Immune magnetic beads, preparation method thereof and cancer antigen chemiluminescence immunoassay kit

Abstract

The present application relates to the technical field of biological detection, in particular to an immunomagnetic bead, a preparation method thereof and a cancer antigen chemiluminescence immunoassay kit; wherein the immunomagnetic bead takes cobalt ferrite CoFe2O4 as a magnetic nanoparticle, polystyrene as a shell coating and carboxyl as a functional group modification; the operation is simple, the separation efficiency is high, the specific surface area is large and the physical stability is good; when the immunomagnetic bead is applied to chemiluminescence immunoassay, the immunomagnetic bead is used as a solid-phase carrier and combined with chemiluminescence immunoassay, under the action of an external magnetic field, the immunomagnetic bead can be quickly separated from a substrate liquid phase; the immunomagnetic bead can significantly reduce the influence of an antigen on the immobilized antibody on the immunomagnetic bead in the immunoassay reaction due to the high separation and enrichment effect. The cancer antigen 15-3 (CA15-3) chemiluminescence immunoassay kit prepared based on the above immunomagnetic bead has high sensitivity, good specificity, high precision, short reaction time and strong anti-interference ability, and is of great significance for the detection of breast cancer.

Description

Technical Field

[0001] This invention relates to the field of biological detection technology, specifically to an immunomagnetic bead and its preparation method, and a chemiluminescent immunoassay kit for cancer antigen detection. Background Technology

[0002] According to global cancer data released by the International Agency for Research on Cancer (IARC) of the World Health Organization in recent years, breast cancer is gradually becoming one of the most common cancers worldwide, accounting for approximately 11.7% of new cancer cases. Cancer antigen 15-3 (CA15-3) is one of the most widely used tumor markers for breast cancer. It belongs to the glycoprotein antigen class, with a molecular weight of 300-500 kDa, and its antigenic determinant is composed of both sugar and polypeptide parts. CA15-3 is closely related to breast cancer, and its specificity for breast cancer is higher than that for carcinoembryonic antigen (CEA). Studies have shown that CA15-3 levels in breast cancer are positively correlated with TNM stage, histological grade, and lymph node metastasis. Dynamic monitoring of stage II and III patients helps in the early detection of recurrence and metastasis. In early-stage breast cancer, the sensitivity of CA15-3 is low, with a positive rate of approximately 20-50%; when metastasis occurs, the positive rate can reach 60-80%. Currently, in clinical practice, CA15-3 and CEA are often used simultaneously to significantly improve detection sensitivity. Therefore, developing a highly sensitive detection kit for cancer antigen detection is of great significance. Summary of the Invention

[0003] To address the technical problems in the prior art, the present invention aims to provide an immunomagnetic bead and its preparation method, and to apply it to the development of a CA15-3 chemiluminescent immunoassay kit. The immunomagnetic bead can be used as a carrier sphere for chemiluminescent immunoassay. The provided cancer antigen 15-3 (CA15-3) chemiluminescent immunoassay kit has advantages such as high sensitivity, wide linear range, good specificity, high precision, short reaction time, and strong anti-interference ability.

[0004] To achieve the above objectives, the technical solution provided by the present invention is as follows: The present invention provides an immunomagnetic bead, wherein the immunomagnetic bead uses cobalt ferrite (CoFe2O4) as magnetic nanoparticles, polystyrene as a shell coating, and carboxyl groups as functional groups for modification.

[0005] This invention provides a method for preparing immunomagnetic beads, comprising the following steps: Step 1: Preparation of continuous phase; The initiator benzoyl peroxide is dissolved in a mixed solution of the functional monomer glycidyl methacrylate and the porogen cyclohexanol. Then, sodium oleate, an active free radical control agent, and sodium dodecyl sulfate, a surfactant, are added to the mixture and mixed to obtain a continuous phase. Specifically, the initiator benzoyl peroxide initiates the decomposition of the chlorine atom of the alkylbenzene chlorination adsorbent of the fluorescent agent cyclohexaneyl chloride, generating free radicals that initiate the polymerization of the monomers.

