Monoclonal antibody specifically combined with pertussis toxin and application thereof

Abstract

The invention provides a monoclonal antibody specifically bound with pertussis toxin and application thereof. The monoclonal antibody comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprises an HCDR1, an HCDR2 and an HCDR3 of which the amino acid sequences are respectively shown as SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3; the HCDR1, the HCDR2 and the HCDR3 are respectively shown as SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8; the light chain variable region comprises an LCDR1, an LCDR2 and an LCDR3 of which the amino acid sequences are respectively shown as SEQ ID NO: 4, AAS and SEQ ID NO: 5; or LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 9, LVS and SEQ ID NO: 10 respectively.

Description

Technical Field

[0001] This invention belongs to the field of immunology, specifically relating to monoclonal antibodies that specifically bind to pertussis toxin and their applications. Background Technology

[0002] Pertussis, or whooping cough, is an acute respiratory infectious disease caused by Bordetella pertussis (BP). It poses a global threat to human health, especially to infants and young children, and can be life-threatening in severe cases. Humans are the only host for this bacterium, which spreads between people through aerosol droplets. Since the advent of the pertussis vaccine, it has provided effective active immunization protection for susceptible populations, particularly children under six years of age. However, over time, due to the natural decline in the protective efficacy and persistence of the vaccine, the adaptive evolution of circulating pertussis strains, and the increased sensitivity of detection technologies, a public health problem of "pertussis resurgence" has emerged in recent years.

[0003] Currently, there are no specific drugs or strategies for treating pertussis infection. While antibiotics (such as azithromycin) can effectively clear Bordetella pertussis from the respiratory tract, their mechanism of action means they cannot neutralize the pertussis toxin (PT) already secreted into the body. PT is the most important virulence factor of Bordetella pertussis, causing significant leukocytosis, histamine sensitization, promotion of lymphokine secretion, and local or systemic immunosuppression. It is a key cause of many severe clinical symptoms of pertussis (especially paroxysmal cough, cyanosis, and apnea in infants). Therefore, simply clearing the bacteria without neutralizing the toxin is insufficient to completely control disease progression. Against this backdrop, antibody drugs, due to their high specificity and efficacy in neutralizing the toxin, compensate for the limitations of antibiotic treatment and have become a promising direction for developing treatment strategies after pertussis infection.

[0004] In the prior art, spleen cells from mice immunized with PT after isolation and detoxification are separated and purified, and then fused with mouse myeloma cells using PEG 4000. After screening for positive monoclonal strains, two monoclonal antibodies specifically binding to PT, 3G5 and 3D6, are obtained, and a corresponding ELISA method is established for the detection of PT antigen content in each production step of pertussis vaccine and in the finished product. This prior art has the following defects: (1) The mouse immunization program has not been optimized. For example, gradient small-volume purification of undetoxified PT and detoxified PT are used for cross-immunization. Although it has been proven that pertussis subunit component vaccines detoxified with formaldehyde or glutaraldehyde are safe and effective, the aldehyde group of the detoxifying agent often undergoes a cross-linking reaction with the free amino group in PT, destroying the overall three-dimensional structure of PT and hindering the ability of PT to stimulate strong neutralizing antibodies after immunization. Therefore, this technique only screened two monoclonal antibodies that specifically bind to active PT, and did not obtain monoclonal antibodies with pertussis toxin protein neutralizing activity; (2) PEG 4000 chemical induction method for fusion of hybridoma cells, because PEG 4000 may have a toxic effect on cells, affecting the proliferation of cells after fusion. In addition, PEG cannot control which type of cell fusion, resulting in a higher probability of non-specific fusion cells. Moreover, the fusion efficiency induced by PEG chemical induction is constrained by a variety of factors, including solution pH, temperature, and PEG concentration, and experimental conditions need to be strictly controlled to achieve sufficient fusion efficiency; (3) The murine antibodies obtained by screening using hybridoma technology were not humanized or subjected to defucosylation of the constant region (Fc) of the antibody or mutation modification of Fc silence to enhance the passive immune protection effect of the monoclonal antibody.

[0005] The determination of effective antigen content is of great significance in vaccine quality control: antigen content, as one of the core indicators of vaccine quality, has a decisive impact on vaccine efficacy due to its purity, stability, and immunogenicity. Developing methods to identify the content, purity, and immunogenicity of antigen components in DPT / FHA / PRN / DT / TT vaccines can ensure good immunization effects and assess the integrity and consistency of effective antigen content throughout the pertussis vaccine production process. Currently, enzyme-linked immunosorbent assay (ELISA) is mainly used to identify the effective antigen content in acellular pertussis combined vaccines. Monoclonal antibodies with high specificity and stability can significantly improve the accuracy, stability, and efficiency of the assay by identifying antigen quality, integrity, homogeneity, and batch-to-batch variability.

[0006] Furthermore, the resurgence of pertussis in recent years indicates that pertussis vaccines do not provide long-term immune protection, and there are currently no effective treatments or regimens for pertussis. Antibiotic treatment suffers from a lack of specific targeting; although it can eliminate Bordetella pertussis from the respiratory tract, it cannot specifically neutralize the pertussis toxin antigen. Therefore, developing highly specific monoclonal antibodies with neutralizing activity targeting the pertussis toxin antigen as prodrugs for the treatment of Bordetella pertussis infection, and further humanizing or engineering them into antibodies or bispecific antibodies, could potentially increase therapeutic efficacy and reduce toxic side effects against the pertussis toxin antigen, representing a better strategy for the treatment of Bordetella pertussis infection.

[0007] In summary, there is an urgent need in this field to develop a series of novel, high-affinity, PT-specific binding monoclonal antibodies. These antibodies could not only be used to establish more accurate detection methods, but more importantly, they could provide core candidate molecules for the development of effective therapeutics against pertussis infection. Summary of the Invention

[0008] To address the aforementioned technical problems, the objective of this invention is to develop a monoclonal antibody that specifically binds to pertussis toxin.

