Monoclonal antibody pair for resisting treponema pallidum TP protein and application thereof

Abstract

The invention relates to the technical field of biomedicine, in particular to a monoclonal antibody pair for resisting treponema pallidum TP protein and application of the monoclonal antibody pair. 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 5C12 antibody, and the other monoclonal antibody is named as a 1E8 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 treponema pallidum TP protein, which comprises the monoclonal antibody pair, the 5C12 antibody is used as a labeled antibody, and the 1E8 antibody is used as a capture antibody. The anti-treponema pallidum TP protein monoclonal antibody pair provided by the invention can realize high-specificity and high-sensitivity detection on the syphilis.

Description

Technical Field

[0001] This application relates to the field of biomedical technology, and in particular to a monoclonal antibody pair against Treponema pallidum TP protein and its application. Background Technology

[0002] Syphilis, commonly known as syphilis or venereal disease, is caused by Treponema pallidum (the bacterium that causes syphilis). Treponema pallidum Phlebitis (TP) is a chronic infectious disease caused by sexual intercourse. It is mainly transmitted through sexual contact, and in very rare cases, through kissing, breastfeeding, or contact with the daily necessities of patients with infectious lesions. It can also be transmitted from mother to fetus and remains a significant global public health issue.

[0003] Currently, the diagnosis of syphilis mainly relies on serological testing, with antibody detection against the TP antigen being the core step. TP proteins are the main immunogenic proteins on the surface of the Treponema pallidum membrane, and their family includes members such as TpN15, TpN17, TpN44.5 (TmpA), and TpN47. These proteins can elicit a strong humoral immune response in the host during infection, and are therefore crucial target antigens in syphilis diagnostic kits.

[0004] Third-generation detection technologies based on recombinant TP protein, such as enzyme-linked immunosorbent assay (ELISA) and chemiluminescent immunoassay, have gradually become mainstream methods for screening and diagnosis due to their high degree of automation and objectivity. However, the performance of these detection methods is highly dependent on the quality of the core raw material used—anti-TP protein monoclonal antibodies—including their affinity, specificity, and the nature of their epitope recognition.

[0005] Although there are TP protein-based test kits on the market, there is still room for improvement in their sensitivity and specificity in clinical applications, especially in the diagnosis of latent syphilis, assessment of treatment efficacy, and differential diagnosis from other spirochetal infections. Summary of the Invention

[0006] This application provides a monoclonal antibody pair against Treponema pallidum TP protein and its application. The monoclonal antibody pair against Treponema pallidum TP protein provided in this application can achieve high specificity and high sensitivity detection of syphilis.

[0007] In a first aspect, this application provides a monoclonal antibody pair against Treponema pallidum TP protein, employing the following technical solution: A monoclonal antibody pair against Treponema pallidum TP protein, the antibody pair comprising two monoclonal antibodies; each monoclonal antibody comprising a heavy chain variable region and a light chain variable region; the heavy chain variable region comprising heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3; the light chain variable region comprising light chain CDR1, light chain CDR2, and light chain CDR3; One of the monoclonal antibodies in the antibody pair is named the 5C12 antibody, and the sequence information of the 5C12 antibody is as follows: The heavy chain CDR1 comprises an amino acid sequence as shown in SEQ ID NO: 1, or an amino acid sequence having one or two conserved amino acid substitutions compared to the sequence shown in SEQ ID NO: 1, or an amino acid sequence containing the above sequence. The heavy chain CDR2 comprises an amino acid sequence as shown in SEQ ID NO: 2, or an amino acid sequence having one or two conserved amino acid substitutions compared to the sequence shown in SEQ ID NO: 2, or an amino acid sequence containing the above sequence. The heavy chain CDR3 comprises an amino acid sequence as shown in SEQ ID NO: 3, or an amino acid sequence having one or two conserved amino acid substitutions compared to the sequence shown in SEQ ID NO: 3, or an amino acid sequence containing the above sequence. The light chain CDR1 comprises an amino acid sequence as shown in SEQ ID NO: 4, or an amino acid sequence having one or two conserved amino acid substitutions compared to the sequence shown in SEQ ID NO: 4, or an amino acid sequence containing the above sequence. The light chain CDR2 comprises an amino acid sequence as shown in SEQ ID NO: 5, or an amino acid sequence having one or two conserved amino acid substitutions compared to the sequence shown in SEQ ID NO: 5, or an amino acid sequence containing the above sequence. The light chain CDR3 comprises an amino acid sequence as shown in SEQ ID NO: 6, or an amino acid sequence having one or two conserved amino acid substitutions compared to the sequence shown in SEQ ID NO: 6, or an amino acid sequence containing the above sequence. The other monoclonal antibody in the antibody pair is named 1E8 antibody, and the sequence information of 1E8 antibody is as follows: The heavy chain CDR1 comprises an amino acid sequence as shown in SEQ ID NO: 7, or an amino acid sequence having one or two conserved amino acid substitutions compared to the sequence shown in SEQ ID NO: 7, or an amino acid sequence containing the above sequence. The heavy chain CDR2 comprises an amino acid sequence as shown in SEQ ID NO: 8, or an amino acid sequence having one or two conserved amino acid substitutions compared to the sequence shown in SEQ ID NO: 8, or an amino acid sequence containing the above sequence. The heavy chain CDR3 comprises an amino acid sequence as shown in SEQ ID NO: 9, or an amino acid sequence having one or two conserved amino acid substitutions compared to the sequence shown in SEQ ID NO: 9, or an amino acid sequence containing the above sequence. The light chain CDR1 comprises an amino acid sequence as shown in SEQ ID NO: 10, or an amino acid sequence having one or two conserved amino acid substitutions compared to the sequence shown in SEQ ID NO: 10, or an amino acid sequence containing the above sequence. The light chain CDR2 comprises an amino acid sequence as shown in SEQ ID NO: 11, or an amino acid sequence having one or two conserved amino acid substitutions compared to the sequence shown in SEQ ID NO: 11, or an amino acid sequence containing the above sequence. The light chain CDR3 comprises an amino acid sequence as shown in SEQ ID NO: 12, or an amino acid sequence having one or two conserved amino acid substitutions compared to the sequence shown in SEQ ID NO: 12, or an amino acid sequence containing the above sequence.

