Monoclonal antibody chemiluminescence homogeneous detection method and kit
By activating the catalytic activity of hemin through neighbor identification technology and rolling circle amplification signal amplification strategy, the problem of cumbersome and time-consuming monoclonal antibody detection methods is solved, and high-sensitivity and stable chemiluminescent homogeneous detection is achieved, which is suitable for high-throughput detection on multiple platforms.
Patent Information
- Application Number
- CN202511261174.8
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-09-04
- Publication Date
- 2025-12-09
AI Technical Summary
Existing monoclonal antibody detection methods such as ELISA and Western blot are cumbersome, time-consuming, and consume a lot of reagents, making it difficult to meet the needs of rapid detection and high-throughput screening. Furthermore, the poor water solubility of hemin molecules leads to poor experimental reproducibility.
By employing adjacent site recognition technology combined with rolling circle amplification signal amplification and reversible molecular probe regulation strategy, the rolling circle amplification reaction is initiated through antigen-DNA and antibody-DNA probe immune recognition, and the catalytic activity of hemin is activated by adjacent assembly of DNA, enabling chemiluminescent homogeneous detection.
It achieves high sensitivity and stability detection of monoclonal antibodies, is simple and rapid to operate, requires no washing or separation steps, and is suitable for high-throughput detection on multiple platforms.
Smart Images
Figure CN121087145A_ABST
Abstract
Description
I. TECHNICAL FIELD
[0001] The present application relates to the technical field of chemiluminescence immunoassay, and particularly relates to a chemiluminescence homogeneous detection method and kit of a monoclonal antibody. II. BACKGROUND
[0002] The detection of monoclonal antibodies plays a key role in the production of antibody drugs, biomedical research, disease diagnosis and treatment. With the development of precision medicine, higher requirements are put forward for the sensitivity, specificity, speed and operation convenience of the detection method. However, the currently commonly used detection techniques in the clinic, such as enzyme-linked immunoassay (ELISA) and Western blot, often have problems such as complicated steps, long time consumption, large reagent consumption and the like, which limit their application in rapid detection and high-throughput screening.
[0003] As a simple and convenient method for protein analysis, the ortho-amplification technology has attracted widespread attention in recent years. Its detection principle is to use a pair of DNA-modified affinity probes for sandwich immunorecognition of the target antibody, based on the ortho effect, to make the DNA on the affinity probe undergo ortho hybridization reaction, and then to output the immunorecognition event through mature DNA signal amplification technology (such as hybridization chain reaction, PCR amplification reaction, rolling circle amplification reaction, etc.). Compared with traditional antibody analysis methods, the antibody analysis method based on ortho recognition has higher detection sensitivity and can be used for homogeneous detection, which is simple and convenient to operate.
[0004] Hemin is an iron porphyrin compound with Fe(III) as the center, which has excellent peroxidase mimetic properties as the active catalytic cofactor of various natural enzymes such as horseradish peroxidase and catalase. However, hemin molecules have poor water solubility and tend to self-aggregate in solution to form aggregates with low catalytic activity. Traditional methods usually need to dissolve hemin with an organic solvent first, and then dilute for use, but aggregation of molecules may still occur after dilution, resulting in poor repeatability of experimental results. To solve this problem, the hemin molecules can be covalently linked to DNA sequences, and the catalytic activity thereof can be reversibly regulated by using the programmable properties of DNA. By controlling the assembly and disassembly of DNA sequences, the aggregation and monomer state of hemin molecules can be accurately regulated, so that the catalytic activity thereof can be flexibly regulated. In addition, DNA modification significantly improves the water solubility of hemin, effectively inhibits its aggregation, enhances the stability and repeatability of experiments, and makes it have more application potential in the field of biosensing.
[0005] The present application combines ortho immune recognition technology, rolling circle amplification signal amplification technology and molecular probe reversible regulation strategy, and develops a chemiluminescence homogeneous detection method of monoclonal antibody and a matching kit. The method can realize high sensitivity and high stability detection of the monoclonal antibody. In addition, the method is a homogeneous detection method, without washing and separation steps, and has the advantages of simple operation and rapid detection, and has important application value and development prospect in the fields of biomedical research, disease diagnosis and treatment, and screening and production of antibody drugs. III. SUMMARY
[0006] The present application is to provide a chemiluminescence homogeneous detection method and kit of monoclonal antibody. The method is to perform immune recognition on the target monoclonal antibody by a pair of antigen-DNA and antibody-DNA probes, and then based on the ortho assembly of nucleic acid, to trigger rolling circle amplification reaction and strand displacement reaction of double-stranded DNA6-hemin / DNA7-hemin, so that hemin is converted from a low-activity dimer state to a high-activity monomer state, and finally the high peroxidase catalytic activity of the activated DNA6-hemin and DNA7-hemin is used to catalyze the reaction of luminol and hydrogen peroxide to generate a chemiluminescence signal for detection. The monoclonal antibody detection method has the advantages of simple operation and high detection sensitivity.
