An anti-CTNT antibody pair and its application

Abstract

The present application relates to the technical field of biomedicine, and specifically to an anti-CTNT antibody pair and its application. The antibody pair comprises two monoclonal antibodies; the monoclonal antibodies comprise a heavy chain variable region and a light chain variable region; one monoclonal antibody in the monoclonal antibody pair is designated as the 15F11 antibody, and the other monoclonal antibody is designated as the 8E8 antibody. The present application also discloses nucleic acid molecules encoding the monoclonal antibodies and hybridoma cell lines secreting the monoclonal antibodies. In addition, the present application also discloses a kit for detecting CTNT comprising the monoclonal antibody pair, wherein the 15F11 antibody serves as a capture antibody and the 8E8 antibody serves as a labeling antibody. The anti-CTNT antibody pair provided in the present application has good specificity for CTNT.

Description

Technical Field

[0001] The present application relates to the technical field of biomedicine, and in particular to an anti-CTNT antibody pair and applications thereof. Background Art

[0002] Cardiac troponin (CTN) is a contractile regulatory protein present in skeletal cardiac muscle. The CTN complex is composed of three subunits: CTN I, CTNT, and CTN C. Each CTN subunit plays an independent role, and different fragments may have different biological activities. Only CTNT and CTN1 are myocardium-specific isoforms, the vast majority of which are localized on the contractile thin filaments, while a small portion (3-6%) is located in the myocyte cytoplasm as free protein. CTNT is a 37,000-Da cardiac structural protein with three isoforms: fast skeletal, slow skeletal, and cardiac. Their expression in skeletal and cardiac muscle is regulated by different genes and encoded by different genes. CTNT is highly organ-specific, and elevated blood concentrations often indicate myocardial cell necrosis.

[0003] CTNT has three advantages as a diagnostic indicator: 1. High specificity: Troponin is primarily expressed only in the myocardium, and an elevated level indicates myocardial damage, making it more myocardial-specific than CK-MB. 2. Smaller molecular weight and longer duration: When myocardial damage occurs, it diffuses more easily from myocardial cells, allowing for earlier detection. Furthermore, its long duration allows for the detection of more recent myocardial infarctions. 3. High correlation between concentration and degree of damage: Different concentrations reflect varying degrees of myocardial necrosis, making dynamic monitoring extremely helpful in determining prognosis.

[0004] Troponin test sensitivity is closely linked to treatment effectiveness. Low-sensitivity troponin tests typically only detect higher levels of troponin and may therefore fail to detect minor heart damage in its early stages. High-sensitivity CTNT tests can provide diagnostic information at an earlier stage of heart damage, allowing medical teams to take treatment measures more quickly and reduce unnecessary risks.

[0005] Therefore, it is necessary to find a CTNT detection method with high detection sensitivity. Summary of the Invention

[0006] The present application provides an anti-CTNT antibody pair and applications thereof.

[0007] This application uses hybridoma technology to screen two monoclonal antibodies, named 8E8 and 15F11. Using 8E8 as the labeled antibody and 15F11 as the coating antibody, colloidal gold chromatography strips were prepared, which were more sensitive than the control and did not cross-react with skeletal muscle.

[0008] The anti-CTNT antibody pair provided in the present application is a monoclonal antibody targeting CTNT with higher sensitivity, which can effectively improve the quality of the CTNT detection method.

[0009] In a first aspect, the present application provides an anti-CTNT antibody pair, which adopts the following technical solution:

[0010] An anti-CTNT antibody pair, comprising two monoclonal antibodies; the monoclonal antibodies comprising a heavy chain variable region and a light chain variable region; the heavy chain variable region comprising a heavy chain CDR1, a heavy chain CDR2, and a heavy chain CDR3; the light chain variable region comprising a light chain CDR1, a light chain CDR2, and a light chain CDR3;

[0011] One of the monoclonal antibodies in the antibody pair is named 8E8 antibody, and the sequence information of the 8E8 antibody is as follows:

[0012] The heavy chain CDR1 comprises the amino acid sequence shown in SEQ ID NO: 1, or an amino acid sequence having one or two conservative amino acid substitutions compared to the sequence shown in SEQ ID NO: 1, or an amino acid sequence comprising the above sequence;

[0013] The heavy chain CDR2 comprises the amino acid sequence shown in SEQ ID NO: 2, or an amino acid sequence having one or two conservative amino acid substitutions compared to the sequence shown in SEQ ID NO: 2, or an amino acid sequence comprising the above sequence;

[0014] The heavy chain CDR3 comprises the amino acid sequence shown in SEQ ID NO: 3, or an amino acid sequence having one or two conservative amino acid substitutions compared to the sequence shown in SEQ ID NO: 3, or an amino acid sequence comprising the above sequence;

