Antibodies or antigen-binding fragments that specifically bind to creatine kinase isoenzymes and uses thereof

Abstract

The application discloses antibodies or antigen binding fragments capable of specifically binding with creatine kinase isoenzyme and application thereof. The application discloses a pair of antibodies or antigen binding fragments capable of specifically binding with creatine kinase isoenzyme (CK-MB), both of which have good affinity with CK-MB antigen, can form a double antibody sandwich structure, and can be used for detection of CK-MB and development of a determination kit for diagnosing CK-MB related diseases.

Description

Technical Field

[0001] This invention belongs to the field of in vitro diagnostics and antibody technology, specifically relating to antibodies or antigen-binding fragments that can specifically bind to creatine kinase isoenzyme (CK-MB) and their applications. Background Technology

[0002] Acute myocardial infarction (AMI) is caused by obstruction or spasm of the coronary arteries, leading to acute and persistent ischemic myocardial necrosis. AMI is often accompanied by severe substernal pain, coronary artery sclerosis, rapid myocardial cell necrosis, elevated serum myocardial enzyme levels, arrhythmia, and shock. If treatment is not timely, it can be life-threatening.

[0003] AMI (Acute Myocardial Infarction) can be diagnosed using myocardial injury markers. Early detection and treatment through blood tests, when myocardial cell damage is minimal, can save lives. Markers that can serve as early diagnostic markers for AMI include troponin I (cTnI), creatine kinase isoenzyme (CK-MB), and myoglobin (MYO). Creatine kinase (CK) has four isoenzymes: hybrid (MB), muscle (MM), brain (BB), and mitochondrial (MiMi). MB isoenzymes are mainly found in cardiomyocytes, MM in skeletal muscle tissue, BB in brain tissue, gastrointestinal tract, and uterine smooth muscle, and MiMi in cardiac and skeletal muscle mitochondria. During a myocardial infarction, serum creatine kinase levels rise rapidly 6 hours after onset, reaching a peak after 24 hours. CK-MB activity can increase to 10-25 times the normal level. CK-MB, as a marker of myocardial injury, has high sensitivity, specificity, and diagnostic accuracy.

[0004] Currently, there are many methods for detecting CK-MB in clinical practice, such as enzyme-linked immunosorbent assay (ELISA), immunosuppression assay, electrophoresis, immunoassay, and chemiluminescence assay. The drawbacks of these methods include long detection time, large amount of raw materials used, and high cost of instruments and equipment.

[0005] Because acute myocardial infarction (AMI) has a rapid onset, patients need rapid diagnosis to facilitate prompt treatment. Immunochromatographic colloidal gold assays are characterized by rapid detection, portability, and ease of operation, and are widely used in infectious diseases, drug use, and early pregnancy detection. However, this method has low sensitivity and cannot provide precise quantification. Summary of the Invention

[0006] In view of the shortcomings of the prior art, the first objective of the present invention is to provide a pair of antibody or antigen-binding fragments that can specifically bind to CK-MB, specifically recognize CK-MB, and form a double-antibody sandwich pattern, with good affinity, specificity and activity.

[0007] A pair of antibody or antigen-binding fragments that can specifically bind to CK-MB, including:

[0008] Antibody B4-3:

[0009] The amino acid sequence of the CDR1 sequence in the light chain variable region is shown in SEQ ID NO:3;

[0010] The amino acid sequence of the CDR2 sequence in the light chain variable region is shown in SEQ ID NO:4;

[0011] The amino acid sequence of the CDR3 sequence in the light chain variable region is shown in SEQ ID NO:5;

[0012] The amino acid sequence of the heavy chain variable region CDR1 is shown in SEQ ID NO:6;

[0013] The amino acid sequence of the heavy chain variable region CDR2 is shown in SEQ ID NO:7;

[0014] The amino acid sequence of the heavy chain variable region CDR3 is shown in SEQ ID NO:8;

[0015] as well as,

[0016] Antibody H6-4:

[0017] The amino acid sequence of the CDR1 sequence in the light chain variable region is shown in SEQ ID NO:9;

[0018] The amino acid sequence of the CDR2 sequence in the light chain variable region is shown in SEQ ID NO:10;

[0019] The amino acid sequence of the CDR3 sequence in the light chain variable region is shown in SEQ ID NO:11;

[0020] The amino acid sequence of the heavy chain variable region CDR1 is shown in SEQ ID NO:12;

[0021] The amino acid sequence of the heavy chain variable region CDR2 is shown in SEQ ID NO:13;

[0022] The amino acid sequence of the heavy chain variable region CDR3 is shown in SEQ ID NO:14.