[0006] Step 2: Preparation of the dispersed phase; Acrylic acid and N-isopropylacrylamide are added to an aqueous solution of calcium chloride and mixed evenly to prepare the dispersed phase; Calcium chloride reacts with the carboxyl and amide groups in the monomer to generate a dispersed phase with a high degree of gelation; Step 3: Preparation of advanced porous microspheres; The dispersed phase obtained in Step 2 is added dropwise to the continuous phase in Step 1, and advanced porous microspheres with a porous structure are obtained through free radical polymerization. The mixture is stirred to form an emulsion. The emulsion is then transferred to a chromatographic column, and the column is heated in a water bath or air bath at 65-75℃ to initiate the polymerization reaction. After the reaction is complete, the microspheres are washed with deionized water by centrifugation, and ethylenediamine is added. The mixture is stirred at 80℃ to obtain advanced porous microspheres. The mass ratio of ethylenediamine to the reaction solution is 2:1. The dropping rate of the dispersed phase is controlled at 0.5-1 mL / min, the stirring speed is 300 r / min, and the stirring time is 15-30 min. The polymerization reaction time is 12-18 h.

[0007] Step 4: Magnetic nanoparticles were prepared using a co-precipitation method. Ferrous nitrate (Fe(NO3)2) and cobalt nitrate (Co(NO3)2) were mixed and heated to 90°C. Ammonia and citric acid were rapidly added while stirring. The mixture was stirred and then cooled to room temperature. Magnets were able to adsorb the mixture. The nanoparticles were then washed repeatedly with deionized water to obtain citric acid-modified magnetic nanoparticles with a large number of negative charges on their surface. The stirring speed was adjusted to 300 rpm and the stirring time was 2 hours. Ferrous nitrate and cobalt nitrate reacted with ammonia to undergo a co-precipitation reaction. Citric acid, as a surface modifier, was responsible for modifying the surface of the magnetic nanoparticles and endowing them with negatively charged surface properties.

[0008] Step 5: Preparation of porous magnetic microspheres; The advanced porous microspheres prepared in Step 3 and the citric acid-modified magnetic nanoparticles prepared in Step 4 are mixed and adsorbed by shaking at room temperature to obtain porous magnetic microspheres with magnetic nanoparticles adsorbed on the surface; the shaking adsorption time is 2 hours. Step Six: Modification of porous magnetic microspheres; (1) Add porous magnetic microspheres to a flask, add N,N-dimethylformamide and ethylenediamine, connect the condenser tube and assemble the instrument, place it on a magnetic stirrer and stir at a constant temperature of 80℃; after stirring, wash the obtained substance with deionized water and dry it to obtain amino magnetic microspheres. (2) Place the amino magnetic microspheres in a clean flask, add triethylamine and succinic anhydride, connect the condenser tube and assemble the instrument, place it on a magnetic stirrer and stir at a constant temperature of 70°C; after stirring, wash the obtained substance with deionized water and dry it to obtain carboxyl magnetic microspheres, i.e. immunomagnetic beads.

[0009] Based on the above technical solution, in step one, the mass ratio of benzoyl peroxide: glycidyl methacrylate: cyclohexanol: sodium oleate: sodium dodecyl sulfate is 1:20-30:60-70:2-3:10-20.

[0010] Based on the above technical solution, in step two, the mass ratio of benzoyl peroxide: acrylic acid: N-isopropylacrylamide is 1.2-1.5:1:1-1.5; and the mass fraction of calcium chloride aqueous solution is 5-10%. Based on the above technical solution, in step six, the volume ratio of N,N-dimethylformamide and ethylenediamine is 1:1, and the volume ratio of triethylamine and succinic anhydride is 1:1.

[0011] First, cobalt ferrite (CoFe2O4), acting as the magnetic nanoparticle (magnetic core), possesses a large specific surface area and high magnetism, which improves the separation efficiency and stability of immunomagnetic beads. Simultaneously, CoFe2O4 also exhibits good optical properties, serving as a beneficial luminescent agent in chemiluminescent immunoassay. Second, polystyrene, as the shell material, possesses excellent physical and chemical stability and biocompatibility, effectively protecting the magnetic core and extending the lifespan of the beads. Furthermore, the carboxyl functional group modification of the polystyrene shell enhances the hydrophilicity of the immunomagnetic bead surface, improving stability and dispersibility during application. Moreover, this invention employs a simple and rapid method for preparing immunomagnetic beads, effectively reducing preparation costs and time. The preparation process utilizes a simple and easy-to-implement solid-phase coating technique to coat the prepared immunomagnetic beads with antibodies, ensuring full antibody utilization and increasing the specificity and sensitivity of the immunomagnetic beads. Simultaneously, the mild reaction conditions during preparation effectively prevent damage and inactivation of the antibodies.