[0015] The light chain CDR1 comprises the amino acid sequence shown in SEQ ID NO: 4, or an amino acid sequence having one or two conservative amino acid substitutions compared to the sequence shown in SEQ ID NO: 4, or an amino acid sequence comprising the above sequence;

[0016] The light chain CDR2 comprises the amino acid sequence shown in SEQ ID NO: 5, or an amino acid sequence having one or two conservative amino acid substitutions compared to the sequence shown in SEQ ID NO: 5, or an amino acid sequence comprising the above sequence;

[0017] The light chain CDR3 comprises the amino acid sequence shown in SEQ ID NO: 6, or an amino acid sequence having one or two conservative amino acid substitutions compared to the sequence shown in SEQ ID NO: 6, or an amino acid sequence comprising the above sequence;

[0018] The other monoclonal antibody in the antibody pair is named 15F11 antibody, and the sequence information of the 15F11 antibody is as follows:

[0019] The heavy chain CDR1 comprises the amino acid sequence shown in SEQ ID NO: 7, or an amino acid sequence having one or two conservative amino acid substitutions compared to the sequence shown in SEQ ID NO: 7, or an amino acid sequence comprising the above sequence;

[0020] The heavy chain CDR2 comprises the amino acid sequence shown in SEQ ID NO: 8, or an amino acid sequence having one or two conservative amino acid substitutions compared to the sequence shown in SEQ ID NO: 8, or an amino acid sequence comprising the above sequence;

[0021] The heavy chain CDR3 comprises the amino acid sequence shown in SEQ ID NO: 9, or an amino acid sequence having one or two conservative amino acid substitutions compared to the sequence shown in SEQ ID NO: 9, or an amino acid sequence comprising the above sequence;

[0022] The light chain CDR1 comprises the amino acid sequence shown in SEQ ID NO: 10, or an amino acid sequence having one or two conservative amino acid substitutions compared to the sequence shown in SEQ ID NO: 10, or an amino acid sequence comprising the aforementioned sequence;

[0023] The light chain CDR2 comprises the amino acid sequence shown in SEQ ID NO: 11, or an amino acid sequence having one or two conservative amino acid substitutions compared to the sequence shown in SEQ ID NO: 11, or an amino acid sequence comprising the aforementioned sequence;

[0024] The light chain CDR3 comprises the amino acid sequence shown in SEQ ID NO: 12, or an amino acid sequence having one or two conservative amino acid substitutions compared to the sequence shown in SEQ ID NO: 12, or an amino acid sequence comprising the above sequence.

[0025] The heavy chain sequence of the 8E8 antibody includes the amino acid sequence shown in SEQ ID NO: 13.

[0026] The light chain sequence of the 8E8 antibody includes the amino acid sequence shown in SEQ ID NO: 15.

[0027] The heavy chain sequence of the 15F11 antibody includes the amino acid sequence shown in SEQ ID NO: 17.

[0028] The light chain sequence of the 15F11 antibody includes the amino acid sequence shown in SEQ ID NO: 19.

[0029] In a second aspect, the present application provides a nucleic acid molecule, which adopts the following technical solution:

[0030] A nucleic acid molecule encoding the amino acid sequence of the monoclonal antibody.

[0031] The nucleic acid molecule encoding the heavy chain sequence of the 8E8 antibody includes the nucleotide sequence shown in SEQ ID NO: 14. The nucleic acid molecule encoding the light chain sequence of the 8E8 antibody includes the nucleotide sequence shown in SEQ ID NO: 16.

[0032] The nucleic acid molecule encoding the heavy chain sequence of the 15F11 antibody includes the nucleotide sequence shown in SEQ ID NO: 18; the nucleic acid molecule encoding the light chain sequence of the 15F11 antibody includes the nucleotide sequence shown in SEQ ID NO: 20.

[0033] In a third aspect, the present application provides a hybridoma cell line using the following technical solution:

[0034] A hybridoma cell line secretes the above-mentioned monoclonal antibody.

[0035] In a fourth aspect, the present application provides a kit for detecting CTNT, which adopts the following technical solution:

[0036] A kit for detecting CTNT, comprising the above-mentioned anti-CTNT antibody pair.

[0037] Optionally, the 15F11 antibody is used as a capture antibody, and the 8E8 antibody is used as a labeling antibody.

[0038] In a fifth aspect, the present application provides the use of the above-mentioned antibody pair, the above-mentioned nucleic acid molecule or the above-mentioned kit in the preparation of a reagent for detecting CTNT.

[0039] In summary, this application has the following beneficial effects:

[0040] The present application provides an anti-CTNT antibody pair, designated as 15F11 antibody and 8E8 antibody, wherein 15F11 antibody is used as a capture antibody and 8E8 antibody is used as a labeling antibody, both of which have good specificity and sensitivity to CTNT. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1This is the color development result of Example 5.

