Anti-CTNT antibody pair and application thereof

Abstract

The invention relates to the technical field of biomedicine, in particular to an anti-CTNT antibody pair and application thereof. The antibody pair comprises two monoclonal antibodies; the monoclonal antibody comprises a heavy chain variable region and a light chain variable region; one monoclonal antibody in the monoclonal antibody pair is named as a 15F11 antibody, and the other monoclonal antibody is named as a 8E8 antibody. The invention also discloses a nucleic acid molecule for coding the monoclonal antibody and a hybridoma cell strain for secreting the monoclonal antibody. In addition, the invention also discloses a kit for detecting CTNT, which comprises the monoclonal antibody pair, the 15F11 antibody is used as a capture antibody, and the 8E8 antibody is used as a labeled antibody. The antibody pair for resisting the CTNT, provided by the invention, has relatively good specificity on the 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 regulatory contraction protein present in cardiac striated muscle. The CTN complex is composed of three subunits: CTN I, CTNT, and CTN C. Each CTN subunit plays an independent functional role, and different fragments may have different biological activities. Only CTNT and CTNl are myocardial specific subtypes, most of which are located on thin filaments with contractile function, and a small part (3-6%) is located in the cytoplasm of myocytes in the form of free protein. Among them, CTNT is a 37000D myocardial structural protein, which has three isomers, namely fast skeletal muscle subtype, slow skeletal muscle and myocardial subtype. Their expression forms in skeletal muscle and myocardium are regulated by different genes and encoded by different genes. CTNT has high organ specificity, and its increased blood concentration often indicates myocardial cell necrosis.

[0003] CTNT has three advantages as a diagnostic indicator: 1. High specificity: Troponin is basically only expressed in the myocardium, and an increase in it indicates myocardial damage, which is more myocardial specific than CK-MB. 2. Smaller molecular weight and longer duration: When the myocardium is damaged, it is easier to diffuse out of the myocardial cells and can be detected earlier. At the same time, the advantage of long duration allows more recent myocardial infarctions to be not missed. 3. High correlation between concentration and degree of damage: Different concentrations reflect different degrees of myocardial necrosis, and its dynamic detection is very helpful in judging the prognosis.

[0004] The sensitivity of troponin testing is closely related to the effectiveness of treatment. Low-sensitivity troponin testing methods usually only detect higher levels of troponin, so they may not be able to detect smaller heart damage in the early stages. High-sensitivity CTNT testing methods can provide diagnostic information at an earlier stage of heart damage so that the medical team can take treatment measures faster 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] The present application screened two monoclonal antibodies through hybridoma technology, named 8E8 antibody and 15F11 antibody respectively. 8E8 was used as the labeled antibody and 15F11 was used as the coating antibody to prepare colloidal gold chromatography paper strips, which had higher sensitivity 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, using the following technical solution:

[0010] An anti-CTNT antibody pair, the 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 monoclonal antibody 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 as shown in SEQ ID NO: 1, or an amino acid sequence having 1 or 2 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 as shown in SEQ ID NO: 2, or an amino acid sequence having 1 or 2 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 as shown in SEQ ID NO: 3, or an amino acid sequence having 1 or 2 conservative amino acid substitutions compared to the sequence as 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 as shown in SEQ ID NO: 4, or an amino acid sequence having 1 or 2 conservative amino acid substitutions compared to the sequence as 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 as shown in SEQ ID NO: 5, or an amino acid sequence having 1 or 2 conservative amino acid substitutions compared to the sequence as 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 as shown in SEQ ID NO: 6, or an amino acid sequence having 1 or 2 conservative amino acid substitutions compared to the sequence as 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 as shown in SEQ ID NO: 7, or an amino acid sequence having 1 or 2 conservative amino acid substitutions compared to the sequence as 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 as shown in SEQ ID NO: 8, or an amino acid sequence having 1 or 2 conservative amino acid substitutions compared to the sequence as 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 as shown in SEQ ID NO: 9, or an amino acid sequence having 1 or 2 conservative amino acid substitutions compared to the sequence as 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 as shown in SEQ ID NO: 10, or an amino acid sequence having 1 or 2 conservative amino acid substitutions compared to the sequence as shown in SEQ ID NO: 10, or an amino acid sequence comprising the above sequence;