[0012] This invention also provides the application of immunomagnetic beads in chemiluminescent immunoassay for cancer antigens. In chemiluminescent immunoassay, immunomagnetic beads containing the anti-glycoprotein antigen CA15-3, exhibiting high sensitivity, high specificity, and low background signal, are used. By coating the surface of the immunomagnetic beads with CA15-3 antibodies, CA15-3 protein can be captured in body fluids. During the detection process, the addition of acrid ester labeling and a chemical substrate triggers a luminescent reaction, thereby measuring the CA15-3 content in the sample. In chemiluminescent immunoassay, the combination of immunomagnetic beads as a solid-phase carrier and chemiluminescent immunoassay enables rapid and efficient capture of antigen molecules. Furthermore, due to their efficient separation and enrichment effects, the influence of antigens on immobilized antibodies can be significantly reduced. Therefore, the immunomagnetic beads prepared in this application exhibit high sensitivity, specificity, and stability in chemiluminescent immunoassay, enabling more accurate detection of the CA15-3 biomarker in breast cancer.

[0013] Based on the above technical solution, the cancer antigen chemiluminescent immunoassay kit includes magnetic microparticle reagent Ra, acridine ester reagent Rb, buffer Rc, calibrators, and quality control materials. Based on the above technical solution, the magnetic microparticle reagent Ra is an immunomagnetic bead coated with an anti-carbohydrate antigen CA15-3 antibody: 0.2-0.4 g / L, HEPES buffer: 10 mmol / L, ProClin 300: 0.5 g / L; Acridinium ester reagent Rb is an acridinium ester label for the anti-carbohydrate antigen CA15-3 antibody: 2-4 mg / L, MES buffer: 10 mmol / L, ProClin 300: 0.5 g / L; The buffer Rc is TRIS buffer: 10 mmol / L, ProClin 300: 0.5 g / L; The calibrators contained different amounts of CA15-3 antigen, TRIS buffer: 10 mmol / L, ProClin 300: 0.5 g / L; The quality control materials contained different amounts of CA15-3 antigen, TRIS buffer: 10 mmol / L, ProClin 300: 0.5 g / L.

[0014] The antibody used in the anti-glycoprotein antigen CA15-3 antibody is mouse monoclonal antibody IgG; the calibrators and quality control products contain different amounts of antigen compared to the Ra reagent.

[0015] Based on the above technical solution, the preparation method of the magnetic microparticle reagent Ra includes the following steps: (1) Replace and adjust the antibody buffer: Replace the antibody buffer with MES buffer and adjust the concentration of the anti-carbohydrate antigen CA15-3 antibody in the buffer to 2 mg / mL; (2) Immunomagnetic bead activation: The immunomagnetic beads were washed with MES buffer and resuspended in buffer to a concentration of ~100 mg / mL; EDC / Sulfo-NHS was weighed and dissolved in buffer; EDC and Sulfo-NHS were added to the washed immunomagnetic beads, mixed immediately, and activated at 25°C for 30 min. Specifically, the concentration of EDC was 20 mg / mL and the concentration of Sulfo-NHS was 24 mg / mL; 50 μL of EDC and 50 μL of Sulfo-NHS were added to the washed immunomagnetic beads; mixed immediately, and the concentration of immunomagnetic beads was ~50 mg / mL.

[0016] Based on the above technical solution, the preparation method of the acridinium ester reagent Rb includes the following steps: (1) Antibody solution purification: Place the anti-glycoprotein antigen CA15-3 antibody into a centrifuge tube and ensure it is at the bottom of the tube. Add phosphate buffer solution, mix thoroughly, and then add acridinium ester DMF solution. Centrifuge. Seal the centrifuge tube with sealing film and place it in a light-proof box. Then place the box in an air bath constant temperature shaker for mixing. Add lysine blocking solution and mix in an air bath constant temperature shaker for 1 hour. Place the blocked anti-glycoprotein antigen CA15-3 antibody into a dextran gel G250 column for purification at a flow rate of 3 ml / min. Elute with PB buffer and collect in steps. Store the collected anti-glycoprotein antigen CA15-3 antibody solution at 2-8℃. (2) Dilution before use: When using, dilute the purified concentrated solution of anti-glycogenic antigen CA15-3 antibody with 100mM Bistris propane, 0.05% Tween, 0.05% Proclin 300, pH 6.0 buffer to a concentration of 0.2μg / ml, and store at 2-8℃. Centrifuge at room temperature for 20s to ensure that the antibody is at the bottom of the centrifuge tube.