[0009] The present invention adopts the following technical solution: On the one hand, the present invention provides a monoclonal antibody that specifically binds to pertussis toxin, wherein the monoclonal antibody comprises a heavy chain variable region and a light chain variable region; The heavy chain variable region comprises amino acid sequences HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3, respectively; or HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO:6, SEQ ID NO:7, and SEQ ID NO:8, respectively. The light chain variable region comprises the amino acid sequences LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO:4, AAS, and SEQ ID NO:5, respectively; or as shown in SEQ ID NO:9, LVS, and SEQ ID NO:10, respectively.

[0010] According to a specific embodiment of the present invention, the monoclonal antibody comprises HCDR1, HCDR2, and HCDR3 shown in SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3 combined with LCDR1, LCDR2, and LCDR3 shown in SEQ ID NO:4, AAS, and SEQ ID NO:5; or

[0011] The HCDR1, HCDR2 and HCDR3 shown in SEQ ID NO:6, SEQ ID NO:7 and SEQ ID NO:8 are combined with the LCDR1, LCDR2 and LCDR3 shown in SEQ ID NO:9, LVS and SEQ ID NO:10.

[0012] According to a specific embodiment of the present invention, the monoclonal antibody comprises a heavy chain and a light chain, wherein the heavy chain contains an amino acid sequence as shown in SEQ ID NO:11 or SEQ ID NO:13; and the light chain contains an amino acid fragment as shown in SEQ ID NO:12 or SEQ ID NO:14.

[0013] According to a specific embodiment of the present invention, the monoclonal antibody comprises a heavy chain as shown in SEQ ID NO:11 and a light chain as shown in SEQ ID NO:12; or

[0014] The heavy chain shown in SEQ ID NO:13 and the light chain shown in SEQ ID NO:14.

[0015] According to a specific embodiment of the present invention, preferably, the monoclonal antibody further includes a mouse-derived or humanized Fc terminus.

[0016] On the other hand, the present invention provides a polynucleotide that encodes the aforementioned monoclonal antibody.

[0017] According to a specific embodiment of the present invention, preferably, the heavy chain encoding the monoclonal antibody comprises a nucleotide sequence as shown in SEQ ID NO:15 or SEQ ID NO:17; The light chain encoding the monoclonal antibody includes a nucleotide sequence as shown in SEQ ID NO:16 or SEQ ID NO:18.

[0018] According to a specific embodiment of the present invention, preferably, the monoclonal antibody comprises the heavy chain shown in SEQ ID NO:15 and the light chain shown in SEQ ID NO:16; or

[0019] The heavy chain shown in SEQ ID NO:17 and the light chain shown in SEQ ID NO:18.

[0020] On the other hand, the present invention provides a carrier containing the above-mentioned polynucleotides.

[0021] On the other hand, the present invention provides a host cell containing the above-described polynucleotides or the above-described vectors.

[0022] On the other hand, the present invention provides a kit for detecting pertussis toxin, the kit comprising the monoclonal antibody as described above.

[0023] On the other hand, the present invention provides the use of the above-described monoclonal antibody, the above-described polynucleotide, the above-described vector, and the above-described host cell in the preparation of reagents or kits for detecting pertussis toxin.

[0024] On the other hand, the present invention provides the application of the above-described monoclonal antibody, the above-described polynucleotide, the above-described vector, the above-described host cell, and the above-described kit in the detection of pertussis toxin for non-diagnostic purposes.

[0025] On the other hand, the present invention provides the above-described monoclonal antibody, the above-described polynucleotide, the above-described vector, the above-described host cell, and the above-described kit for use in quality testing of vaccines containing pertussis toxin antigen.

[0026] Beneficial effects: This invention provides monoclonal antibodies against pertussis toxin with different neutralizing antigenic epitopes, or their antigen-binding fragments, humanized antibodies, and their applications. This invention yields monoclonal antibodies P9-1 and N50-2 that target the S2 and S4 subunits of the pertussis toxin B oligomer, respectively. These S2 and S4 specific antibodies are novel sequences. Their affinity constants can reach 10. -9 M, possessing strong passive immune protection, has been humanized to serve as a candidate drug for pertussis treatment. Antibodies P9-1 and N50-2, with specific binding activity, can be used to establish methods for detecting the presence and / or amount of pertussis toxin antigen in biological samples. Furthermore, highly binding and conserved epitopes such as S2 and S4 can serve as important targets in pertussis vaccine production, for quantifying and identifying the activity of pertussis vaccine antigens, for developing next-generation pertussis vaccines, for identifying immune relevance, and for passive immunization strategies. Attached Figure Description

[0027] Figure 1 This is a technology roadmap.

[0028] Figure 2 For hybridoma cell electrofusion.

[0029] Figure 3 Screening for positive master clones, subclones, and single clones.

[0030] Figure 4 The preparation and characterization of positive monoclonal antibodies are shown in Figure a, where a represents the affinity chromatography separation of PT monoclonal antibody; and b represents the SDS-PAGE gel image of PT monoclonal antibody.

[0031] Figure 5 SDS-PAGE gel images of the junctions and active monoclonal antibodies that specifically target different subunits of PT.

[0032] Figure 6 This is for the affinity assay of PT monoclonal antibodies.

[0033] Figure 7 This is for the competition of antigenic epitopes for PT monoclonal antibodies.

[0034] Figure 8 Sequence analysis of PT monoclonal antibodies.

[0035] Figure 9 The standard curve for the binding of PT's S2 subunit to monoclonal antibodies is shown.

[0036] Figure 10 The standard curve for the binding of PT's S4 subunit to monoclonal antibodies is shown. Detailed Implementation

[0037] Before further describing specific embodiments of the present invention, it should be understood that the scope of protection of the present invention is not limited to the specific embodiments described below; it should also be understood that the terminology used in the embodiments of the present invention is for describing specific embodiments and not for limiting the scope of protection of the present invention.