[0023] The light chain CDR2 comprises the amino acid sequence as shown in SEQ ID NO: 11, or an amino acid sequence having 1 or 2 conservative amino acid substitutions compared to the sequence shown in SEQ ID NO: 11, or an amino acid sequence comprising the above sequence;

[0024] The light chain CDR3 comprises the amino acid sequence shown in SEQ ID NO: 12, or an amino acid sequence having 1 or 2 conservative amino acid substitutions compared with 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, using the following technical solution:

[0030] A nucleic acid molecule, which encodes 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, which adopts the following technical solution:

[0034] A hybridoma cell strain secretes the above 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, which are named 15F11 antibody and 8E8 antibody, respectively. The 15F11 antibody is used as a capture antibody, and the 8E8 antibody is used as a labeling antibody, 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 terms used herein are only used for the purpose of describing specific embodiments. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by ordinary technicians in the field to which the terms belong.

[0044] In order to make the purpose, technical scheme and advantages of the present application clearer, the technical scheme in the embodiments of the present application will be clearly and completely described below in conjunction with the accompanying drawings. Based on the embodiments of the present application, all other embodiments obtained by those skilled in the art without making creative work belong to the scope of protection of the present application. The embodiments described below are exemplary and are only used to explain the present application, and cannot be understood as limiting the present application.

[0045] If no specific techniques or conditions are specified in the examples, the techniques or conditions described in the literature in the field or the product instructions are used. If no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be purchased commercially.

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

[0047] Example

[0048] Example 1

[0049] This example provides a preparation process and screening process of 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 BALB / C female mice. The immunogen was CTNT expressed by E. coli, with 498 amino acids and a molecular weight of 55 kDa, and the sequence was the amino acid sequence shown in SEQ ID NO: 21.

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

[0054] (1) Preparation of immunogens: CTNT and Freund's complete adjuvant (Cat. No. F5881, Sigma) were mixed in equal volumes to prepare the first immunogen; CTNT and Freund's incomplete adjuvant (Cat. No. F5506, Sigma) were mixed in equal volumes to prepare the second immunogen; CTNT and physiological saline were mixed 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-shock 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 process of cell fusion 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 removed and blood was collected, and the serum of the BALB / c female mice was separated 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 myeloma cells are in the logarithmic growth phase when used) to prepare myeloma cell suspension;

[0066] Preparation of feeder layer cells: One day before cell fusion, blank BALB / c female mouse peritoneal macrophages and spleen cells were taken and added to 96-well plates for culture to obtain a cell plate 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 taken and 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 min and the supernatant was removed.

[0069] The bottom of the centrifuge tube was flicked with a finger to loosen and mix the two cells, and the tube was placed in a beaker filled with 37°C water to keep warm. Within 1 min, 1 mL of 50% PEG1500 (pH 8.0, product number 10783641001, Roche) was added to fuse the cells, and the mixture was shaken while adding. After the addition, the mixture was allowed to stand for 30 s. Serum-free 1640 medium (product number C3010-0500, VivaCell) was added to terminate the fusion, and the tube was centrifuged at 800 rpm for 5 min. The precipitate was suspended in HAT medium and dispensed into a cell plate containing feeder cells to obtain a cell plate containing fused cells and feeder cells, and the plate was placed at 37°C and 5% CO. 2 Cultured in a cell culture incubator;

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

[0071] 3. Screening of positive hybridoma cells

[0072] The cell plate containing the fused cells and feeder layer cells was cultured until the 4th day for half medium replacement, and continued to be cultured until the 7th day for full medium replacement. When the fused cells covered 10-50% of the bottom of the well, the positive wells were screened by 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 0.05 mol / L CB buffer (31.8 g Na 2 CO 3 , 58.8 g of NaHCO 3, and make up to 2 L with ultrapure water), diluted to 2 μg / mL, added to the ELISA plate at 100 μL / well, coated overnight at 4°C and patted dry, blocked with 1% gelatin-PBS buffer at a mass volume ratio of 300 μL / well, blocked at 37°C for 2 h, 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 sampling 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] The positive hybridoma cells screened from the cell plate containing fused cells-feeder layer cells are derived from more than two hybridoma cells, so the antibodies secreted by the screened hybridoma cells are of different nature. 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 in step (1) of "Cell Fusion" to obtain a cell plate containing feeder cells; the positive hybridoma cells obtained by screening were suspended in HT medium and mixed by pipetting, and then inoculated into the cell plate containing feeder cells, and the cells in the wells of the cell plate were diluted to 1 cell per well using HT medium, and placed at 37°C, 5% CO 2 After 7 to 10 days of wet culture, when cloned cells visible to the naked eye appear, antibodies can be detected.

