A method for expanding the carboxyl content on the surface of biomolecules or solid-phase carriers and its application

Abstract

The present invention discloses a labeling method for expanding the carboxyl content on the surface of biomolecules or solid-phase carriers and its application. The method includes activating the carboxyl groups on the biomolecules or solid-phase carriers through EDC and NHS, and then adding an appropriate amount of carboxyl-expanding molecular substance NH 2 -(CH 2 ) n -CH-((CH 2 ) n -COOH) 2 , the amino group of which is covalently bonded to the activated carboxyl group on the biomolecule or solid-phase carrier, obtaining a biomolecule or solid-phase carrier with an expanded carboxyl content. It can effectively reduce the steric hindrance effect between molecules and the multi-stage amplification of detection signals after covalent coupling between one molecule or solid-phase carrier and another biomolecule, and at the same time avoid the technical problem of the possibility of clinical misdiagnosis.

Description

Technical Field

[0001] The present invention relates to the technical field of immunology, and particularly to a labeling method for expanding the carboxyl content on the surface of biomolecules or solid-phase carriers and its application. Background Art

[0002] The basic principle of immunolabeling technology is to utilize the high specificity of antigen-antibody reactions and the measurability of various labels to conveniently and sensitively detect various trace bioactive substances in the body. Immunolabeling technology is the most active and fastest-developing important branch in clinical immunoassay technology.

[0003] Biomolecules include enzymes, antibodies, antigens, tag polypeptides, etc., and solid-phase carriers include latex particles, magnetic bead particles, colloidal gold particles, silica particles, time-resolved fluorescence microspheres, and quantum dots, etc., and groups such as carboxyl or amino groups are derivatized on their surfaces. In labeling methods, the current covalent coupling techniques for labeling enzymes, antibodies, antigens, or other types of biomolecules B on biomolecule A or solid-phase carriers are roughly divided into three categories. The first category is to use an activator to activate carboxyl and amino groups to form an amide bond. The second category is to use sugar molecules to form aldehydes after oxidation and then combine with primary amino groups to form secondary amine bonds. The third category is to use an activator to activate amino groups and thiol groups to form thioether bonds. The coupling efficiencies of the three labeling methods are all very high. If it is the first or second labeling method, a tightly connected complex is formed between biomolecule A or solid-phase carrier and biomolecule B, and there is no linker between biomolecule A or solid-phase carrier and biomolecule B, and there will be a certain steric hindrance effect at this time. If it is the third method of forming thioether bonds, there is at least an 8-atom linker between biomolecule A or solid-phase carrier and biomolecule B, which can effectively reduce the steric hindrance effect between the two ends of the linker. At the same time, in the above labeling methods, one group on biomolecule A or solid-phase carrier can only covalently connect one molecule of biomolecule B. In most cases, the sensitivities of the kits prepared by these methods can effectively meet the current clinical detection requirements. However, for the detection of certain extremely trace human markers, higher-sensitivity reagents are needed to capture the extremely trace markers in human specimens.

[0004] In order to effectively reduce the steric hindrance effect between biomolecules and between biomolecules, technicians have developed a variety of new labeling methods. 1) Biotin-avidin system. Biotin (B) and avidin (A) or streptavidin (SA), whose affinity is at least one million times higher than the antigen-antibody binding force, are the substances with the strongest affinity found in nature so far. Advantages: Since both biotin and avidin are relatively stable, and one molecule of avidin can bind four molecules of biotin, the detection signal can be amplified at multiple levels. At the same time, the long-arm connection of biotin can also keep the binding site of the antibody to the antigen effectively exposed, which is a relatively commonly used labeling method in current chemiluminescence detection. Disadvantages: Biotin is one of the vitamins existing in the organism and is sometimes used as a drug. It is particularly common in small intestine tissue, kidney tissue, brain tissue, and liver tissue. Using the biotin-avidin system in the above tissues will result in negative signals unrelated to the detection itself, that is, there may be a misdiagnosis of false negatives. 2) Spacer arm system of thioether bond. As described in CN11209864B, the invention provides a method for labeling fluorescent protein and / or conjugated protein with monoclonal antibody. mainly, the amino group of the fluorescent protein and / or conjugated protein binds to SMCC to convert the amino group into a maleimide group. The amino group on the monoclonal antibody extends a thiol group through a cross-linking agent. After the two are mixed, the maleimide group reacts with the thiol group to form a thioether bond, thereby forming a spacer arm of at least 8 atoms between the fluorescent protein and / or conjugated protein and the monoclonal antibody. Advantages: There is a spacer arm connection between the antibody and the fluorescent protein and / or conjugated protein, which can ensure the effective exposure of the binding site of the antibody to the antigen and greatly reduce the steric hindrance effect. Disadvantages: Only one molecule of fluorescent protein and / or conjugated protein can be coupled to a certain amino group on an antibody molecule after extension, and multiple signal molecules cannot be connected to achieve multi-level amplification of the detection signal.