[0017] The beneficial effects of the technical solution provided by this invention are as follows: 1. This invention provides an immunomagnetic bead and its preparation method. The preparation method is simple to operate; the immunomagnetic beads offer high separation efficiency, a large specific surface area, and good physical stability. These immunomagnetic beads, used as a solid-phase carrier in conjunction with chemiluminescent immunoassay, can rapidly separate from the substrate liquid phase under the action of an external magnetic field. The magnetic beads, with their efficient separation and enrichment effects, significantly reduce the influence of antigens on the antibodies immobilized on the beads during the immunoassay reaction. Furthermore, the immunomagnetic beads of this invention exhibit high specificity and sensitivity, enabling rapid and efficient capture of antigen molecules, and possess good stability, thus playing an important role in chemiluminescent immunoassay.

[0018] 2. The cancer antigen 15-3 (CA15-3) chemiluminescent immunoassay kit prepared based on the above-mentioned immunomagnetic beads exhibits high sensitivity, good specificity, high precision, short reaction time, and strong anti-interference ability. This chemiluminescent immunoassay method can rapidly and accurately detect the diagnosis and treatment effects of breast cancer, providing significant assistance to clinical practice. Furthermore, the self-made immunomagnetic beads in this kit possess excellent performance, exhibiting higher sensitivity, specificity, and stability compared to other similar products, providing a more reliable guarantee for breast cancer detection. Attached Figure Description

[0019] Figure 1 This is the linear test curve of the detection reagent in Embodiment 1 of the present invention; Detailed Implementation The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0020] Unless otherwise defined, all technical and scientific terms used in this specification have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used in this specification is for the purpose of describing particular embodiments only and is not intended to limit the invention. The term "and / or" as used in this specification includes any and all combinations of one or more of the associated listed items. It should be understood that, unless otherwise specified, all the various materials used in this invention are commercially available.

[0021] Example 1 This embodiment provides an immunomagnetic bead and its preparation method; The immunomagnetic beads use cobalt ferrite (CoFe2O4) as magnetic nanoparticles, polystyrene as a shell coating, and carboxyl groups as functional groups for modification.

[0022] The method for preparing the immunomagnetic beads includes the following steps: Step 1: Preparation of continuous phase; Initiator benzoyl peroxide is dissolved in a mixed solution of functional monomer glycidyl methacrylate and porogen cyclohexanol. Then, sodium oleate, an active free radical control agent, and sodium dodecyl sulfate, a surfactant, are added to the mixture and mixed to obtain a continuous phase. The mass ratio of benzoyl peroxide: glycidyl methacrylate: cyclohexanol: sodium oleate: sodium dodecyl sulfate is 1:20:60:2:20.

[0023] Step 2: Preparation of dispersed phase; add acrylic acid and N-isopropylacrylamide to calcium chloride aqueous solution, mix evenly to prepare dispersed phase; the mass ratio of benzoyl peroxide:acrylic acid:N-isopropylacrylamide is 1.2:1:1, and the mass fraction of calcium chloride aqueous solution is 10%.

[0024] Step 3: Preparation of advanced porous microspheres; The dispersed phase obtained in Step 2 is added dropwise to the continuous phase in Step 1, and stirring is continued to obtain an emulsion; The emulsion is then transferred to a chromatographic column, and the column is heated in a water bath or air bath at 65-75℃ to initiate the polymerization reaction; After the reaction is completed, the microspheres are washed with deionized water by centrifugation, ethylenediamine is added, and the mixture is stirred at 80℃ to obtain advanced porous microspheres; The mass ratio of ethylenediamine to the reaction solution is 2:1. The dropping rate of the dispersed phase is controlled at 1 mL / min, the stirring speed is 300 r / min, and the stirring time is 15 min; The polymerization reaction time is 15 h.