[0008] The heavy chain sequence of the 5C12 antibody includes the amino acid sequence shown in SEQ ID NO: 13.

[0009] The light chain sequence of the 5C12 antibody includes the amino acid sequence shown in SEQ ID NO: 15.

[0010] The heavy chain sequence of the 1E8 antibody includes the amino acid sequence shown in SEQ ID NO: 17.

[0011] The light chain sequence of the 1E8 antibody includes the amino acid sequence shown in SEQ ID NO: 19.

[0012] Secondly, this application provides a nucleic acid molecule, employing the following technical solution: A nucleic acid molecule that encodes the amino acid sequence of the aforementioned monoclonal antibody.

[0013] The nucleic acid molecule encoding the heavy chain sequence of the 5C12 antibody includes the nucleotide sequence shown in SEQ ID NO: 14.

[0014] The nucleic acid molecule encoding the light chain sequence of the 5C12 antibody includes the nucleotide sequence shown in SEQ ID NO: 16.

[0015] The nucleic acid molecule encoding the 1E8 antibody heavy chain sequence includes the nucleotide sequence shown in SEQ ID NO: 18.

[0016] The nucleic acid molecule encoding the light chain sequence of the 1E8 antibody includes the nucleotide sequence shown in SEQ ID NO: 20.

[0017] Thirdly, this application provides a hybridoma cell line, which adopts the following technical solution: A hybridoma cell line that secretes the aforementioned monoclonal antibody.

[0018] Fourthly, this application provides a kit for detecting Treponema pallidum TP protein, employing the following technical solution: A kit for detecting Treponema pallidum TP protein, the kit comprising the above-mentioned monoclonal antibody pair against Treponema pallidum TP protein.

[0019] Optionally, the 5C12 antibody is used as a labeling antibody, and the 1E8 antibody is used as a capture antibody.

[0020] Fifthly, this application provides the application of the above-mentioned antibody pairs, nucleic acid molecules, or kits in the detection of Treponema pallidum TP protein.

[0021] In summary, this application has the following beneficial effects: This application provides a pair of monoclonal antibodies against Treponema pallidum TP protein, named 5C12 antibody and 1E8 antibody, respectively. The 5C12 antibody serves as a labeling antibody, and the 1E8 antibody serves as a capture antibody. The monoclonal antibody pair against Treponema pallidum TP protein provided in this application can achieve high specificity and high sensitivity for the detection of syphilis. Attached Figure Description

[0022] Figure 1 For colorimetric cards.

[0023] Figure 2 This is the result of antibody sensitivity testing. Detailed Implementation

[0024] Before describing the embodiments of this application in detail, it should be understood that the terminology used herein is for the purpose of describing a particular embodiment only. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the term pertains.

[0025] To make the objectives, technical solutions, and advantages of this application clearer, the technical solutions in the embodiments of this application will be clearly and completely described below with reference to the accompanying drawings. All other embodiments obtained by those skilled in the art based on the embodiments of this application without inventive effort are 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 construed as limiting this application.

[0026] Where specific techniques or conditions are not specified in the examples, they shall be performed in accordance with the techniques or conditions described in the literature in this field or in accordance with the product instructions. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products.

[0027] The present application will be further described in detail below with reference to the embodiments and test results.

[0028] Example 1

[0029] This embodiment provides the preparation and screening process of hybridoma cell lines. After screening, a total of 10 hybridoma cell lines were obtained in this embodiment.