[0042] Figure 2 This is the color development result of Example 6. DETAILED DESCRIPTION

[0043] Before describing the embodiments of the present application in detail, it should be understood that the terminology used herein is only for the purpose of describing particular embodiments. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the term belongs.

[0044] To make the purpose, technical solutions, and advantages of this application more clear, the technical solutions in the embodiments of this application will be clearly and completely described below with reference to the accompanying drawings. Based on the embodiments of this application, all other embodiments obtained by those skilled in the art without making any creative work shall fall within the scope of protection of this application. The embodiments described below are exemplary and are only used to explain this application, and should not be understood as limiting this application.

[0045] If no specific techniques or conditions are specified in the examples, the experiments were carried out according to the techniques or conditions described in the literature in the field or according to the product instructions. All reagents or instruments used without specifying the manufacturer are commercially available conventional products.

[0046] The present application is further described in detail below in conjunction with the examples and test results.

[0047] Example

[0048] Example 1

[0049] This example provides a preparation process and screening process for hybridoma cell lines. After screening, a total of 10 hybridoma cell lines were obtained in this example.

[0050] The above process specifically includes the following steps:

[0051] 1. Animal Immunization

[0052] The immunized subjects were 8-week-old female BALB / C mice. The immunogen was CTNT expressed in E. coli, which has 498 amino acids, a molecular weight of 55 kDa, and an amino acid sequence as shown in SEQ ID NO: 21.

[0053] The immunization process specifically includes the following steps:

[0054] (1) Preparation of immunogens: CTNT was mixed with Freund's complete adjuvant (Cat. No. F5881, Sigma) in equal volumes to prepare the first immunogen; CTNT was mixed with Freund's incomplete adjuvant (Cat. No. F5506, Sigma) in equal volumes to prepare the second immunogen; CTNT was mixed with physiological saline in equal volumes to prepare the third immunogen.

[0055] (2) Immune process:

[0056] First immunization: 100 μg of the first immunogen was injected subcutaneously at 3-4 points on the back of BALB / C female mice;

[0057] Two weeks later, the second immunization was performed by injecting 100 μg of the second immunogen subcutaneously at 3–4 points on the back of BALB / C female mice;

[0058] Two weeks later, the third immunization was performed by injecting 100 μg of the second immunogen subcutaneously at 3–4 points on the back of BALB / C female mice;

[0059] Two weeks later, the fourth immunization—the surge immunization—was performed by intraperitoneal injection of 100 μg of the third immunogen into BALB / C female mice;

[0060] The fifth immunization was performed 24 hours later by injecting 50 μg of the third immunogen into the tail vein of BALB / C female mice.

[0061] 2. Cell Fusion

[0062] Cell fusion began on the third day after the fifth immunization of BALB / C female mice.

[0063] The cell fusion process specifically includes the following steps:

[0064] (1) Preparation of spleen cell suspension: On the third day after the fifth immunization of BALB / c female mice, the eyeballs of the BALB / c female mice were enucleated and blood was collected. The serum of the BALB / c female mice was isolated and used as a positive control for antibody detection. At the same time, the BALB / c female mice were killed by cervical dislocation, and the spleens were removed to prepare spleen cell suspension.

[0065] Preparation of myeloma cell suspension: Resuscitate myeloma cells (ATCC, catalog number BNCC100908) two weeks in advance (to ensure that the myeloma cells are in the logarithmic growth phase at the time of use) to prepare a myeloma cell suspension;

[0066] Preparation of feeder layer cells: One day before cell fusion, peritoneal macrophages and spleen cells of blank BALB / c female mice were taken and cultured in 96-well plates to obtain cell plates containing feeder layer cells (cell concentration was 1×10 4 / well) to prepare feeder layer cells.

[0067] (2) Cell fusion process:

[0068] Polyethylene glycol (PEG) was used to mediate cell fusion. Splenocyte suspension and myeloma cell suspension were mixed in serum-free 1640 medium (Cat. No. C3010-0500, VivaCell) at a cell number ratio of 5:1. The mixture was centrifuged at 1200 rpm for 5 minutes, and the supernatant was removed.

[0069] Gently tap the bottom of the centrifuge tube with your finger to loosely mix the two cell types. Place the tube in a beaker filled with 37°C water and keep warm. Add 1 mL of 50% PEG1500 (pH 8.0, Catalog No. 10783641001, Roche) over 1 minute to fuse the cells while shaking. After addition, let it stand for 30 seconds. Add serum-free 1640 medium (Catalog No. C3010-0500, VivaCell) to terminate fusion. Centrifuge at 800 rpm for 5 minutes. Resuspend the pellet in HAT medium and distribute it into a cell plate containing feeder cells. The resulting cell plate containing fused cells and feeder cells is cultured in a cell culture incubator at 37°C and 5% CO2.

