: 100mM MES pH4.0;

[0073] Blocking solution: 0.5% bovine serum albumin, 0.5% Tween 20 phosphate buffer;

[0074] Preservative solution: 0.5% bovine serum albumin, 0.5% Tween 20, 1% proclin-300 phosphate buffer.

[0075] Step 2: Mix the 2μm carboxyl magnetic beads with a vortex mixer. Take the amount of magnetic beads as needed, place them in an EP tube, add coupling buffer to make the final concentration of magnetic beads 10mg / mL, mix with a vortex mixer, remove the supernatant by magnetic separation, and wash once with coupling buffer.

[0076] Step 3: Prepare aqueous solutions of EDC and NHS, with a final concentration of 1 mg / mL for both EDC and NHS, and a final concentration of 10 mg / mL for the magnetic beads. First, add the coupling buffer and vortex to mix. Then, add the EDC and NHS, vortex again to mix, and suspend at room temperature in the dark for 1 hour.

[0077] Step 4: After the magnetic beads are activated, the supernatant is removed by magnetic separation. First, add coupling buffer to make the final concentration of the magnetic beads 10 mg / mL, vortex to mix, then add the anti-ovarian antibody recombinant protein. The mass ratio of magnetic beads to antibody is 1:50. Vortex to mix again and suspend at room temperature in the dark for 1 hour.

[0078] Step 5: After the coupling reaction is completed, the supernatant is removed by magnetic separation and a blocking solution is added. The final concentration of the magnetic beads is 10 mg / mL. The mixture is then suspended at room temperature for 2 hours.

[0079] Step 6: After blocking, wash three times with phosphate buffer. Finally, store the magnetic beads in preservation solution. At this point, the working concentration of the amino magnetic beads conjugated with the recombinant anti-ovarian antibody protein is 0.2 mg / ml. The remaining steps are the same as in Example 1. This example yields the anti-ovarian antibody chemiluminescent detection kit 4.

[0080] Example 5: Performance testing was performed using the anti-ovarian antibody chemiluminescent immunoassay kits from each example. The photon quantity and signal-to-noise ratio (S / N) of different concentrations of anti-ovarian antibody calibrators were measured using the Unit IA-260 fully automated chemiluminescence immunoassay analyzer. A two-step procedure was followed: 50 μL of magnetically coated anti-ovarian antibody recombinant protein and 20 μL of the test sample were added to a reaction tube. After mixing and incubation for 10 min, unbound antibody protein was removed using magnetic separation with the Unit IA-260 fully automated chemiluminescence immunoassay analyzer. Then, 50 μL of enzyme-labeled antibody working solution diluted with enzyme-labeled diluent was added, mixed, and incubated for 10 min. Unbound enzyme-labeled antibody was then removed using magnetic separation. Finally, 200 μL of luminescent substrate was added, and the results were measured.

[0081] 1. Linearity test

[0082] The anti-ovarian antibody calibrators at concentrations of 1 RU / mL, 10 RU / mL, 50 RU / mL, 100 RU / mL, 500 RU / mL and 1000 RU / mL were tested according to Example 5, and linearity analysis was performed to calculate the linear correlation coefficient. The results are shown in Table 1.

[0083] Table 1 Linearity Test Results

[0084]

[0085] The results in Table 1 show that the signal-to-noise ratio of kit 1 (3.31) is greater than that of kits 2-4, and the correlation coefficients of kits 1 and 3 are better than those of kits 2 and 4. This indicates that the long-chain crosslinking agent used in the amino magnetic beads in the kits prepared in this invention can improve linearity.

[0086] 2. Stability test of coated magnetic beads

[0087] The coated magnetic beads from different reagent kits were divided into two parts and placed at 2-8℃ / 37℃ for 7 days. The accelerated stability of the different magnetic beads was then tested according to Example 5, and the results are shown in Table 2.

[0088] Table 2 Accelerated stability results

[0089]

[0090] The results in Table 2 show that after 7 days of accelerated treatment at 37°C, the photon signal retention rate of the coated magnetic beads in Kit 1 is above 95%, which is higher than that of other kits. The stability of the carboxyl magnetic beads in Kit 4 is not as good as that of the amino magnetic beads, meaning that Kit 1 has the best stability.

[0091] 3. Reagent kit repeatability test

[0092] The different reagent kits were tested 10 times each for four different concentrations of calibrators according to the method in Example 5, and the coefficient of variation (CV) was calculated. The results are shown in Table 3.

[0093] Table 3 Repeatability Test Results

[0094]

[0095] According to the data in Table 3, the coefficients of variation of the test results of kits 1 to 4 are all within 5%. Among them, the repeatability test results of kit 1 are slightly better than those of kits 2 and 3.

