Anti-troponin I monoclonal antibody and application thereof

Abstract

The invention relates to the field of antibodies, in particular to an anti-troponin I monoclonal antibody and application thereof, performance analysis of a purified electrophoretogram of the antibody and a kit coupled with a monoclonal antibody HK401 coated magnetic particle component shows that the monoclonal antibody has excellent performance in cTni detection, and the cTni detection sensitivity is high. The method can be used as an object for further production and optimization.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of antibodies, in particular to an anti-troponin I monoclonal antibody and application thereof. BACKGROUND

[0002] Myocardial injury is a common and serious condition in cardiovascular diseases, and acute myocardial infarction (AMI) is one of the main causes of death and disability worldwide. Early and accurate diagnosis of myocardial injury is of great significance for timely implementation of effective treatment intervention and improvement of patient prognosis. Among numerous myocardial injury markers, troponin I (cTnI) has become a key indicator for the clinical diagnosis of myocardial infarction and other myocardial injury diseases due to its high specificity and sensitivity for myocardial injury.

[0003] At present, the commonly used cTnI detection methods in clinical practice are mostly based on immunoassay techniques, such as enzyme-linked immunosorbent assay (ELISA), chemiluminescence immunoassay, etc. These methods can achieve quantitative detection of cTnI to some extent, but still have many shortcomings. On the one hand, the polyclonal antibodies or some monoclonal antibodies used in traditional detection methods have cross-reactivity problems, which may react with other heart muscle-related proteins or non-heart muscle-derived proteins, thereby affecting the accuracy of the detection results. For example, in some complex biological samples, there are troponin T or other structurally similar proteins, which may cause false positive results, making the clinical doctors deviate from the judgment of the patient's condition. On the other hand, the detection sensitivity and lower limit of detection of the existing detection methods still need to be improved. In the early stage of myocardial injury, the concentration of cTnI in the blood is relatively low, and some detection methods are difficult to accurately detect trace amounts of cTn protein, leading to missed diagnosis and delay of the best treatment opportunity for patients.

[0004] The development of monoclonal antibody technology provides a new way to solve the above problems. Monoclonal antibodies have high specificity and uniformity, and can accurately recognize specific antigen epitopes. By preparing monoclonal antibodies specific to troponin I, cross-reactions with other unrelated proteins can be effectively avoided, significantly improving the accuracy of detection. In addition, monoclonal antibodies can further improve their affinity and sensitivity by optimizing their preparation process and screening strategy, thereby enabling the detection of lower concentrations of cTnI, which helps to detect changes in the disease at an early stage of myocardial injury. For example, some advanced monoclonal antibody engineering techniques can modify the variable region of the antibody to enhance its binding capacity to the antigen, or by constructing a bispecific monoclonal antibody, achieving simultaneous recognition of multiple epitopes of cTnI, further improving the reliability and effectiveness of detection. However, the existing troponin I monoclonal antibodies on the market still have some technical bottlenecks, such as some antibodies perform poorly in stability and are easily inactivated in long-term storage or complex detection environments, affecting the shelf life and detection performance of the detection reagent; or in large-scale production process, the yield and quality of antibodies are difficult to achieve the ideal balance, resulting in high production cost, which limits its widespread application in clinical practice.

[0005] In summary, the development of a troponin I monoclonal antibody with high specificity, high sensitivity, good stability and easy large-scale production is of great urgency and important practical significance for improving existing myocardial injury diagnosis technology, improving clinical diagnosis level and promoting precise medical treatment of cardiovascular diseases.

[0006] To overcome the above problems, the present application provides a specific and sensitive anti-troponin I monoclonal antibody HK401, characterized as follows:

[0007] The application discloses a monoclonal antibody HK401 which is capable of specifically binding to troponin I, and the monoclonal antibody HK401 comprises three antigen complementarity determining regions of a heavy chain variable region, namely HCDR1, HCDR2 and HCDR3, and three antigen complementarity determining regions of a light chain variable region, namely LCDR1, LCDR2 and LCDR3, and the specific amino acid sequences of the six antigen complementarity determining regions are as follows: the sequence of HCDR1 is shown as SEQ ID NO: 3; the sequence of HCDR2 is shown as SEQ ID NO: 4; the sequence of HCDR3 is shown as SEQ ID NO: 5; the sequence of LCDR1 is shown as SEQ ID NO: 6; the sequence of LCDR2 is shown as SEQ ID NO: 7; and the sequence of LCDR3 is shown as SEQ ID NO: 8; the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO: 1; and the amino acid sequence of the light chain variable region is shown as SEQ ID NO: 2.