[0070] Among them, the following reagents are needed to prepare 500mL of HAT culture medium: 100mL of fetal bovine serum (product number 11011-8611, Sijiqing), 5mL of penicillin-streptomycin double antibody for cell culture (100×, product number E607011-0100, Shanghai Bioengineering), 10mL of HAT culture medium additive (50×, product number H0262, Sigma) and 385mL of 1640 culture medium (product number C3010-0500, VivaCell).

[0071] 3. Screening of positive hybridoma cells

[0072] The cell plate containing the fused cell-feeder layer cells was cultured for half medium change on day 4, and cultured for full medium change on day 7. When the fused cells covered 10-50% of the well bottom, positive wells were screened using conventional indirect ELISA method.

[0073] The indirect ELISA method specifically includes the following steps:

[0074] (1) Coating: CTNT was used as the coating antigen and diluted to 2 μg / mL with 0.05 mol / L CB buffer (31.8 g of Na2CO3, 58.8 g of NaHCO3, and made up to 2 L with ultrapure water) at a pH of 9.6. 100 μL / well of the ELISA plate was added and coated overnight at 4°C, then patted dry. The plate was blocked with 1% gelatin-PBS buffer (mass volume ratio) at 300 μL / well. The plate was blocked at 37°C for 2 h, then patted dry for later use.

[0075] (2) Detection: The cell culture supernatant in the cell plate containing fused cell-feeder layer cells was added to the ELISA plate at 100 μL / well, incubated at 37°C for 60 min, washed three times with 0.01 mol / L PBST buffer containing Tween-20 and patted dry, added with 100 μL / well HRP-labeled goat anti-mouse secondary antibody (Cat. No. TJ-211229CN, Xiamen Taijing), incubated at 37°C for 60 min, washed three times and patted dry, added with 100 μL / well TMB colorimetric solution, incubated at 37°C in the dark for 10 min, and terminated by adding 50 μL / well 1 mol / L HCl.

[0076] At the same time, the BALB / c female mouse serum obtained by eye blood collection and separation in the "cell fusion" was used as a positive control to screen out fusion cells with higher antibody titers, namely positive hybridoma cells.

[0077] 4. Cloning of Positive Hybridoma Cells

[0078] Positive hybridoma cells screened from a cell plate containing fused cells and feeder cells originate from two or more hybridoma cells. Therefore, the antibodies secreted by the screened hybridoma cells are heterogeneous. In order to obtain completely homogeneous monoclonal antibodies, the positive hybridoma cells need to be cloned.

[0079] The day before cloning, feeder cells were prepared and plated according to the method of step (1) in "Cell Fusion" to obtain a cell plate containing feeder cells; the positive hybridoma cells obtained by screening were suspended in HT culture medium and mixed by pipetting, and inoculated into the cell plate containing feeder cells, and the cells in the cell plate wells were diluted to 1 cell per well using HT culture medium, and cultured in a humidified environment at 37°C and 5% CO2 for 7 to 10 days. When cloned cells visible to the naked eye appeared, antibodies could be detected.

[0080] The following reagents are required to prepare 500 mL of HT culture medium: 100 mL of fetal bovine serum (product number 11011-8611, Sijiqing), 5 mL of penicillin-streptomycin double antibody for cell culture (100×, product number E607011-0100, Shanghai Bioengineering), 10 mL of HT culture medium additive (product number H0137, Sigma), and 385 mL of 1640 culture medium (product number C3010-0500, VivaCell).

[0081] Under an inverted microscope, wells with single clones growing and positive by ELISA were marked. These clones were then expanded and established. Ten cell lines were initially screened and the supernatants were used for further functional screening. These 10 hybridoma cell lines were named as shown in Table 1.

[0082] Table 1 Hybridoma cell lines obtained by screening and antibodies produced

[0083]

[0084] Example 2

[0085] This example provides the preparation and screening process of CTNT antibodies.

[0086] The above process specifically includes the following steps:

[0087] 1. Preparation of CTNT Antibody

[0088] (1) Preparation of ascites: 8-12 week old Balb / c mice were intraperitoneally injected with ascites-specific adjuvant (Beijing Biolong, Cat. No. KX0210048). Ten days after injection, the cell line prepared in Example 1 (1×10 6 cells / mouse) were injected into the peritoneal cavity of Balb / c mice, and the ascites of the Balb / c mice were collected with a medical syringe 12 days later.

[0089] (2) Antibody purification: The ascites of Balb / c mice collected in step (1) was poured into a centrifuge tube and centrifuged at 12500 rpm for 20 min. The supernatant was collected and mixed with saturated ammonium sulfate solution at a volume ratio of 1:1. The mixture was centrifuged at 12000 rpm for 5 min. The supernatant was discarded and the precipitate was resuspended and dissolved with the same volume of PBS. The precipitate was then filtered through a 0.22 μm filter membrane and purified by affinity chromatography using a Protein A column (Cat. No. AA301307, Boglon Bio).