[0096] 4. Clinical testing of the reagent kit

[0097] The detection system composed of kits 1-4 from this example and the IA-260 fully automated chemiluminescence immunoassay analyzer from the Urit series was compared with the Kanghua anti-ovarian antibody detection reagent (enzyme-linked immunosorbent assay). The specific experiments are as follows:

[0098] Table 4 Clinical test results of reagent kit 1

[0099]

[0100] Table 5 Clinical trial results of reagent kit 2

[0101]

[0102] Table 6. Clinical test results of reagent kit 3

[0103]

[0104] Table 7 Clinical test results of reagent kit 4

[0105]

[0106] The results in Table 4-7 show that kit 1 has the best clinical concordance rate with the comparison manufacturer's kit.

[0107] Based on the above results, kit 1 exhibits the best overall performance, with the best signal-to-noise ratio, linearity, precision, and acceleration stability. It also demonstrates stable performance and effectively reduces false positives.

[0108] 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 chemiluminescent reagent kit for anti-ovarian antibodies, characterized in that, include: Amino magnetic beads coated with recombinant anti-ovarian antibody protein, enzyme-labeled antibody working solution, anti-ovarian antibody calibrator and chemiluminescent substrate.

2. The anti-ovarian antibody chemiluminescent reagent kit according to claim 1, characterized in that, The anti-ovarian antibody recombinant protein coated amino magnetic beads are coupled with amino magnetic microparticles via SMCC, PEG-4-SMCC, and PEG-12-SMCC anomalous cross-linking agents.

3. The anti-ovarian antibody chemiluminescent reagent kit according to claim 2, characterized in that, The mass ratio of amino magnetic microparticles to recombinant anti-ovarian antibody protein in the amino magnetic beads coated with the recombinant anti-ovarian antibody protein is 1:0.02 to 1:0.

1.

4. The anti-ovarian antibody chemiluminescent reagent kit according to claim 1, characterized in that, The chemiluminescent marker labeled with anti-human immunoglobulin in the enzyme-labeled antibody working solution is alkaline phosphatase-labeled anti-human immunoglobulin, and the mass ratio of anti-human immunoglobulin to chemiluminescent marker is 1:1 to 1:

4. The chemiluminescent marker is selected from horseradish peroxidase, glucose oxidase, pyruvate kinase or alkaline phosphatase.

5. The anti-ovarian antibody chemiluminescent reagent kit according to claim 1, characterized in that, The concentration of the amino group coated with the recombinant anti-ovarian antibody protein is 0.4 mg / ml, the working concentration of the enzyme-labeled antibody working solution is 1 µg / ml, and the concentrations of the anti-ovarian antibody calibrators are solutions of anti-ovarian antibody with concentrations of 1 RU / mL, 10 RU / mL, 50 RU / mL, 100 RU / mL, 500 RU / mL, and 1000 RU / mL.

6. The anti-ovarian antibody chemiluminescent reagent kit according to claim 1, characterized in that, The chemiluminescent substrate is selected from luminol, isoluminol and its derivatives, acridine esters or adamantane-1,2-dioxane and its derivatives.

7. A method for preparing an anti-ovarian antibody chemiluminescent reagent kit, characterized in that, Includes the following steps: Step 1: Coat amino magnetic beads with recombinant anti-ovarian antibody protein to obtain amino magnetic beads coated with recombinant anti-ovarian antibody protein; Step 2: Label anti-human immunoglobulin with alkaline phosphatase to obtain alkaline phosphatase-labeled anti-human immunoglobulin, and then prepare enzyme-labeled antibody working solution. Step 3: Prepare anti-ovarian antibody calibrators; Step 4: Prepare a kit by mixing 5 mL of amino magnetic beads coated with the recombinant anti-ovarian antibody protein obtained in Step 1, 5 mL of enzyme-labeled antibody working solution obtained in Step 2, 1 mL of anti-ovarian antibody calibrator obtained in Step 3, and 100 mL of chemiluminescent substrate.