[0030] The above process specifically includes the following steps: I. Animal Immunization The subjects were 8-week-old female BALB / c mice. The immunogen was a recombinant syphilis TP protein expressed in Escherichia coli, with the amino acid sequence shown in SEQ ID NO: 21.

[0031] The immunization process specifically includes the following steps: (1) Preparation of immunogen: The first immunogen was prepared by mixing the recombinant syphilis TP protein expressed by Escherichia coli with Freund's complete adjuvant (product number F5881, Sigma-Aldrich); the second immunogen was prepared by mixing the recombinant syphilis TP protein expressed by Escherichia coli with Freund's incomplete adjuvant (product number F5506, Sigma-Aldrich); the third immunogen was prepared by mixing the recombinant syphilis TP protein expressed by Escherichia coli with physiological saline.

[0032] (2) Immune process: First immunization: 200 μg of the first immunogen was injected subcutaneously at 3-4 points on the back of female BALB / c mice; Two weeks later, a second immunization was administered by injecting 100 μg of the second immunogen subcutaneously at 3-4 points on the back of female BALB / c mice. Two weeks later, a third immunization was administered by injecting 100 μg of the second immunogen subcutaneously at 3-4 points on the back of female BALB / c mice. Two weeks later, a fourth immunization-shock immunization was performed, with 100 μg of the third immunogen injected intraperitoneally into female BALB / c mice; The fifth immunization was administered 24 hours later, with 50 μg of the third immunogen injected into the tail vein of BALB / C female mice.

[0033] II. Cell Fusion Cell fusion began in BALB / C female mice on the third day after their fifth immunization.

[0034] The cell fusion process specifically includes the following steps: (1) Preparation of spleen cell suspension: On the third day after the fifth immunization of BALB / c female mice, blood was collected by removing the eyeballs of BALB / c female mice, and the serum of BALB / c female mice was separated as a positive control for antibody detection; at the same time, BALB / c female mice were killed by cervical dislocation, and their spleens were taken to prepare spleen cell suspension. Preparation of myeloma cell suspension: Myeloma cells (ATCC, catalog number BNCC100908) were revived two weeks in advance (to ensure that the myeloma cells are in the logarithmic growth phase when used) to obtain myeloma cell suspension. Preparation of feeder cells: One day before cell fusion, peritoneal macrophages and spleen cells from blank BALB / c female mice were added to 96-well plates for culture to obtain cell plates containing feeder cells (cell concentration of 1×10⁻⁶). 4 / hole), to obtain feeder layer cells.

[0035] (2) Cell fusion process: Polyethylene glycol (PEG) was used to mediate cell fusion. Spleen cell suspension and myeloma cell suspension were taken and mixed in serum-free 1640 medium (Catalog No. C3010-0500, VivaCell) at a cell ratio of 5:1. The mixture was centrifuged at 1200 rpm for 5 min and the supernatant was removed. Gently tap the bottom of the centrifuge tube with your finger to loosely mix the two types of cells. Place the tube in a beaker containing 37°C water and incubate. Add 1 mL of 50% PEG1500 (pH 8.0, catalog number 10783641001, Roche) fusion cells over 1 minute while shaking. After adding, let it stand for 30 seconds. Add serum-free 1640 medium (catalog number C3010-0500, VivaCell) to terminate the fusion. Centrifuge at 800 rpm for 5 minutes. Resuspend the pellet in HAT medium and aliquot it into cell plates containing feeder cells to obtain cell plates containing fusion cells and feeder cells. Incubate the plates in a 37°C, 5% CO2 cell culture incubator. The following reagents are required to prepare 500 mL of HAT medium: 100 mL fetal bovine serum (product number 11011-8611, Sijiqing), 5 mL of penicillin-streptomycin antibiotics for cell culture (100×, product number E607011-0100, Shanghai Sangon Biotech), 10 mL of HAT medium additive (50×, product number H0262, Sigma-Aldrich), and 385 mL of 1640 medium (product number C3010-0500, VivaCell).

[0036] III. Screening for positive hybridoma cells The cell plates containing the fused cells-feeder cells were cultured until day 4, at which point the medium was partially changed, and then cultured until day 7, at which point the medium was completely changed. When the fused cells covered 10-50% of the bottom of the wells, positive wells were screened using the conventional indirect ELISA method.

[0037] The indirect ELISA method specifically includes the following steps: (1) Plate coating: The syphilis TP protein recombinantly expressed by Escherichia coli was used as the coating antigen. It was diluted to 2 μg / mL with 0.05 mol / L CB buffer (31.8 g Na2CO3, 58.8 g NaHCO3, and made up to 2 L with ultrapure water) at pH 9.6. 100 μL / well was added to the microplate and coated overnight at 4℃. After drying, it was blocked with 1% gelatin-PBS buffer at 300 μL / well at 37℃ for 2 h and then dried for later use.