[0038] When numerical ranges are given in the embodiments, it should be understood that, unless otherwise stated in the present invention, both endpoints of each numerical range and any value between the two endpoints may be selected. Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art. In addition to the specific methods, apparatus, and materials used in the embodiments, based on the knowledge of the prior art possessed by one of ordinary skill in the art and the description of this invention, any prior art methods, apparatus, and materials similar to or equivalent to those described, apparatus, and materials in the embodiments of this invention may be used to implement the present invention.

[0039] Unless otherwise stated, the experimental methods, detection methods and preparation methods disclosed in this invention all adopt conventional techniques in this technical field.

[0040] In some specific embodiments of the present invention, hybridoma screening technology is used to obtain novel monoclonal antibodies with high specificity and strong affinity for the PT single subunit: This invention utilizes hybridoma fusion technology to obtain high-affinity, single-epitope antibodies through multiple screenings. Hybridoma fusion technology involves fusing sensitized B cells capable of secreting specific antibodies with myeloma cells possessing unlimited proliferative capacity to create a B-cell hybridoma. These hybridoma fusion cells can survive and proliferate in HAT medium. Subsequently, enzyme-linked immunosorbent assay (ELISA) is used to obtain monoclonal cell lines targeting different antigenic epitopes of PT. Monoclonal antibodies can then be prepared through in vitro expansion culture. The basic process mainly includes antigen preparation, animal immunization, cell electrofusion, hybridoma and subclonal screening, and characterization and preparation of specific antibodies. The specific technical route is as follows: Figure 1 The specific steps are as follows: 1. Antigen preparation The antigen is derived from the purified PT obtained from the bacterial culture of Bordetella pertussis fermented in the production tank. The working generation seed of Bordetella pertussis is revived, and after shaking flask culture, the supernatant is collected, sterilized, filtered and clarified, and purified in multiple steps to obtain the purified PT antigen. After detoxification with formaldehyde / glutaraldehyde, the PT stock solution is obtained.

[0041] 2. Animal immunization

[0042] Female Balb / c mice aged 6-8 weeks were used. The antigen and Freund's adjuvant were mixed in equal volumes and emulsified. The mixture was then administered via intraperitoneal and subcutaneous immunization at multiple sites on days 0, 14, 28, and 42. Detoxified pre-transfer (PT) immunization was performed at 50 μg / dose, while non-detoxified PT immunization was performed at 5 μg / dose. Blood was collected from the orbital sinus after four immunizations to determine serum titers. At a 16000-fold dilution, goat anti-mouse IgG-HRP was used as the secondary antibody, and OD was measured using an ELISA reader. 450-630 Mice with a blood glucose level of 2.0 or higher were given a booster immunization via tail vein. Three days later, whole blood and spleen tissue were collected. The spleen was homogenized and erythrocytes were lysed to obtain single B cells for later use.

[0043] 3. Cell electrofusion

[0044] Mouse spleen B cells were electrofused with myeloma SP2 / 0 cells using an electrofusion apparatus to prepare hybridoma fusion cell lines. First, positive master clones capable of recognizing the target antigen PT were screened using indirect ELISA. After multiple limiting dilutions, the supernatant was collected, and monoclonal hybridoma cell lines capable of secreting the target antigen, i.e., positive cell lines, were screened using indirect ELISA. These positive cell lines were then transferred from 96-well plates to 24-well plates for further expansion culture. After continued culture, the supernatant was further screened and verified using indirect ELISA. The cells were then progressively expanded to monolayer or bilayer cell factories for the harvesting and validation of monoclonal antibodies.

[0045] 4. Hybridoma-positive monoclonal screening

[0046] When the fused cells in the 96-well plate reach 25% confluence, the supernatant is collected and screened by indirect ELISA to obtain cell lines that can secrete and express cells that can highly bind to the target antigen PT. These are the positive master clones. Positive monoclonal cell lines are further screened by multiple limiting dilutions to obtain highly expressed and stable monoclonal positive cell lines.

[0047] 5. Preparation and purification of monoclonal antibodies

[0048] Positive monoclonal antibodies obtained from hybridoma screening were purified through multiple stages using methods such as affinity chromatography and ultrafiltration concentration, achieving a purity of over 95%.

[0049] 6. Physicochemical characterization of monoclonal antibodies

[0050] Molecular weight, purity, and homogeneity of monoclonal antibodies: This invention identifies the molecular weight, purity, and homogeneity of monoclonal antibodies using SDS-PAGE, size exclusion chromatography (SEC), and dynamic light scattering (DLS).

[0051] SDS-PAGE uses a gel imaging system to visually analyze the purity and molecular weight of antibodies. After SDS-PAGE electrophoresis, antibodies will show two bands: heavy chain and light chain. The molecular weight of the heavy chain of IgG is about 50-70 kDa, and the molecular weight of the light chain is about 25 kDa. If there are no other bands besides the heavy chain and light chain, the purity of the antibody can be preliminarily judged.

[0052] Size exclusion chromatography (SEC) determines molecular weight based on elution time; smaller molecules retain longer in the column than larger molecules. The relative molecular weight of antibodies is determined by establishing a linear range of the molecular weight-elution volume curve of a standard protein. The purity, homogeneity, or aggregation state of antibodies is assessed by observing the presence of other signal peaks besides the antibody peak. The steps for analyzing monoclonal antibodies using SEC in the AKTA protein chromatography system are as follows: Turn on the AKTA instrument and computer, open the software, and prepare the program. Remove the pump head from 20% ethanol, rinse the pump head with filtered water, and place the pump head in a bottle containing filtered water. Set the system pressure and column pressure to a maximum of 1.8 MPa. Rinse the tubing with filtered water, gradually increasing the flow rate to 5 mL / min until the system is balanced. Load the column with filtered water under flowing conditions, adjusting the flow rate to 0.3 mL / min before loading. Equilibrate at least two column volumes, completely replacing the ethanol in the SEC column with filtered water. Pause the system, rinse the pump head with 10 mM PBS at a flow rate of 0.5 mL / min. Once the system is fully equilibrated, prepare for sample loading. Draw the corresponding volume of sample into the loading loop using a syringe and inject it into the system. Select "inject". Wait for UV... 280 When the value rises, collect the sample into a 50 mL centrifuge tube. After collection, wash the column with 10 mM PBS at a flow rate of 0.5 mL / min until equilibration, then wash with pure water until equilibration. Next, wash the column with 20% ethanol until equilibration, at a flow rate of 0.3 mL / min. Once the column is full, remove it and store it at 4°C.