[0008] The application further provides a method for coupling a magnetic micro-particle with the monoclonal antibody, and the method specifically comprises the following steps:

[0009] 1) 0.1ml of 100mg / ml magnetic micro-particles (JSR company, MS160 / Tosyl) with a particle size of 1.5um and 0.2mg of the monoclonal antibody HK401 are added into 1ml of 0.5M borate buffer solution;

[0010] 2) 1ml of 2M ammonium sulfate buffer solution is added;

[0011] 3) after 24h of reaction at 37 degrees, 0.1ml of 10% BSA is added, and 12h of reaction is carried out at 37 degrees;

[0012] 4) after magnetic separation, the magnetic micro-particles coated with the monoclonal antibody HK401 are obtained by using 1ml of 50mM PBS buffer solution containing 10% BSA for storage.

[0013] In addition, the application further discloses a chemiluminescence kit capable of specifically detecting cTnI.

[0014] The application provides the monoclonal antibody HK401 capable of specifically binding to troponin I, and the detection kit prepared by using the antibody has good accuracy and the detection sensitivity can reach 0.01ng / mL. BRIEF DESCRIPTION OF DRAWINGS

[0015] Figure 1 It is a purification diagram of the anti-cTnI monoclonal antibody HK401.

[0016] Figure 2A standard curve graph for detecting cTnl standard of the kit. Figure 3 A standard curve graph for sample correlation. DETAILED DESCRIPTION

[0017] Example 1 Preparation of monoclonal antibody

[0018] Preparation of materials and reagents: Select 6-8 weeks old Balb / c mice as experimental animals, use recombinant human troponin I (cTnl) with a purity of more than 95% as antigen, and SP2 / 0 myeloma cell line for experiment. Freund's complete adjuvant, Freund's incomplete adjuvant, polyethylene glycol (PEG), HAT selection medium, HT medium, calf serum and ELISA kit and other reagents, as well as centrifuge, cell incubator, clean bench, microplate reader and inverted microscope are also needed in the experiment.

[0019] Immunization procedure: First, emulsify recombinant cTnl with Freund's complete adjuvant at a ratio of 1:1, and then immunize the mice, with each mouse receiving 100 μg of emulsified antigen intraperitoneally. On the 21st day after the first immunization, use recombinant cTnl emulsified with Freund's incomplete adjuvant at a ratio of 1:1 for the first booster immunization, and then every 14 days for a booster immunization, a total of 3-4 times. Three to four days before cell fusion, perform the last booster immunization by injecting 50 μg of recombinant cTnl through the tail vein.

[0020] Cell fusion: Three to four days after the last booster immunization, aseptically remove the spleen of the immunized mouse and prepare a spleen cell suspension. At the same time, prepare the SP2 / 0 myeloma cells in logarithmic growth phase. Mix the spleen cells and myeloma cells at a ratio of 5:1-10:1, induce fusion with PEG, and after termination of PEG action, resuspend the cells with HAT selection medium and inoculate in 96-well plates for culture.

[0021] Screening of positive hybridoma cells: Seven to ten days after cell fusion, screen the positive hybridoma cells secreting anti-cTnl antibodies by indirect ELISA method. Clone the positive hybridoma cells obtained by initial screening to obtain pure clones of single cells. Common cloning methods include limited dilution method and soft agar plate method, and the limited dilution method is used in this embodiment.

[0022] Mass production of monoclonal antibody: The selected positive hybridoma cells were prepared in large quantities using either in vivo induction or in vitro culture methods. In vivo induction involved inoculating positive hybridoma cells into the peritoneal cavity of syngeneic Balb / c mice, collecting the ascites fluid, and then purifying the antibody. In vitro culture involved expanding the positive hybridoma cells in cell culture flasks or bioreactors, collecting the supernatant, and then purifying the antibody.

[0023] Identification of monoclonal antibodies: The titer of monoclonal antibodies was determined by indirect ELISA. Antibody specificity was identified by Western blotting and immunohistochemistry. Antibody subclasses were determined using a mouse monoclonal antibody subclass identification kit, and antibody affinity was determined by ELISA competition assay.

[0024] Table 1. Results of titer assay for anti-cTnI monoclonal antibody HK401

[0025] Dilution factor 1:1000 1:3000 1:9000 1:27000 1:81000 1:243000 1:729000 Results (P / N) 10.21 9.33 7.76 4.91 2.12 1.01 0.2

[0026] A positive result is defined as P / N > 2 in the titer test. As shown in Table 1, the titer of the HK401 monoclonal antibody reached over 1:81000, indicating that the monoclonal antibody has good activity and can be used for subsequent experiments. The purified antibody SDS-PAGE electrophoresis image is shown in the attached instructions. Figure 1 As shown.