[0090] The specific steps of purification using Protein A column affinity chromatography are as follows:

[0091] Column loading: Add 5 mL of Protein A Resin medium to the chromatography column and let it stand. Rinse the column with 10 column volumes of ultrapure water.

[0092] Equilibration: Use 10 column volumes of pre-cooled Protein A column equilibration solution (50 mM Tris-HCl, 100 mM NaCl, solvent is water, pH = 8.0) to equilibrate the chromatography column;

[0093] Sample loading: The sample filtered through a 0.22 μm filter was loaded at a flow rate of 5 mL / min.

[0094] Washing: Wash the column with 10 column volumes of pre-cooled Protein A column equilibration solution;

[0095] Elution: Elute the antibody with an eluent (100 mM Glycine, 150 mM NaCl, water, pH = 3.0) to obtain an elution buffer containing the antibody; immediately after elution, add a neutralization buffer (2 M Tris-HCl, water, pH = 9.0) to the elution buffer until the solution reaches a neutral pH;

[0096] Dialysis: The eluted antibodies were dialyzed three times in PBS (pH=7.4) solution with a volume of 1000 times the elution volume to obtain 10 purified CTNT antibodies, whose specific names are shown in Table 1.

[0097] 2. Preparation of HRP-antibody conjugated complex protein

[0098] (1) Dilute 10 CTNT antibodies to 2 mg / mL using 0.05 mol / L CB buffer at pH 9.6. Select a dialysis membrane and cut it to an appropriate length based on the target protein molecular weight and actual retention requirements and dialysis volume. Pre-treat the dialysis membrane with 0.05 mol / L CB buffer at pH 9.6, then replace it with 0.05 mol / L CB buffer at pH 9.6 and continue soaking and washing the dialysis membrane once. Transfer 1 mL of the antibody protein solution to the dialysis membrane. Stir and dialyze in 0.05 mol / L CB buffer at pH 9.6 at 4°C, changing the buffer every 1 h for a total of 5 dialysis cycles.

[0099] (2) HRP (product number RS20220118, Rise Reagent) was dissolved in ultrapure water to prepare an HRP solution with a concentration of 20 mg / mL, and NaIO4 was dissolved in ultrapure water to prepare a NaIO4 solution with a concentration of 20 mg / mL. After vortexing to fully dissolve, the HRP solution and the NaIO4 solution were mixed in a volume ratio of 1:1, that is, the NaIO4 solution was slowly added to the HRP solution, and the centrifuge tube was immediately wrapped with tin foil. The HRP was activated at 4°C for 30 minutes in the dark.

[0100] (3) Slowly add ethylene glycol dropwise to the HRP-activated centrifuge tube while gently shaking (add 1 μL ethylene glycol for every 1 mg of HRP). Continue to incubate at 4°C in the dark for 30 min to terminate HRP activation.

[0101] (4) Add the terminated HRP solution to the antibody dialysis membrane (1 mg of antibody plus 1 mg of HRP and 1 mg of NaIO4) and couple in 0.05 mol / L CB buffer at pH 9.6 at 4°C in the dark overnight.

[0102] The next day, the solution was replaced with 0.05 mol / L CB buffer (pH = 9.6) and dialysis was continued for 2 hours. After dialysis, the coupled dialysate was transferred to a centrifuge tube to obtain the antibody-HRP coupled solution.

[0103] (5) Prepare a 20 mg / mL NaBH4 solution with pure water and add it to the antibody-HRP coupling solution in step (4). Add 2 μL of NaBH4 solution for every 1 mg of HRP. React at 4°C for 2 h. Mix by inverting the solution several times every 0.5 h to obtain a coupled protein solution.

[0104] (6) The coupled complex protein was precipitated with 50% ammonium sulfate (i.e., an equal volume of saturated concentration of ammonium sulfate and the coupled complex protein solution obtained in step (5) were mixed in a volume ratio of 1:1), precipitated at 4°C for 15 minutes, centrifuged at 10,000 rpm for 10 minutes, and the supernatant was removed to obtain 10 strains of HRP-antibody coupled complex proteins, whose specific names are shown in Table 1.

[0105] 3. Screening of CTNT Antibody Pairs

[0106] The 10 CTNT antibodies obtained in step 1 and the 10 HRP-CTNT antibody-coupled complex proteins obtained in step 2 were paired in pairs, and the antibodies were screened using the double antibody sandwich ELIS method.

