A labeling method for expanding the amino content of biological molecules and its application

Abstract

The present invention discloses a labeling method and application of an extended amino group content on a biological molecule, belonging to the technical field of immunoassay. The specific technical scheme is as follows: (1) a biological molecule and an amino extended molecule COOH-(CH2) n ‑CH‑((CH2) n (2) adding an appropriate amount of EDC and blocking it to obtain a biomolecule with an expanded amino content. The present invention also provides an application of the labeling method in immunoassay. By using the method of the present invention, the coupling efficiency can be increased by more than 1 times, and the labeling efficiency can be increased by 97.28%.

Description

Technical Field

[0001] The invention relates to the technical field of immunology, and in particular to a labeling method for extending the amino content on biological molecules and an application thereof. Background Art

[0002] As an important branch of clinical immunoassay technology, labeled immunoassay technology is the most active and fastest growing. Its basic principle is to utilize the high specificity of antigen-antibody reaction and the measurability of various markers to achieve the purpose of convenient and sensitive detection of various trace bioactive substances in the body.

[0003] Biomolecules include enzymes, antibodies, antigens, label peptides, etc. Among the labeling methods, the covalent coupling technology currently used for biomolecule A to label enzymes, antibodies, antigens or other types of biomolecules B can be roughly divided into three categories. The first category is to use an activator to activate the carboxyl group and the amino group to form an amide bond, the second category is to use sugar molecules to form aldehydes after oxidation and then combine with primary amine groups to form secondary amine bonds, and the third category is to use an activator to activate the amino group and the thiol group to form a thioether bond. The coupling efficiency of the three types of labeling methods is very high. If it is the first or second type of labeling method, a tightly connected complex is formed between biomolecule A and biomolecule B, and there is no connecting arm between biomolecule A and biomolecule B. At this time, there will be a certain steric hindrance effect; if it is the third type of thioether bond formation method, there are at least 8 atoms of connecting arms between biomolecule A and biomolecule B, which can effectively reduce the steric hindrance effect between the two ends of the connector. At the same time, in the above labeling methods, one group of biomolecule A can only covalently connect to one molecule of biomolecule B. In most cases, the sensitivity of the test kits prepared by these methods can effectively meet the current clinical testing needs. However, for the detection of certain extremely trace human markers, it is necessary to rely on more sensitive reagents to capture extremely trace markers in human specimens.

[0004] In order to effectively reduce the steric hindrance effect between biological molecules, technicians have developed a variety of new labeling methods. 1) Biotin-avidin system. Biotin (biotin, B) and avidin (avidin, A) or streptavidin (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. Advantages: Since both biotin and avidin are relatively stable, and one molecule of avidin can bind to four molecules of biotin, the detection signal can be amplified in multiple stages. At the same time, the long arm connection of biotin can also maintain the effective exposure of the binding site of the antibody binding antigen. It is a more commonly used labeling method in current chemiluminescence detection. Disadvantages: Biotin is one of the vitamins present in organisms and is sometimes used as a drug. It is especially common in small intestinal tissue, kidney tissue, brain tissue and liver tissue. The use of the biotin-avidin system will cause negative signals unrelated to the detection itself in the above tissues, that is, the possibility of false negative misdiagnosis. 2) Thioether bond spacer arm system. As described in CN11209864B, the invention provides a method for labeling fluorescent proteins and / or coupled proteins with monoclonal antibodies, which mainly involves combining the amino group of the fluorescent protein and / or coupled protein with SMCC, converting the amino group into a maleimide group, and extending the amino group on the monoclonal antibody to form 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 coupled protein and the monoclonal antibody. Advantages: The antibody and the fluorescent protein and / or coupled protein are connected by a spacer arm, which can ensure the effective exposure of the binding site of the antibody binding to the antigen and greatly reduce the steric hindrance effect. Disadvantages: After extension, a certain amino group on an antibody molecule can only be coupled to one molecule of fluorescent protein and / or coupled protein, and it is impossible to connect multiple signal molecules to achieve multi-stage amplification of the detection signal. Summary of the invention

[0005] The purpose of the present invention is to provide a labeling method for expanding the amino content on biological molecules and its application, by expanding the amino content on biological molecules, thereby greatly improving the probability and efficiency of coupling between the modified biological molecules and other biological molecules, while effectively reducing the steric hindrance effect between molecules.