[0053] Monoclonal antibody stability: This invention uses a differential scanning calorimeter (DSC), a differential scanning fluorimetry (DSF), and an all-purpose protein stability analyzer (Uncle) to detect the Tm and Tagg values ​​of monoclonal antibodies to determine the strength of monoclonal antibody stability.

[0054] DSC calculates the Tm value by monitoring the change in heat capacity (Cp) of a monoclonal antibody as it transitions from natural (folding) to denaturation (unfolding) with temperature changes. DSF calculates the Tm value by monitoring the emission spectral shift (change in fluorescence signal) of the monoclonal antibody's intrinsic fluorescence or its binding to hydrophobic dyes during unfolding. Uncle, on the other hand, integrates full-spectrum fluorescence, static light scattering (SLS), and dynamic light scattering (DLS) technologies to identify monoclonal antibody stability in a high-throughput manner. Full-spectrum fluorescence detection (250nm-720nm) determines the Tm value by measuring the area under the fluorescence intensity curve of the antibody's intrinsic fluorescence (tryptophan) or SYPRO™ Orange dye within a specific wavelength range during thermal denaturation. Simultaneously, SLS can be used to detect the aggregation temperature (Tagg) in real time by monitoring the intensity of the intrinsic fluorescence or scattered light from the SYPRO™ Orange dye during antibody thermal denaturation. DLS can also detect particle size and particle size distribution.

[0055] Affinity and spatial competition epitopes of monoclonal antibodies: Surface plasmon resonance (SPR) is a physical optical phenomenon. Based on the different resonance angles generated by ligands coupled to a metal surface and their binding to the analyte, changes in these resonance angles can be used to infer information such as the affinity, affinity constant, and binding kinetics of biomolecular interactions. Using the SPR method, no antigen-antibody labeling is required; analyzing the affinity constants of screened PT monoclonal antibodies confirms the antigen-antibody interaction mode.

[0056] Sequence analysis of monoclonal cell lines

[0057] Hybridoma cells are prone to chromosome loss, leading to cell degeneration. Reconstruction is time-consuming and labor-intensive. Sequencing these cells allows for the acquisition of antibody gene sequences, enabling long-term preservation and on-demand production. The highly specific binding activity monoclonal cell lines targeting S2 and S4 obtained through the above screening were used to extract RNA from positive clone cell lines, obtain cDNA using RT-PCR, and amplify the light and heavy chain genes of the antibodies. The antibody genes were ligated into a T vector, and positive clones were selected. Sequencing yielded the antibody gene sequences CDR1, CDR2, and CDR3.

[0058] Example 1 Antigen preparation and animal immunization

[0059] Antigen preparation

[0060] The antigen is derived from the purified PT obtained from the bacterial culture of Bordetella pertussis fermented in the production tank. The working generation seed of Bordetella pertussis is revived, and after shaking flask culture, the supernatant is collected, sterilized, filtered and clarified, and purified in multiple steps to obtain the purified PT antigen. After detoxification with formaldehyde / glutaraldehyde, the PT stock solution is obtained.

[0061] Animal Immunization

[0062] Female Balb / c mice aged 6-8 weeks were used. The antigen and Freund's adjuvant were mixed in equal volumes and emulsified. The mixture was then administered via intraperitoneal and subcutaneous immunization at multiple sites on days 0, 14, 28, and 42. Pre-immunization (PT) was 50 μg / dose, and non-detoxified PT was 5 μg / dose. Blood was collected from the orbital sinus after four immunizations to determine serum titers. At a 16000-fold dilution, goat anti-mouse IgG-HRP was used as the secondary antibody, and OD was measured using an ELISA reader. 450-630 Mice with a blood glucose level of 2.0 or higher were given a booster immunization via tail vein. Three days later, whole blood and spleen tissue were collected. The spleen was homogenized and erythrocytes were lysed to obtain single B cells for later use.

[0063] Example 2: Screening for positive monoclonal antibodies using electrofusion hybridoma technology

[0064] Mouse spleen B cells were electrofused with myeloma SP2 / 0 cells using an electrofusion apparatus to prepare a hybridoma fusion cell line, such as... Figure 2 As shown, larger myeloma SP2 / 0 cells and smaller spleen B cells fuse and grow under external voltage, as... Figure 2 The single-clonal cell line (10×) is shown.

[0065] When the fused cells in the 96-well plate reach 25% confluence, the supernatant is collected and screened using indirect ELISA to identify cell lines that secrete and highly bind to the target antigen PT. These are the positive master clones. Further screening of positive monoclonal cell lines is performed through multiple limiting dilutions to obtain highly expressed and stable monoclonal positive cell lines. The specific steps for screening positive monoclonal antibodies using indirect ELISA are as follows: 1) Preparation of coating solution: Weigh 1.59 g of anhydrous sodium carbonate and 2.93 g of sodium bicarbonate and dissolve them in 1000 mL of deionized water until fully dissolved. Store at 4 ℃ for later use. This is the coating solution, a 50 mM pH 9.6 sodium carbonate buffer solution.

[0066] 2) Coating: Dilute the antigen (detoxified PT, purified PT, S1 / S4, S2 / S4, S3 / S4) protein with coating buffer to 1 μg / mL, add 100 uL / well, seal the plate, and incubate overnight at 2-8 ℃.

[0067] 3) Blocking: Discard the liquid in each well, wash the plate 4 times with PBST (0.05% Tween 20 PBS), 200 μL / well / wash. Block the plate with PBS blocking buffer (2% BSA), 200 μL / well, and incubate at 37 °C for 1 hour.