[0107] The specific steps are as follows: 10 CTNT antibodies were used as capture antibodies and coated on an ELISA plate respectively. Then, CTNT (the same immunogen as in Example 1) was added to the ELISA-labeled wells, and unbound CTNT was washed away after incubation. Then, HRP-CTNT antibody-coupled complex protein was added as a labeled antibody, and unbound HRP-CTNT antibody-coupled complex protein was washed away after incubation. Finally, a color developing solution was added for color development, and the absorbance was measured at 450 nm using a spectrophotometer. The test results are shown in Table 2.

[0108] Test result interpretation: If color can be developed, it indicates that the labeled antibody and the capture antibody can recognize different epitopes of CTNT, indicating that the capture antibody and the labeled antibody are a paired antibody pair. If no color is developed, it indicates that the labeled antibody cannot bind to CTNT and is eluted, indicating that the capture antibody and the labeled antibody are not paired antibodies.

[0109] Table 2 Test results of capture antibody and labeling antibody pairing

[0110]

[0111] As shown in Table 2, the test results above indicate that the 15F11 and 8E8 antibody pair has high titers and can be used for CTNT detection. The 15F11 antibody is the capture antibody, and the 8E8 antibody is the labeling antibody. The hybridoma cell line producing the 15F11 antibody was designated the 15F11 cell line, and the hybridoma cell line producing the 8E8 antibody was designated the 8E8 cell line.

[0112] Example 3

[0113] In this example, the antibodies obtained by the above screening were sequenced.

[0114] 10G10 cell line and 11F8 cell line were expanded and cultured separately, and 5×10 6 The cells were placed in a centrifuge tube, the supernatant was aspirated, frozen, and sent on dry ice to Nanjing Detai Bioengineering Co., Ltd. for hybridoma cell sequencing.

[0115] The sequencing results are as follows:

[0116] (1) 8E8 antibody

[0117] (1) Heavy chain sequence information

[0118] The full-length base sequence of the heavy chain includes the nucleotide sequence shown in SEQ ID NO: 14.

[0119] Heavy chain full-length amino acid sequence: includes the amino acid sequence shown in SEQ ID NO: 13.

[0120] The heavy chain variable region includes a heavy chain CDR1, a heavy chain CDR2, and a heavy chain CDR3. The heavy chain CDR1 includes the amino acid sequence shown in SEQ ID NO: 1. The heavy chain CDR2 includes the amino acid sequence shown in SEQ ID NO: 2. The heavy chain CDR3 includes the amino acid sequence shown in SEQ ID NO: 3.

[0121] (2) Light chain sequence information

[0122] The full-length base sequence of the light chain includes the nucleotide sequence shown in SEQ ID NO: 16.

[0123] Light chain full-length amino acid sequence: includes the amino acid sequence shown in SEQ ID NO: 15.

[0124] The light chain variable region includes a light chain CDR1, a light chain CDR2, and a light chain CDR3. The light chain CDR1 includes the amino acid sequence set forth in SEQ ID NO: 4. The light chain CDR2 includes the amino acid sequence set forth in SEQ ID NO: 5. The light chain CDR3 includes the amino acid sequence set forth in SEQ ID NO: 6.

[0125] (2) 15F11 antibody

[0126] (1) Heavy chain sequence information

[0127] The full-length base sequence of the heavy chain includes the nucleotide sequence shown in SEQ ID NO: 18.

[0128] Heavy chain full-length amino acid sequence: includes the amino acid sequence shown in SEQ ID NO: 17.

[0129] The heavy chain variable region includes a heavy chain CDR1, a heavy chain CDR2, and a heavy chain CDR3. The heavy chain CDR1 includes the amino acid sequence set forth in SEQ ID NO: 7. The heavy chain CDR2 includes the amino acid sequence set forth in SEQ ID NO: 8. The heavy chain CDR3 includes the amino acid sequence set forth in SEQ ID NO: 9.

[0130] (2) Light chain sequence information

[0131] The full-length base sequence of the light chain includes the nucleotide sequence shown in SEQ ID NO: 20.

[0132] Light chain full-length amino acid sequence: includes the amino acid sequence shown in SEQ ID NO: 19.

[0133] The light chain variable region includes a light chain CDR1, a light chain CDR2, and a light chain CDR3. The light chain CDR1 includes the amino acid sequence set forth in SEQ ID NO: 10. The light chain CDR2 includes the amino acid sequence set forth in SEQ ID NO: 11. The light chain CDR3 includes the amino acid sequence set forth in SEQ ID NO: 12.

[0134] Example 4

[0135] This embodiment provides a colloidal gold test strip for detecting CTNT. The colloidal gold test strip is prepared using 15F11 antibody and 8E8 antibody.

[0136] The preparation process of the test strip specifically includes the following steps:

[0137] 1. Preparation of the test pad

[0138] (1) Preparation of coating buffer: Weigh 2.901 g of Na2HPO4·12H2O, 0.2914 g of NaH2PO4·2H2O, 8.5 g of NaCl, and 25 g of trehalose, dissolve in 1000 mL of ultrapure water, adjust the pH to 7.4, and store at 4°C until use.