[0038] (2) Detection: 100 μL of cell culture supernatant from cell plate containing fused cell-feeder layer cells was added to ELISA plate and incubated at 37°C for 60 min. The plate was then washed three times with 0.01 mol / L PBST buffer containing Tween-20 and patted dry. 100 μL of HRP-labeled goat anti-mouse secondary antibody (product number TJ-211229CN, Xiamen Taijing) was added to each well and incubated at 37°C for 60 min. The plate was then washed three times and patted dry. 100 μL of TMB chromogenic solution was added to each well and the plate was incubated at 37°C in the dark for 10 min. The reaction was terminated by adding 50 μL of 1 mol / L HCl to each well.

[0039] Meanwhile, BALB / c female mouse serum obtained from blood collection and separation from the eyeballs during "cell fusion" was used as a positive control to screen out fusion cells with high antibody titers, which were identified as positive hybridoma cells.

[0040] IV. Cloning of positive hybridoma cells Positive hybridoma cells obtained from cell plates containing fusion cell-feeder layer cells originate from more than two hybridoma cell lines; therefore, the antibodies secreted by the selected hybridoma cells are heterogeneous. To obtain completely homogeneous monoclonal antibodies, the positive hybridoma cells need to be cloned.

[0041] The day before cloning, feeder cells were prepared and plated according to step (1) in “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. The cells were then seeded into the cell plate containing feeder cells. The cells in the wells of the cell plate were diluted to 1 cell per well using HT medium. The cells were then placed in a humidified environment at 37°C and 5% CO2 for 7-10 days. When clonal cells were visible to the naked eye, antibodies could be detected.

[0042] The following reagents are required to prepare 500 mL of HT medium: 100 mL of fetal bovine serum (product number 11011-8611, Sijiqing), 5 mL of penicillin-streptomycin antibiotics for cell culture (100×, product number E607011-0100, Shanghai Sangon Biotech), 10 mL of HT medium additive (product number H0137, Sigma-Aldrich), and 385 mL of 1640 medium (product number C3010-0500, VivaCell).

[0043] Under an inverted microscope, wells showing only a single clone and a positive ELISA result were marked. These wells were then expanded to establish new cell lines, and 10 cell lines were obtained through preliminary screening. The supernatant was collected for further functional screening. The 10 hybridoma cell lines were named as shown in Table 1.

[0044] Table 1. Hybridoma cell lines obtained through screening and antibodies produced.

[0045] Example 2

[0046] This embodiment provides the preparation and screening process of monoclonal antibody pairs against Treponema pallidum TP protein.

[0047] The above process specifically includes the following steps; I. Preparation of monoclonal antibody pairs against Treponema pallidum TP protein (1) Preparation of ascites: Balb / c mice aged 8-12 weeks were intraperitoneally injected with a special adjuvant for ascites (product number KX0210048, Beijing Bio-Long). On the 10th day after injection, the cell line (1×10⁶ cells / mL) prepared in Example 1 was used to prepare the ascites fluid. 6 (1 cell / mouse) was injected into the peritoneal cavity of Balb / c mice, and the ascites fluid of Balb / c mice was collected with a medical syringe 12 days later.

[0048] (2) Antibody purification: The ascites fluid collected in step (1) was poured into a centrifuge tube, centrifuged at 12500 rpm for 20 min, and the supernatant was collected. The ascites supernatant was mixed with saturated ammonium sulfate solution at a volume ratio of 1:1, centrifuged at 12000 r / min for 5 min, and the supernatant was discarded. The precipitate was resuspended and dissolved in the same volume of PBS, filtered through a 0.22 μm filter membrane, and purified by affinity chromatography using a Protein A column (product number AA301307, Bogelon Biotech).

[0049] The specific steps for purification using Protein A column affinity chromatography are as follows: Column packing: Add 5 mL of Protein A Resin medium to the chromatography column and let it stand. Rinse the chromatography column with 10 column volumes of ultrapure water. Equilibration: The chromatography column was equilibrated using 10 column volumes of pre-cooled Protein A column equilibration buffer (50 mM Tris-HCl, 100 mM NaCl, water as solvent, pH=8.0). Sample loading: Load the sample after filtration through a 0.22 μm filter membrane at a flow rate of 5 mL / min; Washing: Wash the chromatography column with 10 column volumes of pre-cooled Protein A column equilibration buffer; Elution: Elute the antibody with elution buffer (100 mM Glycine, 150 mM NaCl, water as solvent, pH=3.0) to obtain an elution buffer containing the antibody; immediately after elution, add neutralization buffer (2 M Tris-HCl, water as solvent, pH=9.0) to the elution buffer until the solution is at neutral pH. Dialysis: The eluted antibodies were dialyzed three times in 1000 times the elution volume of PBS (pH=7.4) to obtain 10 purified antibodies against Treponema pallidum TP protein, and their specific names are shown in Table 1.