[0005] Therefore, it is necessary to further research a method that can accurately label trace markers and amplify the detection signal to achieve the detection purpose. Summary of the Invention

[0006] The object of the present invention is to provide a method for expanding the carboxyl content on the surface of biomolecules or solid-phase carriers.

[0007] Another object of the present invention is to provide the application of a method for expanding the carboxyl content on the surface of biomolecules or solid-phase carriers.

[0008] The technical solution adopted to achieve the object of the present invention is as follows:

[0009] In order to achieve the above object, it is realized through the following experimental steps:

[0010] (1) The carboxyl groups on biomolecules or solid-phase carriers are activated by EDC and NHS;

[0011] (2) An appropriate amount of carboxyl-extended molecule NH 2 -(CH 2 ) n -CH-((CH 2 ) n -COOH) 2 is added, and its amino group covalently binds to the activated carboxyl groups on the biomolecules or solid-phase carriers to obtain biomolecules or solid-phase carriers with an increased carboxyl content.

[0012] In step (1), the mass ratio of the biomolecule to EDC and NHS is 100:(1 - 10):(1 - 10), preferably 100:5:5.

[0013] In step (1), the mass ratio of the solid-phase carrier to EDC and NHS is 10:(1 - 10):(1 - 100), preferably 10:5:5.

[0014] In step (2), for the carboxyl-extended molecule NH 2 -(CH 2 ) n -CH-((CH 2 ) n -COOH) 2 n is between 3 and 100, preferably 3 - 8, and more preferably n = 6.

[0015] In step (2), the mass ratio of the carboxyl-extended molecule NH 2 -(CH 2 ) n -CH-((CH 2 ) n -COOH) 2 to the biomolecule is 20000:(1 - 100), preferably 20000:(1 - 10), and more preferably 20000:8.5.

[0016] In step (2), the mass ratio of the carboxyl-extended molecule NH 2 -(CH 2 ) n -CH-((CH 2 ) n -COOH) 2 to the solid-phase carrier is 20000:(1 - 100), preferably 20000:(1 - 10), and more preferably 20000:8.5.

[0017] The present invention has the following beneficial effects: According to the present invention, a method capable of expanding the carboxyl content on the surface of biomolecules or solid-phase carriers is provided, which can solve the problem that after covalent coupling between a biomolecule or solid-phase carrier and another biomolecule in the existing labeling method, the steric hindrance is large and affects the detection signal. It can achieve multi-stage amplification of the detection signal, and the detection signal or sensitivity can be increased by 2-4 times, avoiding the technical problem of the possibility of clinical misdiagnosis. Detailed implementation mode

[0018] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the attached tables in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0019] It should be noted that, without conflict, the embodiments in the present invention and the features in the embodiments can be combined with each other.

[0020] Example 1 Covalent labeling of an antibody with an expanded carboxyl content and alkaline phosphatase

[0021] 1) Preparation of a monoclonal antibody with an expanded carboxyl content

[0022] 1 mg of CEA monoclonal antibody (purchased from Nanjing Kaitai'er) was dissolved in 1 mL of 50 mM MES solution with pH = 6.0. 0.05 mg of NHS and 0.05 mg of EDC were successively added, and stirred at room temperature for 20 min. The EDC and NHS were removed using an ultrafiltration tube with a molecular cut-off of 50 KD. The antibody treated by ultrafiltration was dissolved in 1 mL of MES, and then 0.000425 mg of NH 2 -(CH 2 ) 6 -CH-((CH 2 ) 6 -COOH) 2 was added, and stirred at room temperature for 30 min. Dialysis was carried out in 50 mM MES with pH = 6.0 for 24 hours (cut-off molecular weight 70000), and the dialysis solution was changed every 12 h, thus obtaining 1 mg of CEA monoclonal antibody with an expanded carboxyl content.