[0007] The present application is realized by the following technical solutions:
[0008] [1] A chemiluminescence homogeneous detection method of monoclonal antibody, comprising the following three steps: Figure 1 ):
[0009] Step 1: adding the sample to be tested into reaction reagent 1 for immune recognition reaction, and reaction reagent 1 contains antigen-DNA1, antibody-DNA2, double-stranded DNA3 / DNA4, circular DNA5, phi29 polymerase and deoxyribonucleotide triphosphate;
[0010] Step 2: adding reaction reagent 2 to the solution reacted in step 1 for hemin activation reaction, and reaction reagent 2 is a low-activity double-stranded DNA6-hemin / DNA7-hemin solution containing hemin dimer;
[0011] Step 3: adding detection reagent 1 and detection reagent 2 to the solution reacted in step 2 at the same time for chemiluminescence detection, detection reagent 1 is a luminol solution containing imidazole, and detection reagent 2 is a hydrogen peroxide solution.
[0012] [2] The detection method according to the above [1], wherein the antigen-DNA1 is a covalent conjugate of antigen and DNA1 capable of specifically binding to the target monoclonal antibody.
[0013] [3] According to the detection method described in [1] or [2] above, wherein the antibody-DNA2 is a covalent conjugate of an anti-target monoclonal antibody and DNA2.
[0014] [4] According to any one of [1] to [3] above, the circular DNA5 can hybridize with free DNA3 single strands and undergo rolling circle amplification reaction in the presence of phi29 polymerase and deoxyribonucleoside triphosphate.
[0015] [5] According to any one of [1] to [4] above, in step 1, after the sample to be tested is added to the reaction reagent 1, the target monoclonal antibody is recognized by antigen-DNA1 and antibody-DNA2 to form a sandwich immune complex. Based on the ortho-position effect, DNA1 and DNA2 hybridize and assemble and hybridize with DNA4 in double-stranded DNA3 / DNA4, thereby releasing DNA3 to bind to circular DNA5, triggering rolling circle amplification reaction, and generating long single-stranded repeating sequences ( Figure 2 ).
[0016] [6] According to any one of [1] to [5] above, the double-stranded DNA6-hemin / DNA7-hemin is obtained by annealing and hybridizing DNA6-hemin and DNA7-hemin at a molar ratio of 1:1, and the hemin is in a dimerized state and has low peroxidase catalytic activity.
[0017] [7] According to any one of [1] to [6] above, in step 2, double-stranded DNA6-hemin / DNA7-hemin can undergo a strand substitution reaction with the long single strand of the repetitive sequence, so that hemin is transformed from a low-activity dimer state to a high-activity monomer state, and the activated DNA6-hemin and DNA7-hemin have high peroxidase catalytic activity ( Figure 3 ).
[0018] [8] A kit for a chemiluminescent homogeneous detection method for monoclonal antibodies, comprising reaction reagent 1, reaction reagent 2, detection reagent 1 and detection reagent 2.
[0019] Compared with the prior art, the present invention has the following characteristics:
[0020] (1) The present invention designs a detection method based on the strategy of activating hemin catalytic activity by neighbor recognition, which can perform homogeneous detection of monoclonal antibodies without washing and separation steps. It is simple to operate and easy to achieve rapid detection.
[0021] (2) The present invention utilizes rolling circle amplification for signal amplification, which has high detection sensitivity and the detection limit for monoclonal antibodies can be as low as 1 fg / mL.
[0022] (3) The detection method proposed in this invention has excellent platform compatibility and can realize high-throughput chemiluminescence imaging detection of monoclonal antibodies on various platforms such as 96-well plates and microfluidic chips, showing good potential for clinical application expansion. IV. Description of the attached drawings
[0023] Figure 1 A schematic diagram of the detection process of the monoclonal antibody chemiluminescence homogeneous detection method of the present invention.
[0024] Figure 2 A schematic diagram of the rolling circle amplification reaction initiated by neighbor recognition-induced nucleic acid hybridization and strand displacement reaction.
[0025] Figure 3 Schematic diagram of the Hemin activation reaction.
[0026] Figure 4 The chemiluminescence detection method of the present invention provides (a) luminescence intensity and (b) standard curve of different concentrations of anti-proprotein convertase subtilisin 9 antibody (PCSK9-mAb). V. Detailed Implementation Methods
[0027] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. The specific embodiments described herein are preferred embodiments of this invention and are merely used to explain the invention, not to limit it.
[0028] Example 1: Detection of monoclonal antibody PCSK9-mAb.