[0023] Preferably, antibodies B4-3 and H6-4 are monoclonal antibodies.

[0024] Preferably, antibodies B4-3 and H6-4 are murine antibodies.

[0025] A second object of the present invention is to provide a nucleic acid that encodes the aforementioned antibody or antigen-binding fragment.

[0026] A third objective of this invention is to provide a vector comprising the aforementioned nucleic acid.

[0027] A fourth object of the present invention is to provide a cell comprising the above-described carrier.

[0028] A fifth objective of this invention is to provide the application of the aforementioned antibody or antigen-binding fragment in the preparation of early detection products for creatine kinase isoenzyme-related diseases.

[0029] Preferably, the creatine kinase isoenzyme-related disease is acute myocardial infarction (AMI).

[0030] A sixth object of the present invention is to provide a detection kit comprising the above-described antibody or antigen-binding fragment.

[0031] Preferably, antibody B4-3 is used as a labeling antibody and antibody H6-4 is used as a coating antibody.

[0032] The beneficial effects of this invention are:

[0033] The two anti-human CK-MB monoclonal antibodies of the present invention have good affinity and specificity for CK-MB antigen and can form a double antibody sandwich pattern with CK-MB antigen. They can be used to detect the content of CK-MB protein in human serum and to develop detection kits using colloidal gold method, latex method, enzyme-linked immunosorbent assay or immunofluorescence chromatography. Attached Figure Description

[0034] Figure 1 This is an SDS-PAGE image of the purified CK-MB immunogen after expression.

[0035] Figure 2 The image shows the SDS-PAGE of the two anti-CK-MB monoclonal antibodies purified according to the present invention; where label 1: B4-3, 2: H6-4, M: Marker, and protein standard molecular weight.

[0036] Figure 3 The standard curve for the CK-MB calibrator is shown in Figure A, where A is the curve image and B is the corresponding curve parameter. Detailed Implementation

[0037] To make the objectives, technical solutions, experimental methods, and advantages of this invention clearer, the technical solutions of this invention will be further described in detail below with reference to the embodiments and pictures of this invention. Where conditions are indicated in the embodiments, they are performed according to standard experimental conditions or the conditions specified in the manufacturer's instructions. All experimental equipment or instruments used without a specified manufacturer are commercially available, conventional products. The scientific terms used in the embodiments of this invention have the same meaning as commonly understood by a person skilled in the art; any special meanings will be indicated and explained.

[0038] This invention provides a pair of antibody or antigen-binding fragments capable of specifically binding to CK-MB, comprising:

[0039] Antibody B4-3:

[0040] The amino acid sequence of the CDR1 sequence in the light chain variable region is shown in SEQ ID NO:3, specifically:

[0041] ArgThrSerGluSerValGluTyrTyrGlyThrSerLeuMetGln

[0042] The amino acid sequence of the CDR2 sequence in the light chain variable region is shown in SEQ ID NO:4, specifically:

[0043] GlyAlaSerAsnValGluSer

[0044] The amino acid sequence of the CDR3 sequence in the light chain variable region is shown in SEQ ID NO:5, specifically:

[0045] GlnGlnSerArgLysAlaProTrp

[0046] The amino acid sequence of the heavy chain variable region CDR1 is shown in SEQ ID NO:6, specifically:

[0047] SerPheAlaMetSer

[0048] The amino acid sequence of the heavy chain variable region CDR2 is shown in SEQ ID NO:7, specifically:

[0049] ThrIleAsnArgGlyGlyTyrSerThrTyrTyrProAspSerValLysGly

[0050] The amino acid sequence of the heavy chain variable region CDR3 is shown in SEQ ID NO:8, specifically:

[0051] HisLeuGluTyrGlyAsnTyrValAspTyrGluLeuAspTyr

[0052] as well as,

[0053] Antibody H6-4:

[0054] The amino acid sequence of the CDR1 sequence in the light chain variable region is shown in SEQ ID NO:9, and is as follows:

[0055] LysSerSerGlnSerLeuPheAspSerArgThrArgLysAsnTyrLeuAla

[0056] The amino acid sequence of the CDR2 sequence in the light chain variable region is shown in SEQ ID NO:10, and is as follows:

[0057] TrpAlaSerThrArgGluSer

[0058] The amino acid sequence of the CDR3 sequence in the light chain variable region is shown in SEQ ID NO:11, specifically:

[0059] LysGluSerTyrAsnLeuTyrThr

[0060] The amino acid sequence of the heavy chain variable region CDR1 is shown in SEQ ID NO:12, and is as follows:

[0061] ThrTyrAlaMetSer

[0062] The amino acid sequence of the heavy chain variable region CDR2 is shown in SEQ ID NO:13, specifically:

[0063] SerIleIleSerGlyGlyTyrThrTyrTyrProAspSerValLysGly

[0064] The amino acid sequence of the heavy chain variable region CDR3 is shown in SEQ ID NO:14, specifically:

[0065] GlyValAspPheAspVal

[0066] Example 1: Preparation of CK-MB Immunogen

[0067] The amino acid sequence of the CK-MB immunogen of this invention is the sequence published by the National Center for Biotechnology Information (NCBI) in the United States, with the amino acid sequence number EAW57337.1 (SEQ ID NO:1). The complete amino acid fragment of CK-MB was selected, and the amino acid sequence was analyzed and codons optimized by General Biosystems (Anhui) Co., Ltd. Six histidine residues (His) were introduced at the C-terminus of the gene sequence, and the gene was constructed into the pET 22b expression vector. The optimized gene sequence is SEQ ID NO:2.

[0068] SEQ ID NO:1 is as follows:

[0069]

[0070] SEQ ID NO:2 is as follows:

[0071]

[0072] The synthesized and validated CK-MB expression plasmid was transformed into E. coli BL21(DE3) competent host cells and plated onto LB agar plates containing 100 μg / ml ampicillin. The plates were incubated at 37°C for at least 12 hours. Positive single clones were picked and added to LB agar (5 g yeast extract, 10 g tryptone, 10 g sodium chloride), and incubated overnight at 37°C with a shaking incubator. Seed culture was inoculated into 1 L of LB agar at a 1:100 ratio and incubated at 37°C with a shaking incubator at 250 rpm. The absorbance of the bacterial culture was [OD value missing]. 600 Within the range of 0.6-0.8, set the shaker temperature to 16℃ and the rotation speed to 250 rpm. Add isopropyl thiogalactoside (IPTG) to a final concentration of 0.5 mM for induction expression, with an expression time of 18-20 hours (generally overnight). After induction, centrifuge the bacterial culture (8000 rpm, 5 min), and use ultrasonic disruption to disrupt the bacterial cells. Centrifuge at 12000 rpm for 30 min, collect the supernatant, and perform His affinity chromatography on a Cytiva Ni Sepharose 6 Fast Flow column. Purify according to the manufacturer's instructions. The elution method during purification was optimized by changing the imidazole concentration in the elution to 40 mM, which improved the yield of CK-MB immunogen.

[0073] Buffer solution used in the purification process:

[0074] Equilibration buffer: 20mM Tris + 500mM NaCl + 5mM imidazole;

[0075] Wash buffer: 20mM Tris + 500mM NaCl + 40mM imidazole;

[0076] Elution buffer: 20mM Tris + 500mM NaCl + 500mM imidazole;

[0077] The purified CK-MB immunogen was dialyzed into a dialysis buffer of 50 mM Tris + 150 mM NaCl + 1 mM DTT + 1 mM EDTA, pH 8.0, with a sample-to-dialysis buffer ratio of 1:50. Dialysis was performed three times, each time for more than 8 hours. After dialysis, the collected CK-MB protein was analyzed by SDS-PAGE, and the results are as follows. Figure 1 As shown.

[0078] Example 2: CK-MB immunized mice and hybridoma cell screening

[0079] Six- to eight-week-old female Balb / c mice were selected. The purified CK-MB immunogen and Freund's complete adjuvant were emulsified completely in a syringe at a 1:1 ratio for subcutaneous immunization. Each mouse received 500 μL of emulsifier and 50 μg of immunogen. Subsequent immunizations were administered every 14 days, with each mouse receiving 25 μg of CK-MB antigen, alternating between subcutaneous and intraperitoneal immunizations. Cell fusion experiments were performed on day 10 of the fourth immunization.