[0023] 2) Preparation of CEA monoclonal antibody with an expanded carboxyl content covalently labeled with alkaline phosphatase

[0024] In 1 mg of the CEA monoclonal antibody after the carboxyl group is extended, 2 mg of alkaline phosphatase is added, then 0.05 mg of EDC is added, and the mixture is stirred at room temperature for 30 min. Then 10 mg of BSA is added to block for 30 min. 10 μL of Proclin 300 and 1 mL of glycerol are added to obtain the CEA monoclonal antibody with extended carboxyl content covalently labeled with alkaline phosphatase, denoted as labeled antibody 1.

[0025] 3) Preparation of CEA monoclonal antibody with unextended carboxyl content covalently labeled with alkaline phosphatase

[0026] In 1 mg of the CEA monoclonal antibody, 2 mg of alkaline phosphatase is added, then 0.05 mg of EDC is added, and the mixture is stirred at room temperature for 30 min. Then 10 mg of BSA is added to block for 30 min. 10 μL of Proclin 300 and 1 mL of glycerol are added to obtain the CEA monoclonal antibody with unextended carboxyl content covalently labeled with alkaline phosphatase, denoted as labeled antibody 2.

[0027] Example 2 Covalent labeling of magnetic beads with extended carboxyl content and antibodies

[0028] 1) Preparation of magnetic beads with extended carboxyl content

[0029] 10 mg of 1.5 μm carboxyl magnetic beads (purchased from JSR Life Science) are washed twice in 50 mM MES solution at pH = 6.0. 5 mg of NHS and 5 mg of EDC are added successively, and the mixture is stirred at room temperature for 20 min. Then it is washed once in 50 mM MES solution at pH = 6.0 and diluted to a concentration of 1 mg / mL. 0.00425 mg of NH 2 -(CH 2 ) 6 -CH-((CH 2 ) 6 -COOH) 2 is added, and the mixture is stirred at room temperature for 30 min. Then it is washed twice in 50 mM MES at pH = 6.0 and diluted to 2 mg / mL with 50 mM MES at pH = 6.0 to obtain the magnetic beads with extended carboxyl content.

[0030] 2) Preparation of magnetic beads with extended carboxyl content coated with CEA - labeled antibody

[0031] The above-mentioned magnetic beads after the extended carboxyl content of 10 mg were successively added with 5 mg of NHS and 5 mg of EDC, stirred at room temperature for 20 min, washed once with a MES solution of pH = 6.0 and 50 mM, and diluted to a concentration of 1 mg / mL. 0.1 mg of CEA-coated antibody was added, stirred at room temperature for 30 min, and then 10 mg of BSA was added for 30 min of blocking. After magnetic separation to obtain the magnetic beads, they were dissolved in a solution containing 10 mM PBS at pH 7.2, 0.2% BSA, 0.1% Tween-20, and 0.1% Proclin 300, with a concentration of 0.5 mg / mL, thus obtaining magnetic beads labeled with CEA-coated antibody with an extended carboxyl content, which is magnetic bead solution 1.

[0032] 3) Preparation of magnetic beads labeled with CEA-coated antibody without extended carboxyl content

[0033] 10 mg of carboxyl magnetic beads were successively added with 5 mg of NHS and 5 mg of EDC, stirred at room temperature for 20 min, washed once with a MES solution of pH = 6.050 mM, and diluted to a concentration of 1 mg / mL. 0.1 mg of CEA-coated antibody was added, stirred at room temperature for 30 min, and then 10 mg of BSA was added for 30 min of blocking. After magnetic separation to obtain the magnetic beads, they were dissolved in a solution containing 10 mM PBS at pH 7.2, 0.2% BSA, 0.1% Tween-20, and 0.1% Proclin 300, with a concentration of 0.5 mg / mL, thus obtaining magnetic beads labeled with CEA-coated antibody without extended carboxyl content, which is magnetic bead solution 2.

[0034] Detection in Example 3

[0035] The labeled antibody 1 and labeled antibody 2 obtained in Example 1 were diluted to the labeling diluent at a ratio of 1:3000. The components of the labeling diluent are 10 mM PBS at pH 7.2, 0.2% BSA, 0.1% Tween-20, and 0.1% Proclin 300.

[0036] Sample addition method: Mix 10 μL of sample + 50 μL of labeled antibody + 50 μL of magnetic bead solution and incubate for 15 min. After washing 5 times, add 200 μL of luminescent solution for detection.