[0068] 4) Sample addition: Discard the liquid in each well, wash the plate 4 times with washing buffer, 200 μL / well / wash. Discard the liquid in each well, wash the plate 4 times, and pat dry on a wiping cloth. Dilute the hybridoma supernatant with PBS, 50-fold dilution, and add 100 μL / well. Seal the plate and incubate at 37°C for 1–1.5 hours.

[0069] 5) Add secondary antibody: Discard the liquid in each well, wash the plate 4 times with washing buffer, 200 μL / well / wash, and pat dry on wiping paper after the last wash. Add 100 μL / well of HRP-labeled goat anti-mouse antibody diluted 1:10000 with 0.05% BSA in PBS, seal the plate, and incubate at 37 °C for 1 h.

[0070] 6) Color development: Wash the plate 4 times with washing solution, 200 μL / well / wash. Add 50 μL of color development solution A and 50 μL of color development solution B to each well. After gently shaking and mixing, incubate at room temperature in the dark for 10 min for color development.

[0071] 7) Termination and Measurement: Add 50 μL of stop solution (2M H2SO4) to each well, gently vortex to mix and terminate the reaction. Measure the OD value at 450 nm / 630 nm using a microplate reader and calculate the OD value. 450 -OD 630 All OD 450 -OD 630 If the value is more than 5 times that of the negative control, it is preliminarily determined to be a positive clone.

[0072] Positive cell lines were transferred from 96-well plates to 24-well plates for further expansion. After continued culture, the supernatant was further screened and verified using indirect ELISA. The cells were then expanded to T25-T75-T225 monolayer or bilayer cell factories for monoclonal antibody harvesting and verification. Through three limiting dilutions of the electrofused cells, 74 PT-positive master clones, 38 PT-positive subclones, and 48 PT-positive monoclones were screened and cryopreserved. Some results are shown below. Figure 3 As shown.

[0073] Example 3: Preparation and Identification of PT Monoclonal Antibodies

[0074] Positive monoclonal antibodies obtained from hybridoma screening were progressively purified using affinity chromatography and ultrafiltration concentration, achieving a purity of over 95%. The specific preparation protocol for PT monoclonal antibodies is as follows. 1) Sample preparation: Pour the cell culture medium from the T75 culture flask into a 50 mL centrifuge tube, balance the volume, and centrifuge at 4000 rpm for 30 min. Filter the supernatant through a 0.45 μm filter. Add an equal volume of 10 mM PBS, mix well, and place on ice.

[0075] 2) Purification: Place 500 μL of proA beads in a gravity column and wash with 10 mL of pure water. Repeat this step three times to remove residual ethanol. Wash the beads with 10 mL of PBS solution three times. When the liquid is almost dry, add the sample for binding. Flow through the column by gravity. After binding is complete, wash the beads again with 10 mL of PBS solution three times. Add 10 mL of 0.1 M Glycine elution buffer (pH 3.0) for elution, and transfer the flow-through solution to a new centrifuge tube. After elution, add 1 mL of 1 M Tris-HCl (pH 7.4) to the centrifuge tube for neutralization. Wash the beads again with 10 mL of PBS solution three times. Wash the beads with 10 mL of pure water three times. Add 5 mL of 20% ethanol and store the gravity column at 4°C.

[0076] 3) Concentration and Solution Replacement: Take a 30 kDa ultrafiltration tube, pour out the ethanol, rinse three times with pure water, add 10 mM PBS to the ultrafiltration tube, add a high-density filtration membrane, and centrifuge for 5 min (4000 rpm). Pour out the remaining PBS. Pour the purified sample solution into the ultrafiltration tube, centrifuge for 15 min (4000 rpm), remove the ultrafiltration tube and pipette 1 mL to clear the filter membrane and prevent protein accumulation on the membrane, which would affect the concentration rate. When the solution is less than 1.5 mL, add PBS to the top of the ultrafiltration tube (fill with PBS), and repeat the above steps three times. Collect the concentrated sample solution in a 1.5 mL centrifuge tube and measure the sample concentration. Rinse the filter tube three times with pure water, add 20% ethanol, ensuring the liquid level is above the filter membrane. Store the ultrafiltration tube at 4°C.

[0077] The prepared monoclonal antibodies were identified by reducing and non-reducing SDS-PAGE gel electrophoresis. Natural immunoglobulin IgG consists of heavy and light chains linked by interchain disulfide bonds. In reducing SDS-PAGE gels, these chains appear as bands of 55 kDa and 25 kDa, respectively, representing the heavy and light chains of the monoclonal antibodies. Figure 4 The affinity chromatography separation of PT monoclonal antibody and the SDS-PAGE gel image are shown. The PT monoclonal antibody obtained by affinity chromatography purification can have a purity of over 95% and has clear heavy chain and light chain bands.

[0078] Furthermore, this invention uses Western blotting experiments to identify linear epitopes on different subunit surfaces targeted by positive PT monoclonal antibodies, with results as follows: Figure 5 As shown. This invention, through multiple rounds of screening, obtained monoclonal antibodies that specifically target different epitopes of the S2 and S4 subunits. Among them, P9-1 and N50-2 are highly binding antibodies that specifically target the S2 and S4 subunits, respectively.

[0079] Example 4 Affinity characterization of PT monoclonal antibody

[0080] Surface plasmon resonance (SPR) is a physical optical phenomenon. Based on the different resonance angles generated by ligands coupled to a metal surface and their binding to the analyte, changes in these resonance angles can be used to infer information such as the affinity, affinity constant, and binding kinetics of biomolecular interactions. Using the SPR method, no antigen-antibody labeling is required; analyzing the affinity constants of screened PT monoclonal antibodies confirms the antigen-antibody interaction mode.

[0081] The specific SPR plan is as follows: 1) The 10 μM purified PT antigen was immobilized on channels 1 and 2 of the CM5 sensor chip surface using an amino coupling method. Channel 1 was used as the reference channel and channel 2 was used as the sample channel. The binding time was 120 s.

[0082] 2) The PT monoclonal antibody was used as the mobile phase. It was diluted 2-fold from 3.2 μM and analyzed in 8 gradients for the binding and dissociation process. The binding time was 120 s, the dissociation time was 180 s, and the glycine at pH 1.5 was used for regeneration for 30 s. The flow rate was 30 µL / min.