[0025] Step 4: Magnetic nanoparticles were prepared using a co-precipitation method. Ferrous nitrate (Fe(NO3)2) and cobalt nitrate (Co(NO3)2) were mixed and heated to 90°C. Ammonia and citric acid were rapidly added while stirring. The mixture was stirred further and then cooled to room temperature. Magnets were able to adsorb the nanoparticles. The nanoparticles were then washed repeatedly with deionized water to obtain citric acid-modified magnetic nanoparticles with a large number of negative charges on their surface. The stirring speed was adjusted to 300 rpm and the stirring time was 2 hours. Step 5: Preparation of porous magnetic microspheres; The advanced porous microspheres prepared in Step 3 and the citric acid-modified magnetic nanoparticles prepared in Step 4 are mixed and adsorbed by shaking at room temperature to obtain porous magnetic microspheres with magnetic nanoparticles adsorbed on the surface; the shaking adsorption time is 2 hours. Step Six: Modification of porous magnetic microspheres; (1) Add 10g of porous magnetic microspheres to a flask, add 40ml of N,N-dimethylformamide and 40ml of ethylenediamine, connect the condenser tube and assemble the instrument, place it on a magnetic stirrer and stir at a constant temperature of 80℃ for 10h; after stirring, wash the obtained substance with deionized water and dry it to obtain amino magnetic microspheres. (2) Place 10g of amino magnetic microspheres in a clean flask, add 30ml of triethylamine and 30ml of succinic anhydride, connect the condenser tube and assemble the instrument, place it on a magnetic stirrer and stir at a constant temperature of 70℃ for 8 hours; after stirring, wash the obtained substance with deionized water and dry it to obtain carboxyl magnetic microspheres, i.e. immunomagnetic beads.

[0026] Example 2 Based on Example 1 above, in the preparation of immunomagnetic beads, the effect of controlling different amounts of N-isopropylacrylamide on the performance of the magnetic beads during the preparation of the dispersed phase in step three is as follows: The amounts of N-isopropylacrylamide used were 1.0 g, 2.0 g, 4.0 g, and 6.0 g, respectively. The results of the prepared immunomagnetic beads during detection are as follows: Table 1 Effects of N-isopropylacrylamide on immunomagnetic beads

[0027] Therefore, based on the results of this embodiment, the magnetic beads exhibited the best detection capability, stability, and reproducibility when the amount of N-isopropylacrylamide was 4.0 g.

[0028] Example 3 Based on Example 1 above, the effect of controlling different temperatures on the preparation of immunomagnetic beads in steps three and five during the preparation process is discussed. In this embodiment, during the preparation of advanced porous microspheres in step three, the polymerization reaction temperature was set to 55-65℃, and the shaking adsorption temperature during the porous magnetic microsphere preparation in step five was set to 40℃. When the prepared immunomagnetic beads were used for detection, the detection ability of the magnetic beads was optimal when the polymerization reaction temperature was 65-75℃ and the shaking adsorption temperature was room temperature, as well as when the stability and reproducibility were the best. Example 4 This embodiment provides the application of immunomagnetic beads in chemiluminescent immunoassay for cancer antigens, as well as the preparation process of the kit; the cancer antigen chemiluminescent immunoassay kit includes magnetic microparticle reagent Ra, acridine ester reagent Rb, buffer Rc, calibrators, and quality control materials.

[0029] The magnetic microparticle reagent Ra is an immunomagnetic bead coated with an anti-glycogenic antigen CA15-3 antibody: 0.4 g / L, HEPES buffer: 10 mmol / L, ProClin 300: 0.5 g / L; the preparation method of the magnetic microparticle reagent Ra includes the following steps: (1) Antibody buffer replacement and concentration adjustment: replace the antibody buffer with 15 mM MES6.0, and adjust the concentration of anti-glycogenic antigen CA15-3 antibody in the buffer to 2 mg / mL; (2) Immunomagnetic bead activation: 10 g of immunomagnetic beads are washed with MES buffer and resuspended in the buffer until the immunomagnetic bead concentration is 100 mg / mL; weigh EDC / Sulfo-NHS and dissolve it in the buffer: add EDC and Sulfo-NHS to the washed immunomagnetic beads, mix immediately, and activate at 25°C for 30 min. Specifically, the EDC concentration was 20 mg / mL and the Sulfo-NHS concentration was 24 mg / mL; 50 μL of EDC and 50 μL of Sulfo-NHS were added to the cleaned magnetic beads; the mixture was stirred immediately, and the magnetic bead concentration was 50 mg / mL.