[0037] Establishment of the standard curve: CEA standard products with different concentrations (0, 2.5, 7, 20, 50, 150 ng / mL) were prepared as samples. Labeled antibody 1 and magnetic bead solution 1 were used as combination 1, and labeled antibody 2 and magnetic bead solution 2 were used as combination 2. After separate tests, the standard curves were linearly fitted. The standard curve of combination 1 is Y = 10160 + 13450.1*X, and the correlation coefficient r = 0.9999; the standard curve of combination 2 is Y = 6522.4 + 8335.3*X, and the correlation coefficient r = 0.9999. The test results are shown in Table 1.

[0038] Table 1 Detection Results of Example 3

[0039]

[0040]

[0041] Sensitivity evaluation: The standard product with a concentration of 0 ng / mL was detected 20 times, and the average value M + 2 times the standard deviation SD was the analytical sensitivity of the reagent combination. The average value M1 of combination 1 = 0.38, the standard deviation SD1 = 0.23, and the analytical sensitivity of combination 1 = 0.38 + 0.23 * 2 = 0.84; the average value M2 of combination 2 = 0.08, the standard deviation SD2 = 0.06, and the analytical sensitivity of combination 2 = 0.08 + 0.06 * 2 = 0.20. The analytical sensitivity of combination 1, 0.84, is 4.2 times that of combination 2, 0.20, and the sensitivity is increased by 3.2 times. The detection results are shown in Table 2.

[0042] Table 2 Detection Results of Example 3

[0043] Test Serial Number Combination 1 (unit: ng / mL) Combination 2 (unit: ng / mL) 1 0.49 0.07 2 0.25 0.03 3 0.66 0.05 4 0.24 0.04 5 0.10 0.10 6 0.35 0.08 7 0.71 0.04 8 0.30 0.04 9 0.03 0.19 10 0.21 0.07 11 0.20 0.19 12 0.49 0.20 13 0.02 0.04 14 0.52 0.00 15 0.36 0.02 16 0.15 0.11 17 0.48 0.04 18 0.71 0.09 19 0.69 0.10 20 0.70 0.15 Average M 0.38 0.08 SD 0.23 0.06 CV 0.84 0.20

[0044] Covalent Labeling of Antibody with Extended Carboxyl Content and Alkaline Phosphatase in Example 4

[0045] 1) Preparation of Monoclonal Antibody with Extended Carboxyl Content

[0046] 1 mg of CEA monoclonal antibody (purchased from Nanjing Kaitai'er) was dissolved in 1 mL of 50 mM MES solution with pH = 6.0. 0.05 mg of NHS and 0.05 mg of EDC were added successively, and stirred at room temperature for 20 min. EDC and NHS were removed using an ultrafiltration tube with a molecular cut-off of 50 KD. The antibody treated by ultrafiltration was dissolved in 1 mL of MES, and then 0.00005 mg of NH 2 -(CH 2 ) 3 -CH-((CH 2 ) 3 -COOH) 2 was added, and stirred at room temperature for 30 min. Dialysis was carried out in 50 mM MES with pH = 6.0 for 24 hours (cut-off molecular weight 70000), and the dialysis solution was changed every 12 h, thus obtaining 1 mg of CEA monoclonal antibody with extended carboxyl content.

[0047] 2) Preparation of CEA Monoclonal Antibody with Extended Carboxyl Content Covalently Labeled with Alkaline Phosphatase

[0048] In 1 mg of the CEA monoclonal antibody after the extension of carboxyl groups, 2 mg of alkaline phosphatase was added, then 0.05 mg of EDC was added, and the mixture was stirred at room temperature for 30 min. Then 10 mg of BSA was added to block for 30 min. 10 μL of Proclin300 and 1 mL of glycerol were added to obtain the alkaline phosphatase-covalently labeled CEA monoclonal antibody with an extended carboxyl content, denoted as labeled antibody 3.