[0139] (2) Preparation of test line: 15F11 antibody was added to the coating buffer to a final concentration of 1 mg / mL and sprayed onto the nitrocellulose membrane to prepare a test line. The spraying volume was 1.2 μL / cm and the spraying length was 30 cm.

[0140] (3) Preparation of quality control line: Sheep anti-mouse IgG polyclonal antibody (Hangzhou Longji Biotechnology Co., Ltd.) was added to the coating buffer at a final concentration of 1 mg / mL and sprayed onto the nitrocellulose membrane to prepare the quality control line. The spraying volume was 1.2 μL / cm, the spraying length was 30 cm, and the distance between the quality control line and the test line was 5 mm.

[0141] (4) The nitrocellulose membrane coated with the test line and the quality control line is the test pad. The test pad is attached to the PVC backing board and placed in an oven at 50°C to dry for 24±2h.

[0142] 2. Preparation of Gold Label Conjugate

[0143] (1) Preparation of relevant solutions:

[0144] Preparation of 0.2M K2CO3 solution: Weigh 2.7642 g of anhydrous potassium carbonate and dissolve it in 100 mL of pure water.

[0145] Preparation of 20% BSA solution: Weigh 20 g of BSA (Cat. No. V900933, Sigma) and 100 μL of proclin 300, dissolve in 100 mL of ultrapure water, and store at 4° C. for later use.

[0146] Preparation of reconstitution solution: Weigh 0.36 g of Tris, 0.1 g of sodium caseinate, 0.1 g of PEG20000, 100 μL of TW-20, 2 g of sucrose, and 100 μL of proclin300, dissolve in 100 mL of ultrapure water, adjust the pH to 8.5 with concentrated hydrochloric acid, and store at 4°C until use.

[0147] (2) Marking process:

[0148] Take 100mL of 0.04% colloidal gold solution (Cat. No. C805628, McLean) and place it in a clean container. Add 500-2000μL of 0.2M K2CO3 solution and stir evenly; then add 500-1500μg of 8E8 antibody and stir for 10-20min; then add 500μL of 20% BSA solution and stir. Block for 5-10min, centrifuge at 8000-10000r / min for 30min, discard the supernatant, and re-dissolve the precipitate with 10mL of reconstitution solution to obtain the B-type gold-labeled conjugate, which is stored at 4°C for later use.

[0149] 3. Preparation of conjugate pad

[0150] (1) Preparation of gold conjugate solution: 0.362 g of Tris, 0.05 g of sodium caseinate, 0.1 g of PEG20000, 100 μL of TW-20, 5 g of sucrose, and 100 μL of proclin300 were dissolved in 100 mL of ultrapure water, adjusted to pH 8.5 with concentrated hydrochloric acid, and stored at 4°C until use.

[0151] (2) Add the acetyl-gold conjugate to the gold conjugate solution at a concentration of 7-15%, mix well, and evenly coat it on the glass fiber membrane (35 mL / sheet), and place it in an oven at 50°C to dry for 24±2 hours.

[0152] 4. Preparation of sample pad

[0153] (1) Preparation of sample pad treatment solution: 0.242 g of Tris, 0.85 g of NaCl, 0.5 g of sodium caseinate, 2 g of sucrose, and 100 μL of proclin 300 were dissolved in 100 mL of ultrapure water, the pH was adjusted to 8.0, and the solution was stored at 4°C until use.

[0154] (2) Apply 32 mL of sample pad treatment solution evenly on the glass fiber membrane, place in a 50°C oven and dry for 24±2 hours. After passing the test, seal and store at room temperature for later use.

[0155] 5. Assembly of colloidal gold test paper

[0156] Following the colloidal gold test paper structure, attach the sample pad and conjugate pad to the end of the PVC board closest to the test line, with the sample pad partially overlapping the conjugate pad and the conjugate pad partially overlapping the test pad. Attach absorbent paper to the end of the PVC board away from the test line, with the absorbent paper partially overlapping the test pad. Once assembled into a large board, cut it into thin strips with a width of 3 mm.

[0157] Example 5

[0158] This example is used to illustrate the detection sensitivity of the antibody pair for detecting CTNT provided in this application.

[0159] Experimental group: colloidal gold test paper provided in Example 4;

[0160] Control group: mainstream CTNT detection reagent (colloidal gold method) on the market.

[0161] Detection method: Recombinant CTNT protein (Xiamen Tongren Heart, CTNT antigen, ZKP12) was used as the detection object, and diluent (ingredients: 10mM PBS, 0.1% Triaton X-100) was added to the sample pad of the colloidal gold test paper of the experimental group and the control group in the order of 900pg / mL, 300pg / mL, and 100pg / mL, respectively, and the results were observed for 15-20 minutes. The color depth was quantified by the company's internal colorimetric card. The color development results are as follows Figure 1 shown.