[0080] Before performing the cell fusion experiment, mouse myeloma cells SP2 / 0 were expanded and passaged in DMEM complete medium containing 10% fetal bovine serum. Before fusion, it was ensured that SP2 / 0 cells were in the proliferation phase and that there was no contamination by microscopic observation.

[0081] Balb / c female mice were euthanized after immunization. The spleens were removed aseptically after soaking in 75% alcohol for 5 minutes and then ground on a stainless steel mesh to form a spleen cell suspension. SP2 / 0 cells and spleen cells were counted and mixed at a 1:5 cell ratio. SP2 / 0 cells and spleen cells were electrofused using a BTX ECM2001 cell fusion system. The fused cells were seeded into 96-well plates (200 μL per well, 15 plates per well) and cultured in a CO2 incubator. After 7 days of culture, the medium was replaced with HAT selective medium, and cultured for another 7-10 days. Only successfully fused cells can grow normally in HAT selective medium. Cell supernatant was collected for enzyme-linked immunosorbent assay (ELISA). Cells from the wells with the highest positive values ​​were selected for subsequent monoclonal cell selection. This selection process was repeated 3-5 times using limiting dilution until all wells showed positive ELISA results, indicating successful monoclonal cell selection. Six anti-CK-MB hybridoma cell lines were obtained and verified using a double-antibody sandwich method. The antibodies produced by the B4-3 and H6-4 cell lines showed good pairing, and the affinity and specificity were also good during detection.

[0082] Example 3: Expression and purification of recombinant antibodies

[0083] (1) Hybridoma cell sequencing

[0084] Two hybridoma cell lines, B4-3 and H6-4, were sent to a cell sequencing company for antibody gene sequencing. The gene sequences of the antibody light and heavy chains in both cell lines were obtained, and the amino acid sequences were analyzed using software, as follows:

[0085] The amino acid sequence of the B4-3 light chain is SEQ ID NO:15;

[0086] The amino acid sequence of the B4-3 heavy chain is SEQ ID NO:16;

[0087] The H6-4 light chain amino acid sequence is SEQ ID NO:17;

[0088] The amino acid sequence of the H6-4 heavy chain is SEQ ID NO:18.

[0089] SEQ ID NO:15 is as follows:

[0090] AspIleValValThrGlnSerProAlaSerLeuAlaValSerLeuGlyGlnSerValThrIleSerCysArgThrSerGluSerValGluTyrTyrGlyThrSerLeuMetGlnTrpTyrGlnGlnLysProArgGlnProProLysLeuLeuIleAsnGlyAlaSerAsnValGluSerGlyValProAlaArgPheSerGlySerGlySerGlyThrGluPheSerLeuAsnIleHisProValGluGluAspAspIleAlaValTyrPheCysGlnGlnSerArgLysAlaProTrpThrPheGlyGlyGlyThrLysLeuAspIleLysArgThrValAlaAlaProSerValPheIlePheProProSerAspGluGlnLeuLysSerGlyThrAlaSerValValCysLeuLeuAsnAsnPheTyrProArgGluAlaLysValGlnTrpLysValAspAsnAlaLeuGlnSerGlyAsnSerGlnGluSerValThrGluGlnAspSerLysAspSerThrTyrSerLeuSerSerThrLeuThrLeuSerLysAlaAspTyrGluLysHisLysValTyrAlaCysGluValThrHisGlnGlyLeuSerSerProValThrLysSerPheAsnArgGlyGluCys

[0091] SEQ ID NO:16 is as follows:

[0092]

[0093] SEQ ID NO: 17 is as follows:

[0094] AspIleValLeuSerGlnSerProSerSerLeuAlaValSerThrGlyGluLysValThrMetSerCysLysSerSerGlnSerLeuPheAspSerArgThrArgLysAsnTyrLeuAlaTrpTyrGlnGlnLysProGlyGlnSerProLysLeuLeuIleTyrTrpAlaSerThrArgGluSerGlyValProAspArgPheThrGlySerGlySerGlyThrAspPheThrLeuThrValSerSerValGlnAlaGluAspLeuAlaIleTyrTyrCysLysGluSerTyrAsnLeuTyrThrPheGlyGlyGlyThrArgLeuGluIleLysArgAlaAspAlaAlaProThrValSerIlePheProProSerSerGluGlnLeuThrSerGlyGlyAlaSerValValCysPheLeuAsnAsnPheTyrProLysAspIleAsnValLysTrpLysIleAspGlySerGluArgGlnAsnGlyValLeuAsnSerTrpThrAspGlnAspSerLysAspSerThrTyrSerMetSerSerThrLeuThrLeuThrLysAspGluTyrGluArgHisAsnSerTyrThrCysGluAlaThrHisLysThrSerThrSerProIleValLysSerPheAsnArgAsnGluCys

[0095] SEQ ID NO: 18 is as follows:

[0096]

[0097] (2) Construction of recombinant antibody vector

[0098] The recombinant antibody plasmids for B4-3 and H6-4 were constructed based on the pcDNA3.1 eukaryotic expression vector. Light and heavy chain expression plasmids were constructed separately, with NheI and XhoI restriction sites at both ends of the genes, respectively. Amino acid sequence analysis and codon optimization were performed by General Biosystems (Anhui) Co., Ltd., and the light and heavy chain genes of B4-3 and H6-4 were synthesized and constructed into the pcDNA3.1 eukaryotic expression vector. After successful gene synthesis and verification, the constructed B4-3 and H6-4 antibody expression plasmids were transformed into TOP10 *E. coli* for amplification culture. High-concentration, high-purity plasmids were extracted according to the endotoxin-free plasmid extraction kit instructions for subsequent recombinant antibody expression.

[0099] (3) Transient expression of recombinant antibodies

[0100] The mammalian cells used to express the recombinant antibodies B4-3 and H6-4 were HEK-293, and the culture medium was KOP-293 (Zhuhai Kerry Biotechnology). The culture conditions for HEK-293 were 37℃ and 120 rpm, until the cell density reached 2 × 10⁶ cells / year. 6 When the cell viability is >95%, transfection with expression plasmids is performed. During transfection, the mass ratio of light chain to heavy chain plasmid is 3:2. Add 100 μg of plasmid and 500 μL of transfection reagent TA-293 to every 100 ml of cell suspension. Add the transfection reagent to the prepared plasmid mixture, mix well, and let stand at room temperature for 10 min. Then add the mixture to the HEK-293 cell suspension, shaking the cell suspension while slowly adding the transfection reagent and plasmid mixture. Continue culturing in a shaker at 37℃ and 120 rpm. 24 hours after transfection, add 600 μL of cell protein expression enhancer (KE-293, Kairui Biotechnology) and 2 ml of transient transfection nutrient additive (KT-Feed 50×, Kairui Biotechnology) to every 100 ml of cell suspension. Expression ends on day 6 after transfection. Centrifuge the cell suspension (8000 rpm, 15 min) and retain the cell supernatant for antibody purification.

[0101] (4) Purification of recombinant antibody

[0102] Recombinant antibody purification was performed using AT Protein A Diamond Plus affinity chromatography column. The collected cell supernatant was loaded onto the purification column for purification. After purification, the B4-3 and H6-4 recombinant antibodies were validated by SDS-PAGE, and the results are as follows: Figure 2 As shown, the heavy chain molecular weight of both antibodies is around 50KD, and the light chain molecular weight is around 25KD.

[0103] Example 4: Detection of CK-MB using immunofluorescence chromatography strips

[0104] This invention uses fluorescent microspheres as labeled mesons and employs a double-antibody sandwich immunoassay technique, along with a dedicated reader, to prepare CK-MB fluorescent test strips. A calibration curve is established to achieve quantitative detection of CK-MB. This method is simple to operate and exhibits excellent accuracy and stability.

[0105] After activation, fluorescent microspheres are added with CK-MB recombinant antibody (B4-3) for labeling. Following centrifugation, blocking, and sonication, the microspheres are resuspended in storage buffer to obtain CK-MB-labeled recombinant antibody immunomicrospheres. The labeled fluorescent immunomicrospheres are then sprayed onto a polyester membrane at an appropriate concentration and dried before use. Recombinant antibody (H6-4) on the T line and goat anti-mouse polyclonal antibody on the C line are evenly spread onto a nitrocellulose membrane (NC) and dried before use. The sample pad, fluorescent microsphere polyester membrane, NC membrane, and absorbent paper are sequentially attached to the adhesive test strip base. After assembly, the large plate is cut into narrow strips and inserted into the cartridge of a compatible fluorescence reader, ready for use in detecting CK-MB-related samples.