[0049] Example 5 Covalent Labeling of Magnetic Beads with Extended Carboxyl Content and Antibody

[0050] 1) Preparation of magnetic beads with extended carboxyl content

[0051] 10 mg of 1.5 μm carboxyl magnetic beads (purchased from JSR Life Science) were washed twice in a 50 mM MES solution at pH = 6.0, and then 5 mg of NHS and 5 mg of EDC were added successively. The mixture was stirred at room temperature for 20 min, washed once in a 50 mM MES solution at pH = 6.0, and diluted to a concentration of 1 mg / mL. 0.0005 mg of NH 2 -(CH 2 ) 3 -CH-((CH 2 ) 3 -COOH) 2 was added, and the mixture was stirred at room temperature for 30 min. It was washed twice in MES at pH = 6.0 50 mM and diluted to 2 mg / mL with MES at pH = 6.0 50 mM to obtain the magnetic beads with an extended carboxyl content.

[0052] 2) Preparation of magnetic beads with extended carboxyl content labeled with CEA-coated antibody

[0053] For the above 10 mg of magnetic beads with extended carboxyl content, 5 mg of NHS and 5 mg of EDC were added successively, and the mixture was stirred at room temperature for 20 min, washed once in a 50 mM MES solution at pH = 6.0, and diluted to a concentration of 1 mg / mL. 0.1 mg of CEA-coated antibody was added, and the mixture was stirred at room temperature for 30 min. Then 10 mg of BSA was added to block for 30 min. After magnetic separation to obtain the magnetic beads, they were dissolved in a solution containing 10 mM PBS at pH 7.2, 0.2% BSA, 0.1% Tween-20, and 0.1% Proclin300 at a concentration of 0.5 mg / mL to obtain the magnetic beads with extended carboxyl content labeled with CEA-coated antibody, which is magnetic bead solution 3.

[0054] Example 6 Covalent Labeling of Antibody with Extended Carboxyl Content and Alkaline Phosphatase

[0055] 1) Preparation of monoclonal antibody with extended carboxyl content

[0056] 1 mg of CEA monoclonal antibody (purchased from Nanjing Kaitai'er) was dissolved in 1 mL of 50 mM MES solution with pH = 6.0. 0.05 mg of NHS and 0.05 mg of EDC were added successively, and stirred at room temperature for 20 min. The EDC and NHS were removed using an ultrafiltration tube with a molecular cut-off of 50 KD. The antibody treated by ultrafiltration was dissolved in 1 mL of MES, and then 0.0005 mg of NH 2 -(CH 2 ) 8 -CH-((CH 2 ) 8 -COOH) 2 was added, and stirred at room temperature for 30 min. Dialyzed in 50 mM MES with pH = 6.0 for 24 h (molecular weight cut-off 70000), and the dialysis solution was changed every 12 h, thus obtaining 1 mg of CEA monoclonal antibody with increased carboxyl group content.

[0057] 2) Preparation of alkaline phosphatase covalently labeled CEA monoclonal antibody with increased carboxyl group content

[0058] In 1 mg of CEA monoclonal antibody with increased carboxyl group content, 2 mg of alkaline phosphatase was added, then 0.05 mg of EDC was added, and stirred at room temperature for 30 min. Then 10 mg of BSA was added to block for 30 min, 10 μL of Proclin300 and 1 mL of glycerol were added, thus obtaining alkaline phosphatase covalently labeled CEA monoclonal antibody with increased carboxyl group content, denoted as labeled antibody 4.

[0059] Example 7 Covalent labeling of magnetic beads with increased carboxyl group content and antibody

[0060] 1) Preparation of magnetic beads with increased carboxyl group content

[0061] 10 mg of 1.5 μm carboxyl magnetic beads (purchased from JSR Life Science) were washed twice in 50 mM MES solution with pH = 6.0. 5 mg of NHS and 5 mg of EDC were added successively, and stirred at room temperature for 20 min. Washed once in 50 mM MES solution with pH = 6.0, and diluted to a concentration of 1 mg / mL. 0.005 mg of NH 2 -(CH 2 ) 8 -CH-((CH 2 ) 8 -COOH) 2 was added, and stirred at room temperature for 30 min. Washed twice in 50 mM MES with pH = 6.0, and diluted to 2 mg / mL with 50 mM MES with pH = 6.0, thus obtaining magnetic beads with increased carboxyl group content.

[0062] 2) Preparation of magnetic beads with extended carboxyl content labeled with CEA-coated antibody

[0063] The above-mentioned 10 mg of magnetic beads after the extension of carboxyl content were successively added with 5 mg of NHS and 5 mg of EDC, stirred at room temperature for 20 min, washed once in a 50 mM MES solution with pH = 6.0, and diluted to a concentration of 1 mg / mL. 0.1 mg of CEA-coated antibody was added, stirred at room temperature for 30 min, and then 10 mg of BSA was added to block for 30 min. After magnetic separation to obtain the magnetic beads, they were dissolved in a solution containing 10 mM PBS with pH 7.2, 0.2% BSA, 0.1% Tween-20, and 0.1% Proclin300 at a concentration of 0.5 mg / mL, obtaining magnetic beads labeled with CEA-coated antibody with extended carboxyl content, which is magnetic bead solution 4.