[0006] In order to achieve the above purpose, the experimental steps are as follows:

[0007] (1) Biomolecules and amino-extended molecules COOH-(CH2) n -CH-((CH2) n -NH2)2 mixed;

[0008] (2) Add an appropriate amount of EDC and block it to obtain a biomolecule with an expanded amino content.

[0009] In step (1), the biomolecule and the amino-extended molecule COOH-(CH2) n -CH-((CH2) n -NH2)2 mass ratio is 20000:(1-100), preferably 20000:20.

[0010] In step (1), the amino-extended molecule COOH-(CH2) n -CH-((CH2) n -NH2)2 wherein n is between 3 and 100, preferably between 3 and 12, and more preferably n=10.

[0011] The amount of EDC added in step (2) is the same as the mass of the amino-extended molecule.

[0012] In step (2), the blocking agent is selected from one or a combination of glycine, ethanolamine, BSA, casein, and Tris, preferably Tris.

[0013] Compared with the prior art, the beneficial effects of the present invention are: solving the steric hindrance problem and increasing the coupling efficiency by more than 1 times. DETAILED DESCRIPTION

[0014] Example 1 Preparation of Monoclonal Antibodies with Extended Amino Acid Content

[0015] 1mg CHI3L1 monoclonal antibody (purchased from Nanjing Kaiertai) and 0.001mg COOH-(CH2) 10 -CH-((CH2) 10 -NH2)2, dissolved in 1mL pH = 6.5 25mM HEPES solution, added 0.001mg EDC, stirred at room temperature for 30min, then added 0.01mg Tris, stirred at room temperature for 30min, dialyzed in pH = 7.0 25mM HEPES for 24h (molecular weight cutoff 70000), replaced the dialysate every 12h, and finally made up the volume to 1mL with pH = 7.0 25mM HEPES to obtain 1mg CHI3L1 monoclonal antibody with extended amino content, with a concentration of 1mg / mL.

[0016] Example 2 Monoclonal Antibody Coupling Magnetic Beads

[0017] 1) Preparation of magnetic beads coupled to monoclonal antibodies with extended amino content

[0018] 10mg 3.0μm carboxyl magnetic beads (purchased from JSR Life Science) were washed twice in a pH = 7.0 25mM HEPES solution, 5mg NHS and 5mg EDC were added successively, stirred at room temperature for 20min, washed once in a pH = 7.0 25mM MES solution, 0.05mg CHI3L1 monoclonal antibody with extended amino content was added, stirred at room temperature for 30min, and the supernatant was retained after magnetic separation, recorded as supernatant 1, and the volume of the supernatant was 2mL. After the magnetic beads were obtained by magnetic separation, they were dissolved in a solution containing pH 7.2 10mM PBS, 0.2% BSA, 0.1% Tween-20, and 0.1% Proclin300 at a concentration of 0.5mg / mL, and the monoclonal antibody with extended amino content was coupled to the magnetic bead solution, which was magnetic bead solution 1.

[0019] 2) Preparation of magnetic beads coupled to monoclonal antibodies without extended amino content

[0020] 10mg 3.0μm carboxyl magnetic beads (purchased from JSR Life Science) were washed twice in a pH = 7.0 25mM HEPES solution, 5mg NHS and 5mg EDC were added successively, stirred at room temperature for 20min, washed once in a pH = 7.0 25mM MES solution, and diluted to a concentration of 5mg / mL. 0.05mg CHI3L1 monoclonal antibody was added, stirred at room temperature for 30min, and the supernatant was retained after magnetic separation, recorded as supernatant 2, and the volume of the supernatant was 2mL. After obtaining the magnetic beads by magnetic separation, they were dissolved in a solution containing pH 7.2 10mM PBS, 0.2% BSA, 0.1% Tween-20, and 0.1% Proclin300 at a concentration of 0.5mg / mL to obtain a monoclonal antibody-coupled magnetic bead solution, which is magnetic bead solution 2.

[0021] 3. Detection of labeling efficiency

[0022] The protein concentration of supernatant 1 and supernatant 2 obtained in Example 2 was determined by BCA method, and the content of residual monoclonal antibody in the supernatant was calculated. The calculation formula of labeling efficiency is labeling efficiency = (total feed amount 0.05 mg-residual protein in supernatant) / total feed amount 0.05 mg*100%.