[0027] Example 2: Preparation of magnetic microparticles conjugated with monoclonal antibody HK401.

[0028] 0.1 ml of 100 mg / ml magnetic microparticles (JSR, MS160 / Tosyl) with a particle size of 1.5 μm and 0.2 mg of monoclonal antibody HK401 were added to 1 ml of 0.5 M borate buffer, followed by 1 ml of 2 M ammonium sulfate buffer. After reacting at 37 °C for 24 hours, 0.1 ml of 10% BSA was added, and the reaction was continued at 37 °C for 12 hours. Finally, after magnetic separation, the microparticles were stored in 1 ml of 50 mM PBS buffer containing 10% BSA, thus obtaining the magnetic microparticles coated with monoclonal antibody HK401.

[0029] Example 3: Performance testing of the cTnI reagent kit.

[0030] The magnetic microparticles coupled with the monoclonal antibody HK401 are diluted to 0.5 mg / ml and used as component 1, the purchased anti-cTnI monoclonal antibody coupled with alkaline phosphatase is diluted and used as component 2, and the recombinant cTnI antigen is diluted into detection calibrators with concentration gradients of 0 ng / ml, 0.05 ng / ml, 0.1 ng / ml, 0.3 ng / ml, 1 ng / ml, 3 ng / ml, 10 ng / ml, 20 ng / ml and 40 ng / ml. The standard curve is drawn by taking the antigen concentration as the abscissa, the luminescence value of each point of the calibrators as the ordinate and the four-parameter method, and the standard curve is shown in Fig. 2 of the specification.

[0031] Minimum detection limit confirmation.

[0032] In the experimental method, the zero-concentration calibrator is selected as the sample for detection. The specific operation is to repeatedly determine the sample for 20 times, and record the RLU value (relative luminescence value) obtained after each determination. Then, according to the 20 measurement results, the average value (M) and the standard deviation (SD) are calculated. Then, the RLU value corresponding to M + 2SD is determined, and according to the concentration-luminescence value data between the zero-concentration calibrator and the adjacent calibrators, two-point regression fitting is carried out to obtain a linear equation. Finally, the RLU value corresponding to M + 2SD obtained before is substituted into the linear equation to solve the corresponding concentration value, which is the minimum detection limit of this experiment. The calculation shows that the minimum detection limit is 0.003 ng / ml.

[0033] The sample detection correlation is shown in the specification Figure 3 The correlation with the Siemens reagent value is R=0.9961.

[0034] Through the electrophoretogram after the purification of the antibody and the performance analysis of the kit of the component of the magnetic microparticles coated with the monoclonal antibody HK401, it can be seen that the monoclonal antibody in the application has excellent performance in the detection of cTnI and can be used as an object for further production and optimization.

Claims

1. A monoclonal antibody HK401 that binds to troponin I with high affinity, characterized in that, The monoclonal antibody comprises three antigen complementarity determining regions of a heavy chain variable region, namely HCDR1, HCDR2 and HCDR3, and three antigen complementarity determining regions of a light chain variable region, namely LCDR1, LCDR2 and LCDR3, and the specific amino acid sequences of the six antigen complementarity determining regions are as follows: the sequence of HCDR1 is as shown in SEQ ID NO: 3; the sequence of HCDR2 is as shown in SEQ ID NO: 4; the sequence of HCDR3 is as shown in SEQ ID NO: 5; the sequence of LCDR1 is as shown in SEQ ID NO: 6; the sequence of LCDR2 is as shown in SEQ ID NO: 7; and the sequence of LCDR3 is as shown in SEQ ID NO:

8.

2. The monoclonal antibody according to claim 1, characterized in that, The amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO: 1; and the amino acid sequence of the light chain variable region is as shown in SEQ ID NO:

2.

3. A gene encoding the monoclonal antibody of claim 1 or 2.

4. An expression vector comprising the gene of claim 3.

5. An antibody-coupled superparamagnetic bead, characterized in that, The conjugated antibody is HK401 of claim 1.

6. A test kit capable of specifically detecting troponin I in human serum, characterized by, The kit comprises the monoclonal antibody HK401 of claim 1.

7. Use of the monoclonal antibody of claim 1 or 2 in the preparation of a troponin I detection reagent.

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