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

[0081] Observe under an inverted microscope, mark the wells where only a single clone grows and is positive in ELISA test, expand the strain, and finally obtain 10 cell strains through preliminary screening, and take the supernatant for the next functional screening. The above 10 hybridoma cell strains are 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 an ascites-specific adjuvant (Beijing Biolon, Catalog No. KX0210048). Ten days after injection, the cell line prepared in Example 1 (1×10 6 The cells / mouse were injected into the peritoneal cavity of Balb / c mice, and the ascites of the Balb / c mice was collected with a medical syringe 12 days later.

[0089] (2) Antibody purification: The Balb / c mouse ascites 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, filtered through a 0.22 μm filter membrane, and purified by affinity chromatography using a Protein A column (Cat. No. AA301307, Boglon Biotech).

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

[0091] Column loading: Add 5 mL of Protein AResin medium to the chromatography column and let it stand, then rinse the column with 10 column volumes of ultrapure water;

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

[0093] Loading: Load the sample after filtering with a 0.22 μm filter membrane 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 elution buffer (100 mM Glycine, 150 mM NaCl, solvent is water, pH = 3.0) to obtain an elution buffer containing the antibody; immediately after elution, add a neutralization buffer (2 M Tris-HCl, solvent is water, pH = 9.0) to the elution buffer until the solution has a neutral pH;

[0096] Dialysis: The eluted antibodies were dialyzed three times in 1000 times the elution volume of PBS (pH=7.4) solution 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 with 0.05 mol / L CB buffer at pH = 9.6; select the dialysis membrane and cut it to an appropriate length according to the molecular weight of the target protein and the actual retention requirements and dialysis volume; soak the dialysis membrane in 0.05 mol / L CB buffer at pH = 9.6 in advance, and replace the 0.05 mol / L CB buffer at pH = 9.6 to 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 solution every 1 hour, for a total of 5 times.

[0099] (2) HRP (Cat. No. RS20220118, Reiss Reagent) was dissolved in ultrapure water to prepare a 20 mg / mL HRP solution. 4 Dissolve in ultrapure water to make a concentration of 20 mg / mL NaIO 4 Solution; after vortexing to fully dissolve, mix HRP solution and NaIO 4 The solutions were mixed in a 1:1 volume ratio, i.e., NaIO 4 The solution was slowly added to the HRP solution, and the centrifuge tube was immediately wrapped with tin foil and activated at 4°C for 30 min in the dark.

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

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

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

[0103] (5) Prepare NaBH with a concentration of 20 mg / mL using pure water 4 The solution was added to the antibody-HRP conjugate solution in step (4) at a rate of 2 μL of NaBH for every 1 mg of HRP. 4 The solution was reacted at 4°C for 2 h, and mixed by inversion every 0.5 h to obtain a coupled complex 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 min, centrifuged at 10000 rpm for 10 min, and the supernatant was removed to obtain 10 strains of HRP-antibody coupled complex proteins, respectively. The 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 strains of CTNT antibodies are used as capture antibodies and coated on the ELISA plate respectively, and then CTNT (the same immunogen as in Example 1) is added to the wells of the ELISA plate, and the unbound CTNT is washed away after incubation; HRP-CTNT antibody-coupled complex protein is added as a labeled antibody, and the unbound HRP-CTNT antibody-coupled complex protein is washed away after incubation, and finally a color developing solution is added for color development, and the absorbance is measured at 450nm using a spectrophotometer. The test results are shown in Table 2.