8. The method for preparing the anti-ovarian antibody chemiluminescent reagent kit according to claim 7, characterized in that, Step 1 is as follows: Step 1.1, prepare the following buffer solutions: Phosphate buffer: composed of 18 mmol / L disodium hydrogen phosphate, 2 mmol / L sodium dihydrogen phosphate, and 150 mM sodium chloride, with pH adjusted to 7.4; Blocking solution: prepared by adding 0.5% bovine serum albumin and 0.5% Tween 20 to phosphate buffer; Preservative solution: prepared based on phosphate buffer, with the addition of 0.5% bovine serum albumin, 0.5% Tween 20 and 1% Proclin-300; Step 1.2, Magnetic bead pretreatment: Take amino magnetic beads with a particle size of 1μm~5μm, mix them with a vortex mixer, place them in an EP tube according to the required amount, add phosphate buffer to make the final concentration of magnetic beads 10mg / mL, mix with a vortex mixer, remove the supernatant by magnetic separation, wash again with phosphate buffer, and finally add phosphate buffer to maintain the final concentration of magnetic beads 10mg / mL to obtain a magnetic bead suspension for later use; Step 1.3, magnetic bead activation reaction: Prepare an aqueous solution of 2-IT and add it to the magnetic bead suspension to make the final concentration of 2-IT 1 mg / ml. After vortexing and mixing, suspend at room temperature in the dark for 1 h. Step 1.4, Antibody activation reaction: Prepare a DMSO solution of PEG-12-SMCC and add it to the recombinant anti-ovarian antibody protein solution to make the final concentration of PEG-12-SMCC 0.5 mg / mL and the concentration of anti-ovarian antibody 2 mg / mL. After vortexing and mixing, suspend at room temperature in the dark for 1 h. The activated anti-ovarian antibody is then desalted using a desalting column. Step 1.5, Magnetic bead antibody conjugation reaction: After the magnetic beads are activated, the supernatant is removed by magnetic separation. First, phosphate buffer is added to make the final concentration of magnetic beads 10 mg / mL. The mixture is vortexed and mixed. Then, the activated and desalted anti-ovarian antibody is added at a mass ratio of 1:0.05 between magnetic beads and antibody. The mixture is vortexed and mixed again. The mixture is then suspended at room temperature in the dark for 2 hours or incubated at 4 degrees for 10 hours. Step 1.6, magnetic bead blocking: After the coupling reaction is completed, the supernatant is removed by magnetic separation, and blocking solution is added to make the final concentration of magnetic beads 10 mg / mL. The mixture is then suspended at room temperature for 1-2 hours. Step 1.7, Cleaning and Preservation: After blocking, add phosphate buffer and wash three times to obtain amino magnetic beads coated with recombinant anti-ovarian antibody protein; Step 1.8, Prepare magnetic bead working solution: Prepare a 0.4 mg / ml solution of amino magnetic beads coated with recombinant anti-ovarian antibody protein obtained in step 1.7 using preservation solution.

9. The method for preparing the anti-ovarian antibody chemiluminescent reagent kit according to claim 7, characterized in that, Step 2 is as follows: Step 2.1, Activation of anti-human immunoglobulin: Prepare a glutaraldehyde aqueous solution with a final concentration of 20%, add anti-human immunoglobulin to make the final concentration of anti-human immunoglobulin 2 mg / mL, vortex to mix, and then let it stand at room temperature in the dark for 30 min to activate. Step 2.2, Enzyme-labeled conjugation and desalting: Alkaline phosphatase was added to the activated anti-human immunoglobulin solution at a mass ratio of 1:

2. After mixing, the mixture was reacted at room temperature in the dark for 2 hours. After the reaction, desalting was performed to obtain alkaline phosphatase-labeled anti-human immunoglobulin. An equal volume of glycerol was added, and the mixture was stored at -20°C for later use. Step 2.3, prepare enzyme-labeled dilution solution: the components and ratios are 50mM Tris, 9g / L sodium chloride, 10g / L glucose, 10g / L BSA, 0.01mM zinc ions, 10mM magnesium ions, 0.5% TW20, 1% proclin-300, and adjust the pH to 7.4; Step 2.4, Prepare enzyme-labeled antibody working solution: Prepare a 1µg / ml solution of alkaline phosphatase-labeled anti-human immunoglobulin obtained in step 2.2 with the enzyme-labeled diluent prepared in step 2.3 to obtain the enzyme-labeled antibody working solution.

10. The method for preparing the anti-ovarian antibody chemiluminescent reagent kit according to claim 7, characterized in that, Step 3 specifically involves: Step 3.1: Prepare a calibrator dilution solution with pH 6.8 using the following raw materials: 50 mmol / L PBS, 150 mM sodium chloride, 10 g / L bovine serum albumin, 0.1% Tween 20, 0.1 g / L glucose, 10 g / L PEG4000, and 1% proclin-300. Step 3.2: Prepare solutions of anti-ovarian antibody with concentrations of 1 RU / mL, 10 RU / mL, 50 RU / mL, 100 RU / mL, 500 RU / mL and 1000 RU / mL using the calibrator diluent prepared in Step 3.1, thereby obtaining anti-ovarian antibody calibrators.

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