[0106] Calibration curves were plotted using CK-MB immunofluorescence test strips with calibrator concentration gradients of 0.25 ng / ml, 0.5 ng / ml, 2.5 ng / ml, 5 ng / ml, 10 ng / ml, 18 ng / ml, 35 ng / ml, 60 ng / ml, and 100 ng / ml. Calibrators were added to the test wells, and after the reaction, the fluorescence values ​​of the T and C lines were measured using a fluorescence reader. Each standard concentration was measured five times, and the specific values ​​are shown in Table 1. A curve was fitted using the calibrator concentrations and the T / C values. R0 2 =0.9969, see Figure 3 .

[0107] Table 1 CK-MB Calibration Test

[0108]

[0109] To verify the accuracy and specificity of the CK-MB kit for quantitative detection, clinical serum samples of CK-MB were collected at different time periods. The concentration of CK-MB in the serum samples was quantitatively detected using Leadman reagent. A total of 60 CK-MB positive clinical serum samples were collected, and the concentration of CK-MB measured by Leadman was compared with that measured by the kit of this invention, as shown in Table 2.

[0110] Table 2. Detection of CK-MB in Clinical Specimens

[0111]

[0112]

[0113]

[0114] According to the results in Table 2, the present invention shows good consistency with Leadman's method for detecting CK-MB, indicating that the immunofluorescence chromatography method established in this invention has good accuracy in detecting CK-MB protein.

[0115] The specific embodiments described above illustrate the present invention in detail, but the present invention is not limited to the embodiments; the CK-MB recombinant paired antibody embodiments of the present invention are applied to immunofluorescence chromatography detection, but are not limited to this method.

Claims

1. A pair of antibodies that can specifically bind to CK-MB, including: Antibody B4-3: The amino acid sequence of the CDR1 sequence in the light chain variable region is shown in SEQ ID NO:3; The amino acid sequence of the CDR2 sequence in the light chain variable region is shown in SEQ ID NO:4; The amino acid sequence of the CDR3 sequence in the light chain variable region is shown in SEQ ID NO:5; The amino acid sequence of the heavy chain variable region CDR1 is shown in SEQ ID NO:6; The amino acid sequence of the heavy chain variable region CDR2 is shown in SEQ ID NO:7; The amino acid sequence of the heavy chain variable region CDR3 is shown in SEQ ID NO:8; as well as, Antibody H6-4: The amino acid sequence of the CDR1 sequence in the light chain variable region is shown in SEQ ID NO:9; The amino acid sequence of the CDR2 sequence in the light chain variable region is shown in SEQ ID NO:10; The amino acid sequence of the CDR3 sequence in the light chain variable region is shown in SEQ ID NO:11; The amino acid sequence of the heavy chain variable region CDR1 is shown in SEQ ID NO:12; The amino acid sequence of the heavy chain variable region CDR2 is shown in SEQ ID NO:13; The amino acid sequence of the heavy chain variable region CDR3 is shown in SEQ ID NO:

14.

2. The pair of antibodies that specifically bind to CK-MB according to claim 1, characterized in that, The antibodies B4-3 and H6-4 are monoclonal antibodies.

3. The pair of antibodies that specifically bind to CK-MB according to claim 1, characterized in that, The antibodies B4-3 and H6-4 are murine antibodies.

4. A nucleic acid, characterized in that, The nucleic acid encodes the antibody according to any one of claims 1-3.

5. A carrier, characterized in that, The vector comprises the nucleic acid described in claim 4.

6. A cell, characterized in that, The cells comprise the carrier as described in claim 5.

7. The use of the antibody according to any one of claims 1-3 in the preparation of an early detection product for creatine kinase isoenzyme-related diseases, characterized in that, The creatine kinase isoenzyme-related disease is acute myocardial infarction (AMI).

8. A test kit, characterized in that, It includes the antibody as described in any one of claims 1-3.

9. A detection kit according to claim 8, characterized in that, The antibody B4-3 is used as the labeling antibody, and the antibody H6-4 is used as the coating antibody.

Images