[0108] Test result judgment: If color can be developed, it means 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 pair of paired antibodies. If color cannot be developed, it means 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 above test results show that the 15F11 antibody and the 8E8 antibody have high titers and can be used in the detection of CTNT. Among them, the 15F11 antibody is a capture antibody and the 8E8 antibody is a labeling antibody. The hybridoma cell producing the 15F11 antibody is named as the 15F11 cell line, and the hybridoma cell producing the 8E8 antibody is named as the 8E8 cell line.

[0112] Example 3

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

[0114] The 10G10 cell line and the 11F8 cell line were expanded and cultured separately, and 5×10 6 The cells / ml 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] (I) 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 heavy chain CDR1, heavy chain CDR2 and 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 shown in SEQ ID NO: 4. The light chain CDR2 includes the amino acid sequence shown in SEQ ID NO: 5. The light chain CDR3 includes the amino acid sequence shown in SEQ ID NO: 6.

[0125] (II) 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 heavy chain CDR1, heavy chain CDR2 and heavy chain CDR3. The heavy chain CDR1 includes the amino acid sequence shown in SEQ ID NO: 7. The heavy chain CDR2 includes the amino acid sequence shown in SEQ ID NO: 8. The heavy chain CDR3 includes the amino acid sequence shown 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 shown in SEQ ID NO: 10. The light chain CDR2 includes the amino acid sequence shown in SEQ ID NO: 11. The light chain CDR3 includes the amino acid sequence shown 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 Na 2 HPO 4 12H 2 O, 0.2914 g of NaH 2 PO 4 ·2H 2O, 8.5 g of NaCl and 25 g of trehalose were dissolved in 1000 mL of ultrapure water, the pH was adjusted to 7.4, and stored at 4°C for later 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 the 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 with 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] 0.2M K 2 CO 3 Preparation of 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 future use.

[0146] Preparation of the complex 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 for later use.

[0147] (2) Marking process:

[0148] Take 100 mL of 0.04% colloidal gold solution (Cat. No. C805628, McLean) and place it in a clean container. Add 500-2000 μL of 0.2 M K 2 CO 3The solution was stirred evenly; 500-1500 μg of 8E8 antibody was added, and the reaction was stirred for 10-20 min; then 500 μL of 20% BSA solution was added and stirred, and the mixture was blocked for 5-10 min. After centrifugation at 8000-10000 r / min for 30 min, the supernatant was discarded, and the precipitate was re-dissolved with 10 mL of reconstitution solution to obtain the β-gold-labeled conjugate, which was stored at 4°C for later use.

[0149] 3. Preparation of conjugate pad

[0150] (1) Preparation of gold-labeled 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 for later use.

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

[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 stored at 4°C for later use.

[0154] (2) 32 mL of sample pad treatment solution is evenly coated on the glass fiber membrane, and the membrane is placed in an oven at 50°C for drying for 24±2 hours. After passing the test, the membrane is sealed and stored at room temperature for future use.

[0155] 5. Assembly of colloidal gold test paper

[0156] According to the structure of colloidal gold test paper, the sample pad and the conjugate pad are sequentially pasted on the end of the PVC board close to the test line, and the sample pad is partially overlapped on the conjugate pad, and the conjugate pad is partially overlapped on the test pad; the absorbent paper is pasted on the end of the PVC board away from the test line, and the absorbent paper is partially overlapped on the test pad. After forming a large board, cut it into thin strips with a width of 3mm.

[0157] Example 5

[0158] This example is used to illustrate the detection sensitivity of the antibody pair for detecting CTNT provided in the present 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 Tongrenxin, CTNT antigen, ZKP12) was used as the detection object, and the diluent (ingredients: 10mM PBS, 0.1% Triaton X-100) was added to the sample pad of the colloidal gold test paper of the above experimental group and control group in sequence to 900pg / mL, 300pg / mL, and 100pg / mL, respectively, and the results were observed for 15 to 20 minutes. The color depth was quantified by the company's internal colorimetric card. The color development results are shown in Figure 1 shown.

[0162] Depend on Figure 1 It can be seen from the test results that the colloidal gold test paper provided in Example 4 of the present application has better detection sensitivity for the antibody pair for detecting CTNT provided in the present application at different recombinant CTNT protein concentrations compared with the control group reagent.