[0029] The detection process of the chemiluminescence detection method of the present invention for PCSK9-mAb is as follows: Figure 1 As shown, the process includes the following three reagent addition steps:
[0030] Step 1: Add 0.5 μL of the sample to be tested (e.g., the sample solution of PCSK9-mAb) to 4.5 μL of reaction reagent 1, mix well, and react at 37°C for a certain time, preferably 30 minutes;
[0031] The reaction reagent 1 is prepared by mixing antigen-DNA1, antibody-DNA2, double-stranded DNA3 / DNA4, circular DNA5, phi29 polymerase, deoxyribonucleoside triphosphate, recombinant albumin, phi29 buffer (10×), and 1×PBS in a certain volume ratio, preferably 10:10:10:5:5:5:1:10:34. The preferred concentrations of antigen-DNA1 and antibody-DNA2 are 1 μg / mL, double-stranded DNA3 / DNA4 are 100 nM, circular DNA5 is 3 μM, phi29 polymerase is 10 U / μL, deoxyribonucleoside triphosphate (containing dATP, dGTP, dCTP, and dTTP) is 10 mM, and recombinant albumin is 20 mg / mL.
[0032] Step 2: Add 3 μL of reaction reagent 2 to the solution after the reaction in step 1, mix, and react at room temperature under light-protected conditions for a certain period of time, preferably 5 minutes;
[0033] Among them, reaction reagent 2 is a double-stranded DNA 6-hemin / DNA 7-hemin solution with a certain concentration. The double-stranded DNA 6-hemin / DNA 7-hemin is prepared by mixing single-stranded DNA 6-hemin and DNA 7-hemin in a 1:1 molar ratio, annealing at 95°C for 5 minutes and then cooling under gradient conditions. The preferred concentration of double-stranded DNA 6-hemin / DNA 7-hemin is 5 μM.
[0034] Step 3: Add 32 μL of detection reagent 1 and 10 μL of detection reagent 2 to the solution after the reaction in step 2, and immediately record the image using a chemiluminescence imaging system (exposure time: 30 seconds). Based on the method of this invention, obtain the standard curve of PCSK9-mAb. Figure 4 ), and determine the PCSK9-mAb in the sample;
[0035] Among them, the detection reagent 1 is prepared by imidazole, luminol and 1×PBS in a certain volume ratio, the preferred volume ratio is 5:20:39, the preferred concentration of imidazole is 100mM, and the preferred concentration of luminol is 3mM.
[0036] Test reagent 2 is prepared with hydrogen peroxide, with a preferred concentration of 10 mM.
[0037] The embodiments described above are merely illustrative, and the terms and expressions used are for illustration rather than limitation, and it is not intended that any equivalents of the features or portions thereof shown and described be excluded in the use of these terms and expressions.
Claims
1. A chemiluminescent homogeneous detection method for monoclonal antibodies, characterized in that... Includes the following steps: Step 1: Add the sample to be tested to reaction reagent 1 containing antigen-DNA1, antibody-DNA2, double-stranded DNA3 / DNA4, circular DNA5, phi29 polymerase and deoxyribonucleoside triphosphate for recognition reaction; Step 2: Add reaction reagent 2 containing double-stranded DNA 6-hemin / DNA 7-hemin to the solution after the reaction in step 1 to carry out the hemin activation reaction; Step 3: Add detection reagent 1 containing imidazole and luminol and detection reagent 2 containing hydrogen peroxide to the solution after the reaction in step 2, and perform chemiluminescence detection. Quantitatively analyze the monoclonal antibody in the sample based on the intensity of the chemiluminescence signal.
2. The detection method according to claim 1, wherein, The antigen-DNA1 is a covalent conjugate of antigen and DNA1 that can specifically bind to the target monoclonal antibody.
3. The detection method according to claim 1 or 2, wherein, The antibody-DNA2 is a covalent conjugate of an anti-target monoclonal antibody and DNA2.
4. The detection method according to any one of claims 1 to 3, wherein, The circular DNA5 can hybridize with free DNA3 single strands and undergo rolling circle amplification in the presence of phi29 polymerase and deoxyribonucleoside triphosphate.
5. The detection method according to any one of claims 1 to 4, wherein, In step 1, after the sample to be tested is added to reaction reagent 1, the target monoclonal antibody is recognized by antigen-DNA1 and antibody-DNA2 to form a sandwich immune complex. Based on the ortho-position effect, DNA1 and DNA2 hybridize and assemble, and then hybridize with DNA4 in double-stranded DNA3 / DNA4, thereby releasing DNA3 to bind to circular DNA5, triggering rolling circle amplification reaction, and generating long single strands of repetitive sequences.
6. The detection method according to any one of claims 1 to 5, wherein, The double-stranded DNA6-hemin / DNA7-hemin was obtained by annealing and hybridizing DNA6-hemin and DNA7-hemin in a 1:1 molar ratio. The hemin was in a dimerized state and had low peroxidase catalytic activity.
7. The detection method according to any one of claims 1 to 6, wherein, In step 2, double-stranded DNA 6-hemin / DNA 7-hemin can undergo a strand substitution reaction with long single strands of repetitive sequences, transforming hemin from a low-activity dimer state to a high-activity monomer state. The activated DNA 6-hemin and DNA 7-hemin have high peroxidase catalytic activity.
8. A kit for use in the detection method according to any one of claims 1 to 7, comprising reaction reagent 1, reaction reagent 2, detection reagent 1, and detection reagent 2.