[0050] II. Preparation of HRP-antibody-coupled complex (1) Antibodies against 10 strains of Treponema pallidum TP protein were diluted to 2 mg / mL with 0.05 mol / L CB buffer at pH 9.6. Dialysis membranes were selected and cut to appropriate lengths according to the actual needs of the target protein molecular weight and the dialysis volume. The dialysis membranes were pre-treated by soaking in 0.05 mol / L CB buffer at pH 9.6, and then soaked and washed once more with 0.05 mol / L CB buffer at pH 9.6. 1 mL of antibody protein solution was transferred to the dialysis membrane. Dialysis was performed at 4°C with stirring in 0.05 mol / L CB buffer at pH 9.6, with the buffer changed every 1 h, for a total of 5 dialysis cycles.

[0051] (2) Dissolve HRP (product number RS20220118, Ruisi Reagent) in ultrapure water to prepare an HRP solution with a concentration of 20 mg / mL, and dissolve NaIO4 in ultrapure water to prepare a NaIO4 solution with a concentration of 20 mg / mL; after vortexing to fully dissolve, mix the HRP solution and NaIO4 solution at a volume ratio of 1:1, that is, slowly add the NaIO4 solution to the HRP solution, immediately wrap the centrifuge tube with tin foil, and activate HRP at 4℃ for 30 min in the dark.

[0052] (3) Slowly add ethylene glycol to the HRP-activated centrifuge tube while gently shaking (add 1 μL of ethylene glycol for every 1 mg of HRP), continue at 4°C in the dark for 30 min, and terminate the HRP activation.

[0053] (4) Add the HRP solution that has been deactivated to the antibody dialysis membrane (1 mg of antibody is added to 1 mg of HRP and 1 mg of NaIO4), and couple it overnight at 4°C in 0.05 mol / L CB buffer at pH 9.6 in the dark.

[0054] The next day, after changing the 0.05 mol / L CB buffer to pH 9.6, dialyzing continued for 2 hours. After dialyzing, the conjugated dialysate was transferred to a centrifuge tube to obtain the antibody-HRP conjugated solution.

[0055] (5) Prepare a NaBH4 solution with a concentration of 20 mg / mL using pure water and add it to the antibody-HRP coupling solution in step (4). The amount added is 2 μL of NaBH4 solution for every 1 mg of HRP. React at 4°C for 2 h, and mix by inverting the solution several times every 0.5 h to obtain the coupling complex protein solution.

[0056] (6) The conjugated protein was precipitated with 50% ammonium sulfate (i.e., equal volume of saturated ammonium sulfate mixed with the conjugated protein solution obtained in step (5) at a volume ratio of 1:1). The precipitation was carried out at 4°C for 15 min, centrifuged at 10000 rpm for 10 min, and the supernatant was removed. Ten strains of HRP-antibody conjugated protein were obtained, and their specific names are shown in Table 1.

[0057] III. Screening of anti-Treponema pallidum TP protein antibody pairs The 10 anti-Treponema pallidum TP protein antibodies obtained in step one and the 10 HRP-antibody conjugate proteins obtained in step two were paired up, and the antibodies were screened using the double-antibody sandwich ELIS method.

[0058] The specific steps are as follows: Ten antibodies against Treponema pallidum TP protein were used as capture antibodies and coated onto an ELISA plate. Then, recombinant syphilis TP protein (the same immunogen as in Example 1) was added to the wells of the ELISA plate. After incubation, the unbound recombinant syphilis TP protein was washed away. HRP-antibody-conjugate protein was then added as a labeling antibody. After incubation, the unbound HRP-antibody-conjugate protein was washed away. Finally, chromogenic 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.

[0059] Interpretation of test results: If color development occurs, it indicates that the labeled antibody and the capture antibody can recognize different epitopes of the recombinant syphilis TP protein, meaning that the capture antibody and the labeled antibody in this group are a paired antibody pair. If color development does not occur, it indicates that the labeled antibody cannot bind to the recombinant syphilis TP protein and is thus eluted, meaning that the capture antibody and the labeled antibody in this group are not paired antibodies.

[0060] Table 2. Test results of capture antibody and labeled antibody pairing

[0061] Table 2 shows that the test results indicate that the 1E8 and 5C12 antibodies have high titers and can be used for the detection of Treponema pallidum or its proteins. The 1E8 antibody is a capture antibody, and the 5C12 antibody is a labeling antibody. Hybridoma cells producing the 1E8 antibody are named the 1E8 cell line, and hybridoma cells producing the 5C12 antibody are named the 5C12 cell line.

[0062] Example 3

[0063] In this embodiment, the antibodies obtained through the above screening were sequenced.