[0083] 3) Design the parameters of Biacore. As the sample is loaded, relevant parameters such as antibody baseline, binding, dissociation and regeneration will appear.

[0084] 4) Simulate the relevant curves to obtain the monoclonal antibody binding rate and dissociation rate, and calculate the KD value of the sample (KD = Ka / Kd).

[0085] The results are as follows Figure 6 As shown in Table 1, after multiple rounds of screening, this embodiment obtained a PT monoclonal antibody with high affinity, with an affinity constant reaching 10. -9 M.

[0086] Table 1. Affinity KD (nM) of PT monoclonal antibodies

[0087] Example 6 Characterization of spatially competing epitopes of PT monoclonal antibodies

[0088] To identify whether the antigenic epitopes of monoclonal antibodies specifically targeting PT overlap, a competitive binding assay was performed on two antibodies using the SPR method. PT was immobilized on the surface of a CM5 sensor chip using amino-coupling, and then the two antibodies were tested. Theoretically, if the two antibodies target the same epitope, the antigenic epitope will be blocked by the first antibody, and the other antibody targeting the same epitope will be unable to bind to the antigen, resulting in no significant response value (RU). If the two antibodies recognize different epitopes, there is no competition between them, and the binding of the first antibody to the antigen will not affect the binding of the second antibody to the antigen, resulting in significant response values ​​(RU) for both antibody injections.

[0089] The specific implementation plan for competing tabletops is as follows: The two paired antibodies flowed sequentially through the chip channel at a concentration of 3200 nM, with a binding time of 240 s, a dissociation time of 60 s, and a regeneration time of 30 s with glycine at pH 1.5. The flow rate was 30 µL / min. After the program completed, the runs were opened using BiacoreInsight Evaluation Software, the data were exported, and then plotted using Graphpad Prism to observe whether there were significant response values ​​after both antibody injections, thus determining whether epitope competition existed between the two antibodies.

[0090] The results are as follows Figure 7 As shown, neither P9-1 nor N50-2 has an epitope competition relationship. Based on the linear epitope analysis of these two monoclonal antibodies, P9-1 is a binding monoclonal antibody that specifically targets S2, and N50-2 is a binding monoclonal antibody that specifically targets S4.

[0091] Example 7 Sequence Analysis of PT Monoclonal Antibody

[0092] Hybridoma cells are prone to chromosome loss, leading to cell degeneration. Reconstruction is time-consuming and labor-intensive. Sequencing these cells allows for the acquisition of antibody gene sequences, enabling long-term preservation and immediate production. The neutralizing monoclonal cell lines targeting S2 and S24 obtained through the above screening were processed by extracting RNA from positive clones, obtaining cDNA via RT-PCR, and amplifying the light and heavy chain genes of the antibodies. The antibody genes were ligated into a T vector, and positive clones were selected. Sequencing yielded the antibody gene sequences CDR1, CDR2, and CDR3.

[0093] The results are as follows Figure 8 As shown, bands 4 and 5 represent the heavy chain bands of P9-1 and N50-2, respectively; bands 9 and 10 represent the light chain bands of P9-1 and N50-2, respectively; and bands 14 and 15 represent the antibody bands of P9-1 and N50-2, respectively. The heavy and light chain variable region sequences of monoclonal antibodies with different antigenic epitopes obtained through multiple rounds of screening were sequenced to obtain the antibody gene sequences, providing a basis for further engineering or humanization of the antibodies.