[0064] Detection in Example 8

[0065] The labeled antibody 3 and labeled antibody 4 obtained in Examples 4 and 6 were diluted to the labeling diluent at a ratio of 1:3000. The components of the labeling diluent are 10 mM PBS with pH 7.2, 0.2% BSA, 0.1% Tween-20, and 0.1% Proclin300.

[0066] Sample addition method: Mix 10 μL of the sample + 50 μL of the labeled antibody + 50 μL of the magnetic bead solution, incubate for 15 min, wash 5 times, and then add 200 μL of the luminescent solution for detection.

[0067] Establishment of the standard curve: CEA standard products with different concentrations (0, 2.5, 7, 20, 50, 150 ng / mL) were prepared as samples. Labeled antibody 3 and magnetic bead solution 3 were used as combination 3, and labeled antibody 4 and magnetic bead solution 4 were used as combination 4. After separate tests, the standard curves were linearly fitted. The standard curve of combination 3 is Y = 12989 + 9635.7*X, and the correlation coefficient r = 0.9998; the standard curve of combination 4 is Y = 9987.9 + 11355*X, and the correlation coefficient r = 0.9999. The test results are shown in Table 3.

[0068] Table 3 Standard curves in Example 8

[0069]

[0070]

[0071] Sensitivity evaluation: The standard product with a concentration of 0 ng / mL was detected 20 times, and the average value M + 2 times the standard deviation SD was taken as the analytical sensitivity of the reagent combination. The average value M2 of combination 3 was 0.08, and the standard deviation SD3 was 0.06. The analytical sensitivity of combination 3 = 0.16 + 0.06 * 2 = 0.28; the average value M4 of combination 4 was 0.38, and the standard deviation SD4 was 0.23. The analytical sensitivity of combination 4 = 0.14 + 0.04 * 2 = 0.22. The analytical sensitivity of combination 2, 0.84, is 3 times that of combination 3, 0.28, and the sensitivity has increased by 2 times; the analytical sensitivity of combination 2, 0.84, is 3.8 times that of combination 4, 0.22, and the sensitivity has increased by 2.8 times. The test results are shown in Table 4.

[0072] Table 4 Analytical sensitivity of Example 8

[0073] Test Serial Number Combination 3 (unit: ng / mL) Combination 4 (unit: ng / mL) 1 0.10 0.10 2 0.23 0.16 3 0.18 0.19 4 0.06 0.16 5 0.22 0.14 6 0.24 0.09 7 0.09 0.10 8 0.07 0.20 9 0.23 0.18 10 0.13 0.19 11 0.20 0.17 12 0.23 0.07 13 0.07 0.07 14 0.07 0.12 15 0.14 0.08 16 0.15 0.16 17 0.20 0.19 18 0.15 0.11 19 0.14 0.17 20 0.24 0.12 Average M 0.16 0.14 SD 0.06 0.04 CV 0.28 0.22

[0074] Based on the experimental data of Examples 3 and 8, the analytical sensitivity of the labeled antibody 1 and the magnetic bead solution 1 as combination 1 is the highest, and the carboxyl-extended molecular substance and process parameters used therein are the optimal choices. At the same time, the above examples also fully verify that after expanding the carboxyl content on the surface of biomolecules or solid-phase carriers using carboxyl-extended molecular substances, the detection signal can be amplified at multiple levels, achieving the purpose of improving the detection sensitivity.

[0075] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. A method for expanding the carboxyl content on the surface of biomolecules or solid-phase carriers, characterized in that, it comprises the following steps: (1) Carboxyl groups on biomolecules or solid-phase carriers are activated by EDC and NHS; (2) An appropriate amount of carboxyl extender molecule NH 2 -(CH 2 ) n -CH-((CH 2 ) n -COOH) 2 is added, where n is 3 - 8, and the amino group of the carboxyl extender molecule is covalently bound to the activated carboxyl group on the biomolecule or solid-phase carrier to obtain a molecule or solid-phase carrier with an extended carboxyl content.