[0163] Example 6

[0164] This example is used to illustrate the detection sensitivity of the antibody pair for detecting CTNT provided in the present 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 test value was used as the test object, and negative serum was diluted to 900pg / mL, 300pg / mL, and 100pg / mL in sequence, and added to the sample pad of the colloidal gold test paper of the above experimental group and control group 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.

[0168] Depend on Figure 2 It can be seen from the test results 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 it. 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 monoclonal antibody in the antibody pair is named 8E8 antibody, and the sequence information of the 8E8 antibody is as follows: The heavy chain CDR1 comprises the amino acid sequence as shown in SEQ ID NO: 1, or an amino acid sequence having 1 or 2 conservative amino acid substitutions compared to the sequence shown in SEQ ID NO: 1, or an amino acid sequence comprising the above sequence; The heavy chain CDR2 comprises the amino acid sequence as shown in SEQ ID NO: 2, or an amino acid sequence having 1 or 2 conservative amino acid substitutions compared to the sequence shown in SEQ ID NO: 2, or an amino acid sequence comprising the above sequence; The heavy chain CDR3 comprises the amino acid sequence as shown in SEQ ID NO: 3, or an amino acid sequence having 1 or 2 conservative amino acid substitutions compared to the sequence as shown in SEQ ID NO: 3, or an amino acid sequence comprising the above sequence; The light chain CDR1 comprises the amino acid sequence as shown in SEQ ID NO: 4, or an amino acid sequence having 1 or 2 conservative amino acid substitutions compared to the sequence as shown in SEQ ID NO: 4, or an amino acid sequence comprising the above sequence; The light chain CDR2 comprises the amino acid sequence as shown in SEQ ID NO: 5, or an amino acid sequence having 1 or 2 conservative amino acid substitutions compared to the sequence as shown in SEQ ID NO: 5, or an amino acid sequence comprising the above sequence; The light chain CDR3 comprises the amino acid sequence as shown in SEQ ID NO: 6, or an amino acid sequence having 1 or 2 conservative amino acid substitutions compared to the sequence as shown in SEQ ID NO: 6, or an amino acid sequence comprising the above sequence; The other monoclonal antibody in the antibody pair is named 15F11 antibody, and the sequence information of the 15F11 antibody is as follows: The heavy chain CDR1 comprises the amino acid sequence as shown in SEQ ID NO: 7, or an amino acid sequence having 1 or 2 conservative amino acid substitutions compared to the sequence as shown in SEQ ID NO: 7, or an amino acid sequence comprising the above sequence; The heavy chain CDR2 comprises the amino acid sequence as shown in SEQ ID NO: 8, or an amino acid sequence having 1 or 2 conservative amino acid substitutions compared to the sequence as shown in SEQ ID NO: 8, or an amino acid sequence comprising the above sequence; The heavy chain CDR3 comprises the amino acid sequence as shown in SEQ ID NO: 9, or an amino acid sequence having 1 or 2 conservative amino acid substitutions compared to the sequence as shown in SEQ ID NO: 9, or an amino acid sequence comprising the above sequence; The light chain CDR1 comprises the amino acid sequence as shown in SEQ ID NO: 10, or an amino acid sequence having 1 or 2 conservative amino acid substitutions compared to the sequence as shown in SEQ ID NO: 10, or an amino acid sequence comprising the above sequence; The light chain CDR2 comprises the amino acid sequence as shown in SEQ ID NO: 11, or an amino acid sequence having 1 or 2 conservative amino acid substitutions compared to the sequence shown in SEQ ID NO: 11, or an amino acid sequence comprising the above sequence; The light chain CDR3 comprises the amino acid sequence shown in SEQ ID NO: 12, or an amino acid sequence having 1 or 2 conservative amino acid substitutions compared with the sequence shown in SEQ ID NO: 12, or an amino acid sequence comprising the above sequence.

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; 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 hybridoma cell line, characterized in that: The hybridoma cell line secretes the monoclonal antibody according to claim 1 or 2.

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

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

8. 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 6 or 7 in preparing a reagent for detecting CTNT.

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