[0023] Establishment of BCA standard curve: Take 500 μg / mL BSA standard solution, dilute 7 standard samples with 10 mM PBS according to the method in Table 1, the concentration range is 0.6-30 μg / mL, take 0.5 mL of standard sample and 0.5 mL and 1 mL of BCA working solution respectively, incubate in 60℃ water bath for 1 hour, cool to room temperature, and measure the A of each tube on the spectrophotometer. 562The linear fitting equation of the standard curve is Y=0.025X+0.0215, the correlation coefficient is r=0.9988, and the test results are shown in Table 2.

[0024] Table 1 Protein concentration gradient of BCA method

[0025] Tube No. 0 1 2 3 4 5 6 7 PBS (μL) 1200 1175 1150 1100 1000 800 400 0 BSA (μL) 0 25 50 10 200 400 800 1200 Final BSA concentration 0 0.6 1.25 2.5 5.0 10 20 30

[0026] Table 2 A of standards and supernatants 562 value

[0027]

[0028] The protein concentration of supernatant 1 is calculated to be 0.68 μg / mL, and the protein concentration of supernatant 2 is 11.01 ng / mL. The residual protein amount of supernatant 1 is 1.36 μg = 0.00136 mg, and the residual protein amount of supernatant 2 is 27.02 μg = 0.02702 mg. The labeling efficiency of magnetic bead solution 1 is 1 = (0.05 mg-0.00136 mg) / 0.05 mg*100% = 97.28%, and the labeling efficiency of magnetic bead solution 2 is 2 = (0.05 mg-0.02702 mg) / 0.05 mg*100% = 45.96%, and the labeling efficiency is increased by 97.28% / 45.96%-1 = 1.17 times.

[0029] The above is only a preferred embodiment of the present invention. It should be pointed out that, for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A labeling method for expanding the amino content of a biomolecule, characterized in that: The steps include: (1) biomolecule and amino-extended molecule COOH-(CH2) n -CH-((CH2) n -NH2)2 mixed, amino extended molecule COOH-(CH2) n -CH-((CH2) n -NH2)2 where n is 3-12; (2) adding an appropriate amount of EDC and sealing it, a biological molecule with an expanded amino content is obtained.

2. The labeling method for expanding the amino group content on a biomolecule according to claim 1, characterized in that: In the step (1), the biomolecule and the amino-extended molecule COOH-(CH2) n -CH-((CH2) n -NH2)2 has a mass ratio of 20000:(1-100).

3. The labeling method for expanding the amino group content on a biomolecule according to claim 2, characterized in that: In the step (1), the biomolecule and the amino-extended molecule COOH-(CH2) n -CH-((CH2) n -NH2)2 has a mass ratio of 20000:

20.

4. The labeling method for expanding the amino group content on a biomolecule according to claim 1, characterized in that: In step (1), the amino-extended molecule COOH-(CH2) n -CH-((CH2) n -NH2)2's n is n=10.

5. The labeling method for expanding the amino group content on a biomolecule according to claim 1, characterized in that: The amount of EDC added in step (2) is the same as the mass of the amino-extended molecule.

6. The labeling method for expanding the amino group content on a biomolecule according to claim 1, characterized in that: In the step (2), the blocking agent is selected from one or a combination of glycine, ethanolamine, BSA, casein, and Tris.

7. Use of the labeling method for expanding the amino group content on a biological molecule as claimed in claim 1 in preparing an immunolabeling reagent.

8. Use of the labeling method for expanding the amino group content on a biomolecule as claimed in claim 7 in preparing an immunolabeling reagent, characterized in that: For the preparation of monoclonal antibodies with extended amino content and magnetic bead immunoassay: 1 mg CHI3L1 monoclonal antibody and 0.001 mg COOH-(CH2) 10 -CH-((CH2) 10 -NH2)2, dissolved in 1mL of pH=6.525mM HEPES solution, added 0.001mg of EDC, stirred at room temperature for 30min, then added 0.01mg of Tris, stirred at room temperature for 30min, dialyzed in pH=7.0 25mM HEPES for 24h, replaced the dialysate every 12h, and finally made up the volume to 1mL with pH=7.025mM HEPES to obtain 1mg of CHI3L1 monoclonal antibody with extended amino content, with a concentration of 1mg / mL.

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