[0094] The amino acid and nucleotide sequences of the heavy and light chains of the S4 subunit monoclonal antibody N50-2 are as follows: 1. N50-2 heavy chain amino acid sequence: EVQLVESGGGLVKPGGSLKLSCAASGFTLSDHYMYWVRQTPEKRLEWVAAIGDGDNYIYYPDSVKGRLTISRDNAKNNLYLQLSSLKSDDTAIYYCARAPLYGNYFDYWGQGTTVTVSSSGGLGGLVDKGQFV (SEQ ID NO: 11) The CDR area is as follows: <----CDR1---> <--CDR2--> <---CDR3---- GFTLSDHY (SEQ ID NO:1)___IGDGDNYI (SEQ ID NO:2..___ARAPLYGNYFDY (SEQ ID NO:3) The FR area is as follows: FR1:EVQLVESGGGLVKPGGSLKLSCAAS (SEQ ID NO:19) FR2:MYWVRQTPEKRLEWVAA (SEQ ID NO:20) FR3: YYPDSVKGRLTISRDNAKNNLYLQLSSLKSDDTAIYYC (SEQ ID NO:21) FR4: WGQGTTVTVSS (SEQ ID NO:22) 2. N50-2 heavy chain nucleotide sequence GAGGTGCAGCTTGTAGAGTCTGGGGGAGGCTTAGTGAAGCCTGGAGGGTCCCTGAAACTCTCCTGTGCAGCCTCTGGATTCACTTTAAGTGACCATTACATGTATTGGGTTCGCCAGACTCCGGAAAAGAGGCTGGAGTGGGTCGCAGCCATTGGTGATGGTGATAATTACATCTACTATCCAGACAGTGTGAAGGGGCGACTCACCATCTCCAGAGACAATGCCAAGAACAACCTGTACCTGCAACTGAGCAGTCTGAAGTCTGATGACACAGCCATTTATTATTGTGCAAGAGCCCCCCTCTATGGTAACTACTTTGACTACTGGGGCCAAGGAACCACAGTCACCGTCTCCTCAGGTGGCCTCGGGGGCCTGGTCGACAAGGGCCAATTCGTTTAA (SEQ ID NO:15) 3. N50-2 light chain amino acid sequence RRGPAGHGRNCAHPVSSNVDAAQPAMADIKMTQSSSSLAVSLGQGATISCRASESVDNYVISFMNWFQQKPGQPPKLLIYAASNQGSGVPTRFSGSGSGTDFSLNIHPMEEDDTAMYFCQQNKEVPYTFGDVGPGWKLKEAAVAAEVAQAV (SEQ ID NO:12) The CDR regions are as follows: ----CDR1---> <--CDR2--> <---CDR3---- ESVDNYVISF (SEQ ID NO:4) ___AAS.......___QQNKEVPYTFGDVGPGWK (SEQ ID NO:5) The FR regions are as follows: FR1: DIKMTQSSSSLAVSLGQGATISCRAS (SEQ ID NO:23) FR2: MNWFQQKPGQPPKLLIY (SEQ ID NO:24) FR3: NQGSGVPTRFSGSGSGTDFSLNIHPMEEDDTAMYFC (SEQ ID NO:25) 4. N50-2 light chain nucleotide sequence CGACGCGGCCCAGCCGGCCATGGCAGAAATTGTGCTCACCCAGTCTCCAGCAATGTCGACGCGGCCCAGCCGGCCATGGCAGACATCAAGATGACACAATCTTCATCTTCTTTGGCTGTGTCTCTAGGGCAGGGGGCCACCATCTCCTGCAGAGCCAGCGAAAGTGTTGATAATTATGTCATTAGTTTTATGAACTGGTTCCAACAGAAACCAGGACAGCCACCCAAA CTCCTCATCTATGCTGCGTCCAACCAAGGTTCCGGGGTCCCTACCAGGTTTAGTGGCAGTGGGTCTGGGACAGACTTCAGCCTCAACATCCATCCTATGGAGGAGGATGATACTGCAATGTATTTCTGTCAGCAAAATAAGGAGGTTCCGTACACGTTCGGAGATGTGGGACCAGGCTGGAAATTAAAGGAGGCGGCGGTAGCGGCGGAGGTGGCTCAGGCGGTG (SEQ ID NO:16) Amino acid and nucleotide sequences of the heavy and light chain variable regions of S2 subunit monoclonal antibody P9-1 1. P9-1 heavy chain amino acid sequence QVQLKESGPELVKPGASMKISCKASGYSFTGYTMNWVKQSHGKDLEWIGLINPYNGVPNYNQKFKGKATLTVDKSSSTAYMELLSLTSEDSAVYYCARRDYDYDSYAMDYWGQGTLVTVSAGGGLGGLVDRNLSHRLLRWPRGPGR (SEQ ID NO: 13) The CDR area is as follows: ----CDR1---> <--CDR2--> <---CDR3---- GYSFTGYT (SEQ ID NO:6)__INPYNGVP (SEQ ID NO:7)..___ARRDYDYDSYAMDY (SEQ ID NO:8) The FR area is as follows: FR1: QVQLKESGPELVKPGASMKISCKAS (SEQ ID NO:26) FR2: MNWVKQSHGKDL (SEQ ID NO:27) FR3: NYNQKFKGKATLTVDKSSSTAYMELLSLTSEDSAVYYC (SEQ ID NO:28) FR4: WGQGTLVTVS (SEQ ID NO:29) 2. Heavy chain nucleotide sequence of P9-1 CAGGTGCAGCTGAAGGAGTCAGGACCTGAGCTGGTGAAGCCTGGAGCTTCAATGAAGATTTCCTGCAAGGCTTCTGGTTACTCATTCACTGGCTACACCATGAACTGGGTGAAACAGAGCCATGGAAAGGACCTTGAGTGGATTGGACTTATTAATCCTTACAATGGTGTTCCTAACTACAACCAGAAGTTCAAGGGCAAGGCCACATTAACTGTAGACAAGTCATCCAGCACAGCCTACATGGAGCTCCTCAGTCTGACATCTGAGGACTCTGCAGTCTATTACTGTGCAAGAAGGGACTATGATTACGACTCCTATGCTATGGACTACTGGGGTCAAGGGACTCTGGTCACTGTCTCTGCAGGTGGCCTCGGGGGCCTGGTCGACAGGAACCTCAGTCACCGTCTCCTCAGGTGGCCTCGGGGGCCTGGTCGAC (SEQ ID NO:17) 3. Light chain amino acid sequence of P9-1 AAQPAMADIEMTQSPASLAVSLGQRATISYRASKSVSTSGYSYMHWSQQKPGQPPRLLICLVSNLESGVPARFSGSGSGTDFTLNIHPVEEEDAATYYCQHIRELTRSEKAQSWKSKEAAVAAEVAQAV (SEQ ID NO:14) The CDR regions are as follows: ----CDR1---> <--CDR2--> <---CDR3---- KSVSTSGYSY(SEQ ID NO:9)___LVS.......___QHIRELTRSEKAQSWKSK(SEQ ID NO:10) The FR area is as follows: FR1: DIEMTQSPASLAVSLGQRATISYRAS (SEQ ID NO:30) FR2:MHWSQQKPGQPPRLLIC (SEQ ID NO:31) FR3: NLESGVPARFSGSGSGTDFTLNIHPVEEEDAATYYC (SEQ ID NO:32) 4. P9-1 light chain nucleotide sequence GCGGCCCAGCCGGCCATGGCAGACATCGAGATGACCCAGTCTCCTGCTTCCTTAGCTGTATCTCTGGGGCAGAGGGCCACCATCTCATACAGGGCCAGCAAAAGTGTCAGTACATCTGGCTATAGTTATATGCACTGGAGCCAACAGAAACCAGGACAGCCACCCAGACTCCTCATCTGTCTTGTATCCAACCTA GAATCTGGGGTCCCTGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACCCTCAACATCCATCCTGTGGAGGAGGAGGATGCTGCAACCTATTACTGTCAGCACATTAGGGAGCTTACACGTTCGGAGAAGGCACAAAGTTGGAAATCAAAGGAGGCGGCGGTAGCGGCGGAGGTGGCTCAGGCGGTG (SEQ ID NO:18) Example 8: Application of PT-specific binding active monoclonal antibody In the production of acellular pertussis vaccines, the international trend has been towards using ELISA to accurately quantify each individual antigenic component and to quantitatively monitor their separation and purification. ELISA, with its advantages of ease of operation, high sensitivity, and strong specificity, is one of the commonly used methods for antigen content determination.