2. The method for expanding the carboxyl content on the surface of biomolecules or solid-phase carriers according to claim 1, characterized in that: In the step (1), the mass ratio of the biomolecule or solid-phase carrier to EDC and NHS is 100:(1 - 10):(1 - 10).

3. The method for expanding the carboxyl content on the surface of biomolecules or solid-phase carriers according to claim 2, characterized in that: In the step (1), the mass ratio of the biomolecule or solid-phase carrier to EDC and NHS is 100:5:

5.

4. The method for expanding the carboxyl content on the surface of biomolecules or solid-phase carriers according to claim 1, characterized in that: In the step (2), the carboxyl-extended molecular NH 2 -(CH 2 ) n -CH-((CH 2 ) n -COOH) 2 has n = 6.

5. The method for expanding the carboxyl content on the surface of biomolecules or solid-phase carriers according to claim 4, characterized in that: In the step (2), the mass ratio of the carboxyl-extended molecule NH 2 -(CH 2 ) n -CH-((CH 2 ) n -COOH) 2 to the biomolecule or solid-phase carrier is 20000:8.

5.

6. The application of the biomolecule or solid-phase carrier with expanded carboxyl groups prepared by the method according to any one of claims 1 to 5 in the preparation of immunolabeling reagents.

7. The application according to claim 6, characterized in that: it comprises the following steps: 1) Preparation of monoclonal antibody with increased carboxyl content: 1 mg of CEA monoclonal antibody was dissolved in 1 mL of 50 mM MES solution with pH = 6.

0. 0.05 mg of NHS and 0.05 mg of EDC were added successively, and the mixture was stirred at room temperature for 20 min. EDC and NHS were removed using an ultrafiltration tube with a molecular cut-off of 50 KD. The ultrafiltered antibody was dissolved in 1 mL of MES, and then 0.000425 mg of NH 2 -(CH 2 ) 6 -CH-((CH 2 ) 6 -COOH) 2 was added. The mixture was stirred at room temperature for 30 min and dialyzed in 50 mM MES with pH = 6.0 for 24 h, and the dialysis solution was changed every 12 h to obtain 1 mg of CEA monoclonal antibody with increased carboxyl content; 2) Prepare a covalently labeled alkaline phosphatase CEA monoclonal antibody with expanded carboxyl content: In 1 mg of the CEA monoclonal antibody with expanded carboxyl groups obtained, add 2 mg of alkaline phosphatase, then add 0.05 mg of EDC, stir at room temperature for 30 min, then add 10 mg of BSA to block for 30 min, add 10 μL of Proclin300 and 1 mL of glycerol, and the covalently labeled alkaline phosphatase CEA monoclonal antibody with expanded carboxyl content is obtained.

8. The application according to claim 6, characterized in that: it comprises the following steps: 1) Preparation of magnetic beads with increased carboxyl content: 10 mg of 1.5 μm carboxyl magnetic beads were washed twice in 50 mM MES solution at pH = 6.0, then 5 mg of NHS and 5 mg of EDC were added successively, and the mixture was stirred at room temperature for 20 min. After washing once in 50 mM MES solution at pH = 6.0, it was diluted to a concentration of 1 mg / mL. 0.00425 mg of NH 2 -(CH 2 ) 6 -CH-((CH 2 ) 6 -COOH) 2 was added, and the mixture was stirred at room temperature for 30 min. After washing twice in 50 mM MES at pH = 6.0, it was diluted to 2 mg / mL with 50 mM MES at pH = 6.0 to obtain the magnetic beads with increased carboxyl content; 2) Prepare magnetic beads labeled with CEA-coated antibody with expanded carboxyl content: Take 10 mg of magnetic beads with expanded carboxyl content, add 5 mg of NHS and 5 mg of EDC successively, stir at room temperature for 20 min, wash once in a 50 mM MES solution with pH = 6.0, and dilute to a concentration of 1 mg / mL; add 0.1 mg of CEA-coated antibody, stir at room temperature for 30 min, then add 10 mg of BSA to block for 30 min. After magnetic separation to obtain the magnetic beads, dissolve them in a solution containing 10 mM PBS with pH 7.2, 0.2% BSA, 0.1% Tween-20, and 0.1% Proclin300, with a concentration of 0.5 mg / mL, and the magnetic beads labeled with CEA-coated antibody with expanded carboxyl content are obtained.

Images