[0030] The acridinium ester reagent Rb is an acridinium ester label for the anti-glycogenic antigen CA15-3 antibody: 4 mg / L, MES buffer: 10 mmol / L, ProClin 300: 0.5 g / L; the preparation method of the acridinium ester reagent Rb includes the following steps: (1) Antibody solution purification: 500 μg of anti-glycogenic antigen CA15-3 antibody is placed in a centrifuge tube and placed at the bottom of the centrifuge tube, phosphate buffer is added, and after thorough mixing, 5 μl of phosphate buffer is added. Centrifuge 2 mg / mL acridinium ester DMF solution; seal the centrifuge tube with sealing film and place it in a light-proof dark box, then place the dark box in an air bath constant temperature shaker for 4 h; then add 1 mL 20% lysine blocking solution, place it in an air bath constant temperature shaker for 1 h; place the blocked anti-glycogen CA15-3 antibody in a dextran gel G250 column for purification at a flow rate of 3 ml / min, elute with PB buffer, and collect in steps; store the collected anti-glycogen CA15-3 antibody solution at 2-8℃; (2) Dilution for use: when using, dilute the purified anti-glycogen CA15-3 antibody concentrated solution with 100 mM Bis-tris propane, 0.05% Tween, 0.05% Proclin 300, pH 6.0 buffer to a concentration of 0.2 μg / ml and store at 2-8℃. The antibody was ensured to be located at the bottom of the centrifuge tube by centrifuging at room temperature for 20 seconds. The buffer Rc is TRIS buffer: 10 mmol / L, ProClin 300: 0.5 g / L; The calibrators contained different amounts of CA15-3 antigen, TRIS buffer: 10 mmol / L, ProClin 300: 0.5 g / L; The quality control materials contained different amounts of CA15-3 antigen, TRIS buffer: 10 mmol / L, ProClin 300: 0.5 g / L.

[0031] Preferably, the antibody against the carbohydrate antigen CA15-3 is a mouse monoclonal antibody IgG; Example 5 This embodiment provides a detection method using a chemiluminescent immunoassay kit for cancer antigens: The detection method of the cancer antigen 15-3 (CA15-3) chemiluminescent immunoassay kit is an indirect method, using a fully automated chemiluminescence analyzer as the detection tool. The instrument sequentially adds 20 μL of sample to the corresponding reaction tube, followed by 50 μL of immunomagnetic beads coated with anti-glycogenic antigen CA15-3 antibody. The reaction is allowed to proceed for 10 min. Under the influence of a magnetic field, the immunomagnetic beads are adsorbed onto the reaction tube wall, while unbound substances are washed away with the washing solution. Then, 60 μL of anti-glycogenic antigen CA15-3 antibody-acrididine ester label is added, and after incubation for 5 min, magnetic separation and washing are performed. The instrument pushes the reaction tube into a dark chamber, and luminescent substrate solutions A (HNO3 solution) and B (NaOH solution) are added sequentially for reaction. The mixture is thoroughly mixed, and the chemiluminescence intensity is measured. The analyzer then automatically calculates the concentration of the anti-glycogenic antigen CA15-3 antibody using a calibration curve.

[0032] Example 6 This example provides the performance test results of the cancer antigen chemiluminescent immunoassay kit: (1) Linearity detection: High-value samples close to the upper limit of the linear range were diluted to 7 concentrations at a certain ratio. Each concentration was tested twice, and the average value was calculated. The average value and the dilution ratio were fitted to a straight line using the least squares method, and the linear correlation coefficient r was calculated. The linear correlation coefficient r = 0.9999, and the linear range was 0.01-500.00 ng / mL. The results are as follows: Figure 1 As shown in Table 2.

[0033] Table 2 Linearity Detection Results

[0034] (2) Minimum detection limit: The minimum detection limit is defined as the average value of 20 luminescence intensity measurements of the zero-value calibrator, minus twice the standard deviation, and the result is substituted into the standard curve to obtain the sensitivity. The minimum detection limit of the cancer antigen 15-3 (CA15-3) chemiluminescent immunoassay kit was calculated to be <0.01 ng / mL. The results are shown in Table 3. Table 3 Luminous intensity detection

[0035] (3) Accuracy: When cancer antigen 15-3 (CA15-3) sample (A) with a concentration of approximately 50 ng / mL (with an allowable concentration deviation of ±20%) was added to the corresponding matrix sample (B) at a volume ratio of (A) / (B) of 1:9, the recovery rate was calculated to be 96.80%, and the accuracy of the kit was 96.80%, as shown in Table 4. Table 4 Recovery rate test results