[0064] The 5C12 and 1E8 cell lines were cultured on a large scale, and 5 × 10⁶ cells were collected. 6 Cells / ml were placed in centrifuge tubes, the supernatant was aspirated, the cells were frozen, and sent to a third-party sequencing company on dry ice for hybridoma cell sequencing.

[0065] The sequencing results are as follows: (a) 5C12 antibody (1) Heavy chain sequence information Heavy chain full-length base sequence: including the nucleotide sequence shown in SEQ ID NO: 14.

[0066] Heavy chain full-length amino acid sequence: including the amino acid sequence shown in SEQ ID NO: 13.

[0067] The heavy chain variable region includes heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3. Heavy chain CDR1 includes the amino acid sequence shown in SEQ ID NO: 1. Heavy chain CDR2 includes the amino acid sequence shown in SEQ ID NO: 2. Heavy chain CDR3 includes the amino acid sequence shown in SEQ ID NO: 3.

[0068] (2) Light chain sequence information Light chain full-length base sequence: including the nucleotide sequence shown in SEQ ID NO: 16.

[0069] Light chain full-length amino acid sequence: including the amino acid sequence shown in SEQ ID NO: 15.

[0070] The light chain variable region includes light chain CDR1, light chain CDR2, and light chain CDR3. Light chain CDR1 includes the amino acid sequence shown in SEQ ID NO: 4. Light chain CDR2 includes the amino acid sequence shown in SEQ ID NO: 5. Light chain CDR3 includes the amino acid sequence shown in SEQ ID NO: 6.

[0071] (ii) 1E8 antibody (1) Heavy chain sequence information Heavy chain full-length base sequence: including the nucleotide sequence shown in SEQ ID NO: 18.

[0072] Heavy chain full-length amino acid sequence: including the amino acid sequence shown in SEQ ID NO: 17.

[0073] The heavy chain variable region includes heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3. Heavy chain CDR1 includes the amino acid sequence shown in SEQ ID NO: 7. Heavy chain CDR2 includes the amino acid sequence shown in SEQ ID NO: 8. Heavy chain CDR3 includes the amino acid sequence shown in SEQ ID NO: 9.

[0074] (2) Light chain sequence information Light chain full-length base sequence: including the nucleotide sequence shown in SEQ ID NO: 20.

[0075] Light chain full-length amino acid sequence: including the amino acid sequence shown in SEQ ID NO: 19.

[0076] The light chain variable region includes light chain CDR1, light chain CDR2, and light chain CDR3. Light chain CDR1 includes the amino acid sequence shown in SEQ ID NO: 10. Light chain CDR2 includes the amino acid sequence shown in SEQ ID NO: 11. Light chain CDR3 includes the amino acid sequence shown in SEQ ID NO: 12.

[0077] Example 4

[0078] This embodiment provides a colloidal gold test strip for detecting Treponema pallidum TP protein. The colloidal gold test strip is prepared using 5C12 antibody and 1E8 antibody.

[0079] The preparation process of this test strip includes the following steps: I. Preparation of the test pad (1) Preparation of coating buffer: Weigh 2.901g of Na2HPO4•12H2O, 0.2914g of NaH2PO4•2H2O, 8.5g of NaCl and 25g of trehalose, dissolve them in 1000mL of ultrapure water, adjust the pH to 7.4, and store at 4℃ for later use.

[0080] (2) Preparation of detection line: Add 1E8 antibody to coating buffer to a final concentration of 1 mg / mL, and spray it onto nitrocellulose membrane to prepare detection line. The spraying amount is 1.2 μL / cm and the spraying length is 30 cm.

[0081] (3) Preparation of control line: Goat anti-mouse IgG polyclonal antibody (Hangzhou Longi Biotechnology Co., Ltd.) was added to the coating buffer to a final concentration of 1 mg / mL and sprayed onto a nitrocellulose membrane to prepare the control line. The spraying amount was 1.2 μL / cm and the spraying length was 30 cm. The distance between the control line and the detection line was 5 mm.

[0082] (4) The nitrocellulose membrane that coats the test line and the quality control line is the test pad. The test pad is attached to the PVC backing and placed in an oven at 50°C to dry for 24±2 hours.

[0083] II. Preparation of Gold-Infused Compounds (1) Preparation of relevant solutions: Preparation of 0.2M K2CO3 solution: Weigh 2.7642g of anhydrous potassium carbonate and dissolve it in 100mL of pure water.

[0084] Preparation of 20% BSA solution: Weigh 20g of BSA (product number V900933, Sigma) and 100μL of proclin300 and dissolve them in 100mL of ultrapure water. Store at 4℃ for later use.