[0095] The monoclonal antibodies used in the ELISA method have the following advantages: (1) They can be produced indefinitely and stably in vitro through hybridoma or recombinant technology, ensuring that different batches of monoclonal antibodies have the same specificity, affinity and biochemical characteristics; (2) They are produced by a single B cell clone, recognize and bind only to a specific antigenic epitope on the antigen, and can distinguish structurally similar substances, with high specificity and accuracy; (3) They have high affinity and high detection sensitivity; (4) Through docking calculation, alanine scanning mutagenesis and surface plasmon resonance, conserved immune sites of antigens can be identified to achieve standardization, ensuring the comparability and reproducibility of test results from different laboratories and at different time points.

[0096] This invention independently developed highly specific binding monoclonal antibodies P9-1 and N50-2 targeting the S2 and S4 subunits of antigen PT, respectively, using hybridoma technology. HRP-labeled secondary antibodies and PT rabbit polyclonal antibodies were prepared using these two monoclonal antibodies to establish a sandwich ELISA method for the determination of PT antigen content. The reproducibility results of the standard curves for S2 and S4 binding monoclonal antibodies are shown below. Figure 9 , Figure 10 .

[0097] Depend on Figure 9 , Figure 10 As shown, the ELISA method established based on monoclonal antibodies targeting the S2 and S4 epitopes of the PT subunits, obtained through independent screening, has linear standard curve quantitation limits ranging from 117-3000 ng / mL and 117-2000 ng / mL, respectively. R 2 All values ​​are greater than 0.97, indicating good robustness of the standard curve.

[0098] Furthermore, as shown in Tables 2 and 3, at concentrations of the target antigen PT higher than 10 times, the OD values ​​for FHA, PRN, DT, and TT were comparable to the blank values. However, when the target antigen PT concentration was 0.3 μg / mL, the OD values ​​for the monoclonal antibodies targeting different epitopes of the S2 and S4 subunits were above 0.2. This indicates that these monoclonal antibodies with high binding activity to different epitopes of the PT subunits have good specificity. Using the self-developed monoclonal antibodies targeting the S2 and S4 subunits as detection antibodies allows for the specific identification of the pertussis subunit integrity in the pertussis vaccine components and enables quantitative analysis.

[0099] Table 2 S2-Specificity Assessment (OD Value)

[0100] Table 3 S4 - Specificity Assessment (OD Value)

[0101] The above description of the embodiments is provided to enable those skilled in the art to understand and use the invention. It will be apparent to those skilled in the art that various modifications can be made to these embodiments, and the general principles described herein can be applied to other embodiments without inventive effort. Therefore, the present invention is not limited to the above embodiments, and any improvements and modifications made by those skilled in the art based on the disclosure of the present invention without departing from the scope of the invention should be within the protection scope of the present invention.

Claims

1. Monoclonal antibodies that specifically bind to pertussis toxin, among which, The monoclonal antibody comprises a heavy chain variable region and a light chain variable region; The heavy chain variable region comprises amino acid sequences HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3, respectively; or HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO:6, SEQ ID NO:7, and SEQ ID NO:8, respectively. The light chain variable region comprises the amino acid sequences LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO:4, AAS, and SEQ ID NO:5, respectively; or LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO:9, LVS, and SEQ ID NO:10, respectively.

2. The monoclonal antibody according to claim 1, wherein, The monoclonal antibody comprises HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3, and LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO:4, AAS, and SEQ ID NO:5; or The HCDR1, HCDR2 and HCDR3 shown in SEQ ID NO:6, SEQ ID NO:7 and SEQ ID NO:8 are combined with the LCDR1, LCDR2 and LCDR3 shown in SEQ ID NO:9, LVS and SEQ ID NO:10; Preferably, the monoclonal antibody comprises a heavy chain and a light chain, wherein the heavy chain contains an amino acid sequence as shown in SEQ ID NO:11 or SEQ ID NO:13; and the light chain contains an amino acid fragment as shown in SEQ ID NO:12 or SEQ ID NO:

14. Preferably, the monoclonal antibody comprises the heavy chain shown in SEQ ID NO:11 and the light chain shown in SEQ ID NO:12; or The heavy chain shown in SEQ ID NO:13 and the light chain shown in SEQ ID NO:

14.

3. A polynucleotide, wherein the polynucleotide encodes the monoclonal antibody of claim 1 or 2.

4. The polynucleotide according to claim 3, wherein, The heavy chain encoding the monoclonal antibody comprises a nucleotide sequence as shown in SEQ ID NO:15 or SEQ ID NO:17; The light chain encoding the monoclonal antibody comprises a nucleotide sequence as shown in SEQ ID NO:16 or SEQ ID NO:18; Preferably, the monoclonal antibody comprises the heavy chain shown in SEQ ID NO:15 and the light chain shown in SEQ ID NO:16; or The heavy chain shown in SEQ ID NO:17 and the light chain shown in SEQ ID NO:

18.

5. A carrier containing the polynucleotide as described in claim 3 or 4.

6. A host cell containing the polynucleotide of claim 3 or 4 or the vector of claim 5.

7. A kit for detecting pertussis toxin, wherein, The kit contains the monoclonal antibody as described in claim 1 or 2.

8. Use of the monoclonal antibody of claim 1 or 2, the polynucleotide of claim 3 or 4, the vector of claim 5, or the host cell of claim 6 in the preparation of reagents or kits for detecting pertussis toxin.

9. The use of the monoclonal antibody of claim 1 or 2, the polynucleotide of claim 3 or 4, the vector of claim 5, the host cell of claim 6, and the kit of claim 7 in the detection of pertussis toxin for non-diagnostic purposes.

10. The monoclonal antibody of claim 1 or 2, the polynucleotide of claim 3 or 4, the vector of claim 5, the host cell of claim 6, and the kit of claim 7 are used for quality testing of vaccines containing pertussis toxin antigen.

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