[0162] Depend on Figure 1 The test results show that the colloidal gold test paper provided in Example 4 of the present application has better detection sensitivity than the control group reagent for detecting CTNT at different recombinant CTNT protein concentrations.

[0163] Example 6

[0164] This example is used to illustrate the detection sensitivity of the antibody pair for detecting CTNT provided in this application.

[0165] Experimental group: colloidal gold test paper provided in Example 4;

[0166] Control group: mainstream CTNT detection reagent (colloidal gold method) on the market.

[0167] Detection method: Clinical serum with high CTNT values ​​was used as the test object, and negative serum was diluted to 900pg / mL, 300pg / mL, and 100pg / mL in sequence, and then dripped onto the sample pad of the colloidal gold test paper in the experimental group and the control group respectively. The results were observed after 15-20 minutes. The color depth was quantified using the company's internal colorimetric card. The color development results are as follows Figure 1 shown.

[0168] Depend on Figure 2 The test results show that the colloidal gold test paper provided in Example 4 of the present application has better detection sensitivity than the control group reagent under different CTNT clinical serum.

[0169] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present application, rather than to limit them. Although the present application has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present application.

Claims

1. An anti-CTNT antibody pair, characterized in that: The antibody pair comprises two monoclonal antibodies; the monoclonal antibodies comprise a heavy chain variable region and a light chain variable region; the heavy chain variable region comprises a heavy chain CDR1, a heavy chain CDR2 and a heavy chain CDR3; the light chain variable region comprises a light chain CDR1, a light chain CDR2 and a light chain CDR3; One of the monoclonal antibodies in the antibody pair is named 8E8 antibody, and the sequence information of the 8E8 antibody is as follows: The amino acid sequence of the heavy chain CDR1 is shown in SEQ ID NO: 1; The amino acid sequence of the heavy chain CDR2 is shown in SEQ ID NO: 2; The amino acid sequence of the heavy chain CDR3 is shown in SEQ ID NO: 3; The amino acid sequence of the light chain CDR1 is shown in SEQ ID NO: 4; The amino acid sequence of the light chain CDR2 is shown in SEQ ID NO: 5; The amino acid sequence of the light chain CDR3 is shown in SEQ ID NO: 6; The other monoclonal antibody in the antibody pair is named 15F11 antibody, and the sequence information of the 15F11 antibody is as follows: The amino acid sequence of the heavy chain CDR1 is shown in SEQ ID NO: 7; The amino acid sequence of the heavy chain CDR2 is shown in SEQ ID NO: 8; The amino acid sequence of the heavy chain CDR3 is shown in SEQ ID NO: 9; The amino acid sequence of the light chain CDR1 is shown in SEQ ID NO: 10; The amino acid sequence of the light chain CDR2 is shown in SEQ ID NO: 11; The amino acid sequence of the light chain CDR3 is shown in SEQ ID NO:

12.

2. The anti-CTNT antibody pair according to claim 1, characterized in that The heavy chain sequence of the 8E8 antibody includes the amino acid sequence shown in SEQ ID NO: 13; the light chain sequence of the 8E8 antibody includes the amino acid sequence shown in SEQ ID NO: 15; the heavy chain sequence of the 15F11 antibody includes the amino acid sequence shown in SEQ ID NO: 17; and the light chain sequence of the 15F11 antibody includes the amino acid sequence shown in SEQ ID NO:

19.

3. A nucleic acid molecule, characterized in that The nucleic acid molecule encodes the amino acid sequence of the monoclonal antibody according to claim 1 or 2.

4. The nucleic acid molecule according to claim 3, characterized in that The nucleic acid molecule encoding the 8E8 antibody heavy chain sequence includes the nucleotide sequence shown in SEQ ID NO: 14; the nucleic acid molecule encoding the 8E8 antibody light chain sequence includes the nucleotide sequence shown in SEQ ID NO: 16; the nucleic acid molecule encoding the 15F11 antibody heavy chain sequence includes the nucleotide sequence shown in SEQ ID NO: 18; the nucleic acid molecule encoding the 15F11 antibody light chain sequence includes the nucleotide sequence shown in SEQ ID NO:

20.

5. A kit for detecting CTNT, characterized in that: The kit comprises the anti-CTNT antibody pair according to claim 1 or 2.

6. The kit according to claim 5, characterized in that The 15F11 antibody was used as a capture antibody, and the 8E8 antibody was used as a labeling antibody.

7. Use of the antibody pair according to claim 1 or 2, the nucleic acid molecule according to claim 3 or 4, or the kit according to claim 5 or 6 in preparing a reagent for detecting CTNT.

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