[0085] Preparation of the reconstitution solution: Weigh 0.36g of Tris, 0.1g of sodium caseinate, 0.1g of PEG20000, 100μL of TW-20, 2g of sucrose and 100μL of proclin300 and dissolve them in 100mL of ultrapure water. Adjust the pH to 8.5 with concentrated hydrochloric acid and store at 4℃ for later use.

[0086] (2) Marking process: Take 100 mL of 0.04% colloidal gold solution (C805628, Maclean) and place it in a clean container. Add 500-2000 μL of 0.2 M K2CO3 solution and stir until homogeneous. Then add 500-1500 μg of 5C12 antibody and stir for 10-20 min. Next, add 500 μL of 20% BSA solution and stir. Block for 5-10 min. Centrifuge at 8000-10000 r / min for 30 min and discard the supernatant. Redissolve the precipitate with 10 mL of reconstitution solution to obtain the syphilis gold conjugate. Store at 4℃ for later use.

[0087] III. Preparation of the binding pad (1) Preparation of gold standard conjugate solution: 0.362g Tris, 0.05g sodium caseinate, 0.1g PEG20000, 100μL TW-20, 5g sucrose and 100μL proclin300 were dissolved in 100mL ultrapure water, and the pH was adjusted to 8.5 with concentrated hydrochloric acid and stored at 4℃ for later use.

[0088] (2) Add the gold-labeled syphilis conjugate to the gold-labeled conjugate solution at a concentration of 7-15%, mix well, and coat evenly on the glass fiber membrane (35mL / sheet), and dry in an oven at 50℃ for 24±2h.

[0089] IV. Preparation of Sample Pads (1) Preparation of sample pad treatment solution: Dissolve 0.242g Tris, 0.85g NaCl, 0.5g sodium caseinate, 2g sucrose and 100μL proclin300 in 100mL ultrapure water, adjust the pH to 8.0, and store at 4℃ for later use.

[0090] (2) Apply 32 mL of sample pad treatment solution per plate evenly to the glass fiber membrane, dry it in an oven at 50°C for 24±2 h, and after the test and identification are qualified, store it in a sealed container at room temperature for later use.

[0091] V. Assembly of Colloidal Gold Test Strips Following the structure of the colloidal gold test strip, the sample pad and conjugate pad are sequentially attached to the end of the PVC board near the detection line, with the sample pad partially overlapping the conjugate pad and the conjugate pad partially overlapping the test pad. The absorbent paper is then attached to the end of the PVC board away from the detection line, with the absorbent paper partially overlapping the test pad. After assembling the large board, it is cut into thin strips with a width of 3mm.

[0092] Example 5

[0093] This embodiment is used to illustrate the detection sensitivity of the antibody pair for detecting syphilis provided in this application.

[0094] Experimental group: Colloidal gold test paper provided in Example 4; Control group: mainstream syphilis test kits on the market (colloidal gold method).

[0095] Detection Method: Clinical serum with high Treponema pallidum detection (Concerta Stimulant Treponema pallidum Antibody Serum (Liquid) Standard Material Certificate No. GBW(E)090085) was used as the test subject. Negative serum (fetal bovine serum, brand: Sijiqing, batch number: 21110401) was diluted sequentially at 1:100, 1:500, and 1:1000, and then added dropwise to the sample pads of the colloidal gold test strips in the experimental and control groups, respectively. Results were observed after 15-20 minutes. Colorimetric depth was quantified using a colorimetric card.

[0096] Colorimeter cards, such as Figure 1 As shown. The test results are as follows. Figure 2 As shown.

[0097] Depend on Figure 2The test results show that the colloidal gold test strip provided in Example 4 of this application has better detection sensitivity than the control group reagent at different concentrations of recombinant syphilis TP protein.

[0098] Example 6

[0099] This embodiment is used to illustrate the detection specificity of the antibody pair for detecting syphilis provided in this application.

[0100] Experimental group: Colloidal gold test paper provided in Example 4; Control group: mainstream syphilis test kits on the market (colloidal gold method).

[0101] Detection method: Using the substances provided in Table 3 as the test objects, the substances were added to the sample pads of the colloidal gold test strips in the experimental and control groups, respectively, and the results were observed after 15-20 minutes. The test results are shown in Table 3.

[0102] Table 3 Specific detection results

[0103] The test results show that the colloidal gold test strip provided in Example 4 does not react with other respiratory pathogens in the table and has good specificity.

[0104] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "example," "specific example," or "some examples," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of this application. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. Moreover, without contradiction, those skilled in the art can combine and integrate the different embodiments or examples described in this specification, as well as the features of different embodiments or examples.

[0105] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of this application, and are not intended to limit them. Although this application has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of this application.