[0036] (4) Repeatability: Samples with concentrations of 5±2 ng / mL and 50±10 ng / mL were tested 10 times each. The coefficients of variation (CV) for repeatability of the two samples were 1.12% and 1.25%, respectively. The results are shown in Table 5. Table 5 Repeatability coefficient of variation test

[0037] (5) Inter-batch variation: Take 3 batches of reagent kits and use samples with two concentrations of 5 ng / ml and 50 ng / ml as test samples. Perform 10 repeated measurements on the test samples. Calculate the coefficient of variation (CV) of the 30 measurements, as shown in Table 6. The inter-batch variation is less than 5%.

[0038] Table 6. Multi-batch coefficient of variation test

[0039] (6) Anti-interference specificity test: The results of tests for conjugated bilirubin, free bilirubin, hemoglobin and ascorbic acid with an added concentration of 20% will not significantly affect the determination of cancer antigen 15-3 (CA15-3).

[0040] (7) Stability: The reagent kit provided in this invention is stored at 2-8℃. After 12 months of testing, the sensitivity, linearity and repeatability of the reagent kit described in the examples are all within the normal range.

[0041] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above, and that the invention can be implemented in other specific forms without departing from its spirit or essential characteristics. Therefore, the embodiments should be considered illustrative and non-limiting in all respects, and the scope of the invention is defined by the appended claims rather than the foregoing description. Thus, all variations falling within the meaning and scope of equivalents of the claims are intended to be included within the present invention. No reference numerals in the claims should be construed as limiting the scope of the claims. Furthermore, it should be understood that although this specification describes embodiments, not every embodiment contains only one independent technical solution. This narrative style is merely for clarity. Those skilled in the art should consider the specification as a whole, and the technical solutions in each embodiment can also be appropriately combined to form other embodiments that can be understood by those skilled in the art.

Claims

1. An immunomagnetic bead, characterized in that, The immunomagnetic beads use cobalt ferrite (CoFe2O4) as magnetic nanoparticles, polystyrene as a shell coating, and carboxyl groups as functional groups for modification; the preparation method of the immunomagnetic beads includes the following steps: Step 1: Preparation of continuous phase; Initiator benzoyl peroxide is dissolved in a mixed solution of functional monomer glycidyl methacrylate and porogen cyclohexanol, and then sodium oleate, active free radical control agent and surfactant sodium dodecyl sulfate are added to the mixture and mixed to obtain continuous phase; Step 2: Preparation of the dispersed phase; add acrylic acid and N-isopropylacrylamide to an aqueous solution of calcium chloride, mix thoroughly to obtain the dispersed phase; Step 3: Preparation of advanced porous microspheres; The dispersed phase obtained in Step 2 is added dropwise to the continuous phase in Step 1, and stirred to obtain an emulsion; The emulsion is then transferred to a chromatographic column, and the column is heated in a water bath or air bath at a temperature of 65-75℃ to initiate the polymerization reaction; After the reaction is completed, the microspheres are washed with deionized water by centrifugation, ethylenediamine is added, and the mixture is stirred at a temperature of 80℃ to obtain advanced porous microspheres; Step 4: Prepare magnetic nanoparticles using the co-precipitation method; mix ferrous nitrate Fe(NO3)2 and cobalt nitrate Co(NO3)2 and heat to 90℃, then rapidly add ammonia and citric acid while stirring, continue stirring and cool to room temperature, magnets can be attracted down, and then deionized water is added for repeated washing; citric acid-modified magnetic nanoparticles with a large number of negative charges on the surface are obtained. Step 5: Preparation of porous magnetic microspheres; The advanced porous microspheres prepared in Step 3 and the citric acid-modified magnetic nanoparticles prepared in Step 4 are mixed and adsorbed by shaking at room temperature to obtain porous magnetic microspheres with magnetic nanoparticles adsorbed on the surface. Step Six: Modification of porous magnetic microspheres; (1) Add porous magnetic microspheres to a flask, add N,N-dimethylformamide and ethylenediamine, connect the condenser tube and assemble the instrument, place it on a magnetic stirrer and stir at a constant temperature of 80℃; after stirring, wash the obtained substance with deionized water and dry it to obtain amino magnetic microspheres. (2) Place the amino magnetic microspheres in a clean flask, add triethylamine and succinic anhydride, connect the condenser tube and assemble the instrument, place it on a magnetic stirrer and stir at a constant temperature of 70°C; after stirring, wash the obtained substance with deionized water and dry it to obtain carboxyl magnetic microspheres, i.e. immunomagnetic beads.