Claims

1. A monoclonal antibody pair against Treponema pallidum TP protein, characterized in that, The antibody pair comprises two monoclonal antibodies; each monoclonal antibody includes a heavy chain variable region and a light chain variable region; the heavy chain variable region includes heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3; the light chain variable region includes light chain CDR1, light chain CDR2, and light chain CDR3. One of the monoclonal antibodies in the antibody pair is named the 5C12 antibody, and the sequence information of the 5C12 antibody is as follows: The heavy chain CDR1 comprises an amino acid sequence as shown in SEQ ID NO: 1, or an amino acid sequence having one or two conserved amino acid substitutions compared to the sequence shown in SEQ ID NO: 1, or an amino acid sequence containing the above sequence. The heavy chain CDR2 comprises an amino acid sequence as shown in SEQ ID NO: 2, or an amino acid sequence having one or two conserved amino acid substitutions compared to the sequence shown in SEQ ID NO: 2, or an amino acid sequence containing the above sequence. The heavy chain CDR3 comprises an amino acid sequence as shown in SEQ ID NO: 3, or an amino acid sequence having one or two conserved amino acid substitutions compared to the sequence shown in SEQ ID NO: 3, or an amino acid sequence containing the above sequence. The light chain CDR1 comprises an amino acid sequence as shown in SEQ ID NO: 4, or an amino acid sequence having one or two conserved amino acid substitutions compared to the sequence shown in SEQ ID NO: 4, or an amino acid sequence containing the above sequence. The light chain CDR2 comprises an amino acid sequence as shown in SEQ ID NO: 5, or an amino acid sequence having one or two conserved amino acid substitutions compared to the sequence shown in SEQ ID NO: 5, or an amino acid sequence containing the above sequence. The light chain CDR3 comprises an amino acid sequence as shown in SEQ ID NO: 6, or an amino acid sequence having one or two conserved amino acid substitutions compared to the sequence shown in SEQ ID NO: 6, or an amino acid sequence containing the above sequence. The other monoclonal antibody in the antibody pair is named 1E8 antibody, and the sequence information of 1E8 antibody is as follows: The heavy chain CDR1 comprises an amino acid sequence as shown in SEQ ID NO: 7, or an amino acid sequence having one or two conserved amino acid substitutions compared to the sequence shown in SEQ ID NO: 7, or an amino acid sequence containing the above sequence. The heavy chain CDR2 comprises an amino acid sequence as shown in SEQ ID NO: 8, or an amino acid sequence having one or two conserved amino acid substitutions compared to the sequence shown in SEQ ID NO: 8, or an amino acid sequence containing the above sequence. The heavy chain CDR3 comprises an amino acid sequence as shown in SEQ ID NO: 9, or an amino acid sequence having one or two conserved amino acid substitutions compared to the sequence shown in SEQ ID NO: 9, or an amino acid sequence containing the above sequence. The light chain CDR1 comprises an amino acid sequence as shown in SEQ ID NO: 10, or an amino acid sequence having one or two conserved amino acid substitutions compared to the sequence shown in SEQ ID NO: 10, or an amino acid sequence containing the above sequence. The light chain CDR2 comprises an amino acid sequence as shown in SEQ ID NO: 11, or an amino acid sequence having one or two conserved amino acid substitutions compared to the sequence shown in SEQ ID NO: 11, or an amino acid sequence containing the above sequence. The light chain CDR3 comprises an amino acid sequence as shown in SEQ ID NO: 12, or an amino acid sequence having one or two conserved amino acid substitutions compared to the sequence shown in SEQ ID NO: 12, or an amino acid sequence containing the above sequence.

2. The monoclonal antibody pair against Treponema pallidum TP protein according to claim 1, characterized in that, The heavy chain sequence of the 5C12 antibody includes the amino acid sequence shown in SEQ ID NO: 13; the light chain sequence of the 5C12 antibody includes the amino acid sequence shown in SEQ ID NO: 15; the heavy chain sequence of the 1E8 antibody includes the amino acid sequence shown in SEQ ID NO: 17; and the light chain sequence of the 1E8 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 5C12 antibody heavy chain sequence includes the nucleotide sequence shown in SEQ ID NO: 14; the nucleic acid molecule encoding the 5C12 antibody light chain sequence includes the nucleotide sequence shown in SEQ ID NO: 16; the nucleic acid molecule encoding the 1E8 antibody heavy chain sequence includes the nucleotide sequence shown in SEQ ID NO: 18; and the nucleic acid molecule encoding the 1E8 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 as described in claim 1 or 2.

6. A kit for detecting Treponema pallidum TP protein, characterized in that, The kit includes a monoclonal antibody pair against Treponema pallidum TP protein as described in claim 1 or 2.

7. The reagent kit according to claim 6, characterized in that, The 5C12 antibody is used as a labeling antibody, and the 1E8 antibody is used as a capture antibody.

8. The use of an antibody pair according to claim 1 or 2, a nucleic acid molecule according to claim 3 or 4, or a kit according to claim 6 or 7 in the detection of Treponema pallidum TP protein.

Images