2. The immunomagnetic bead according to claim 1, characterized in that, In step one, the mass ratio of benzoyl peroxide: glycidyl methacrylate: cyclohexanol: sodium oleate: sodium dodecyl sulfate is 1:20-30:60-70:2-3:10-20.

3. An immunomagnetic bead according to claim 1, characterized in that, In step two, the mass ratio of acrylic acid to N-isopropylacrylamide is 1:1-1.

5.

4. An immunomagnetic bead according to claim 1, characterized in that, In step six, the volume ratio of N,N-dimethylformamide to ethylenediamine is 1:1, and the volume ratio of triethylamine to succinic anhydride is 1:

1.

5. The application of the immunomagnetic beads according to claim 1 in the preparation of a chemiluminescent immunoassay kit for cancer antigen detection.

6. The application of the immunomagnetic beads according to claim 5 in the preparation of a chemiluminescent immunoassay kit for cancer antigens, characterized in that, The chemiluminescent immunoassay kit for cancer antigens includes magnetic microparticle reagent Ra, acridine ester reagent Rb, buffer Rc, calibrators, and quality control materials.

7. The application of the immunomagnetic beads according to claim 6 in the preparation of a chemiluminescent immunoassay kit for cancer antigens, characterized in that, The magnetic microparticle reagent Ra consists of immunomagnetic beads coated with anti-carbohydrate antigen CA15-3 antibody: 0.2-0.4 g / L, HEPES buffer: 10 mmol / L, and ProClin 300: 0.5 g / L; Acridinium ester reagent Rb is an acridinium ester label for the anti-carbohydrate antigen CA15-3 antibody: 2-4 mg / L, MES buffer: 10 mmol / L, ProClin 300: 0.5 g / L; The buffer Rc is TRIS buffer: 10 mmol / L, ProClin 300: 0.5 g / L; The calibrators contained different amounts of CA15-3 antigen, TRIS buffer: 10 mmol / L, ProClin 300: 0.5 g / L; The quality control materials contained different amounts of CA15-3 antigen, TRIS buffer: 10 mmol / L, ProClin 300: 0.5 g / L.

8. The application of the immunomagnetic beads according to claim 7 in the preparation of a chemiluminescent immunoassay kit for cancer antigens, characterized in that, The preparation method of the magnetic microparticle reagent Ra includes the following steps: (1) Replace and adjust the antibody buffer: Replace the antibody buffer with MES buffer and adjust the concentration of the anti-carbohydrate antigen CA15-3 antibody in the buffer to 2 mg / mL; (2) Immunomagnetic bead activation: The immunomagnetic beads were washed with MES buffer and resuspended in buffer to a concentration of 100 mg / mL. EDC / Sulfo-NHS were weighed and dissolved in buffer. EDC and Sulfo-NHS were added to the washed immunomagnetic beads and mixed immediately. The mixture was activated at 25°C for 30 min.

9. The application of the immunomagnetic beads according to claim 7 in the preparation of a chemiluminescent immunoassay kit for cancer antigens, characterized in that, The preparation method of the acridinium ester reagent Rb includes the following steps: (1) Antibody solution purification: Place the anti-glycoprotein antigen CA15-3 antibody into a centrifuge tube and ensure it is at the bottom of the tube. Add phosphate buffer solution, mix thoroughly, and then add acridinium ester DMF solution. Centrifuge. Seal the centrifuge tube with sealing film and place it in a light-proof box. Then place the box in an air bath constant temperature shaker for mixing. Add lysine blocking solution and mix in an air bath constant temperature shaker for 1 hour. Place the blocked anti-glycoprotein antigen CA15-3 antibody into a dextran gel G250 column for purification at a flow rate of 3 ml / min. Elute with PB buffer and collect in steps. Store the collected anti-glycoprotein antigen CA15-3 antibody solution at 2-8℃. (2) Dilution for use: When using, dilute the purified anti-glycogen CA15-3 antibody concentrate with 100mM Bistris propane, 0.05% Tween, 0.05% Proclin 300, pH 6.0 buffer to a concentration of 0.2μg / ml and store at 2-8℃.

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