Humanized monoclonal antibody to helicobacter pylori cytotoxin-associated protein a and uses thereof

Abstract

This invention relates to a humanized Helicobacter pylori cytotoxin-associated protein A monoclonal antibody and its application. The humanized Helicobacter pylori cytotoxin-associated protein A monoclonal antibody includes a heavy chain variable region and a light chain variable region. The nucleic acid sequence of the heavy chain variable region includes CDR1 shown in SEQ ID No. 1 or SEQ ID No. 2, CDR2 shown in SEQ ID No. 3 or SEQ ID No. 4, and CDR3 shown in SEQ ID No. 5 or SEQ ID No. 6. The nucleic acid sequence of the light chain variable region includes CDR1 shown in SEQ ID No. 7 or SEQ ID No. 8, CDR2 with the sequence LVS or KVS, and CDR3 shown in SEQ ID No. 9 or SEQ ID No. 10.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to a humanized Helicobacter pylori cytotoxin-associated protein A monoclonal antibody and its application. Background Technology

[0002] Helicobacter pylori is a pathogenic bacterium that colonizes the human gastric mucosa and is closely associated with the development of chronic gastritis, peptic ulcers, gastric mucosa-associated lymphoid tissue lymphoma, and even gastric cancer. In 1994, the World Health Organization classified it as a Group 1 carcinogen. The pathogenicity of this bacterium varies significantly, primarily depending on the virulence factors it carries. Cytotoxin-associated gene A (CagA) is one of the most important virulence factors; CagA-positive strains are significantly associated with more severe gastric mucosal lesions and a higher risk of gastric cancer. Therefore, highly specific and sensitive accurate detection of CagA protein is crucial for clinical diagnosis, pathogen typing, and research into pathogenic mechanisms.

[0003] In current technologies, antibodies used to detect CagA are typically prepared using traditional hybridoma techniques. While hybridoma technology can obtain naturally paired antibodies, the process is cumbersome, time-consuming, and the resulting antibodies are mostly murine in origin. Direct application to humans can trigger strong human anti-mouse antibody responses, limiting its application in clinical diagnosis and treatment. To address the immunogenicity issue, phage display technology is often used to prepare human anti-CagA monoclonal antibodies. Although this avoids animal immunization and yields fully human antibodies, the core problem lies in the random combination of the antibody's light and heavy chain genes in vitro, disrupting the natural pairing relationships of light and heavy chains selected by B cells in vivo. This often results in antibodies with lower affinity and specificity than natural antibodies, increasing the difficulty and cost of subsequent screening for high-quality clones.

[0004] Humanized antibody technology transplants the complementarity-determining region (CDR) of highly specific animal-derived antibodies onto the framework of human antibodies. While preserving the high affinity and precise targeting ability of the original antibody to the greatest extent, it significantly reduces its immunogenicity in the human body, thereby significantly improving treatment safety and drug half-life. In the diagnostic field, humanized antibodies against human samples can achieve higher detection specificity. This technology uses naturally secreted antibodies in the body as a blueprint, avoiding the blindness of random pairing of light and heavy chains when displaying phages in a fully human antibody library. While ensuring efficacy, it has the engineering advantages of low clinical translation risk and a more robust development path.

[0005] Therefore, how to obtain highly efficient humanized anti-CagA antibodies through humanized antibody technology has become an urgent technical problem to be solved. Summary of the Invention

[0006] To address the aforementioned technical problems, this invention provides a humanized Helicobacter pylori cytotoxin-associated protein A monoclonal antibody and its application. This antibody can specifically recognize and bind to Helicobacter pylori CagA protein, exhibiting good affinity and specificity.

[0007] To achieve this objective, the present invention adopts the following technical solution:

[0008] In a first aspect, the present invention provides a humanized Helicobacter pylori cytotoxin-associated protein A monoclonal antibody, wherein the humanized Helicobacter pylori cytotoxin-associated protein A monoclonal antibody comprises a heavy chain variable region and a light chain variable region;

[0009] The amino acid sequence of the heavy chain variable region includes CDR1 shown in SEQ ID No. 1 or SEQ ID No. 2, CDR2 shown in SEQ ID No. 3 or SEQ ID No. 4, and CDR3 shown in SEQ ID No. 5 or SEQ ID No. 6;

[0010] The amino acid sequence of the light chain variable region includes CDR1 as shown in SEQ ID No. 7 or SEQ ID No. 8, CDR2 as LVS or KVS, and CDR3 as shown in SEQ ID No. 9 or SEQ ID No. 10.

[0011] SEQ ID No.1: GYTFTEYT.

[0012] SEQ ID No. 2: GYSITSGYY.

[0013] SEQ ID No. 3: FNPNNGGT.

[0014] SEQ ID No.4: ISYDGSN.

[0015] SEQ ID No. 5: SKNFDV.

[0016] SEQ ID No. 6: ARISTGAKDY.

[0017] SEQ ID No. 7: KSVSTSGYSY.

[0018] SEQ ID No. 8: QSLVHSNGNTY.

[0019] SEQ ID No. 9: QHIRELTR.

[0020] SEQ ID No. 10: SQSTHIPWT.

[0021] The antibody provided by this invention can directly isolate a single B cell that recognizes CagA from an immune host, ensuring the natural pairing of the antibody light chain and heavy chain, thereby maximizing the preservation of the antibody's high affinity and high specificity; it obtains the sequence information of the antibody gene, providing a basis for subsequent rapid and precise modification, fundamentally solving the immunogenicity problem; the preparation process is efficient and fast, overcoming the shortcomings of long cycle and low efficiency of hybridoma technology.

[0022] Preferably, the amino acid sequence of the heavy chain variable region includes CDR1 shown in SEQ ID No. 1, CDR2 shown in SEQ ID No. 3, and CDR3 shown in SEQ ID No. 5.

[0023] Preferably, the amino acid sequence of the light chain variable region includes CDR1 shown in SEQ ID No. 7, CDR2 with the LVS sequence, and CDR3 shown in SEQ ID No. 9.

[0024] Preferably, the amino acid sequence of the heavy chain variable region includes CDR1 shown in SEQ ID No. 2, CDR2 shown in SEQ ID No. 4, and CDR3 shown in SEQ ID No. 6.

[0025] Preferably, the amino acid sequence of the light chain variable region includes CDR1 shown in SEQ ID No. 8, CDR2 with the S sequence being KVS, and CDR3 shown in SEQ ID No. 10.

[0026] Preferably, the humanized Helicobacter pylori cytotoxin-associated protein A monoclonal antibody further includes a heavy chain constant region and a light chain constant region.

[0027] Preferably, the amino acid sequence of the heavy chain constant region is as shown in SEQ ID No. 11.

[0028] SEQ ID No. 11:

[0029] MGWSCIILFLVATATGVHSEVQLQQSGPELVKPGASVKISCKTSGYTFTEYTMHWVKQSHGKSLEWIGGFNPNNGGTSYNQKFKGKATLTVDKSSSTAYMELRSLTSEDSAVYYY SKNFDVWGAGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVE PKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPI EKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

[0030] Preferably, the amino acid sequence of the light chain constant region is as shown in SEQ ID No. 12.

[0031] SEQ ID No. 12:

[0032] RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC.

[0033] Secondly, the present invention provides a nucleic acid molecule that encodes the humanized Helicobacter pylori cytotoxin-associated protein A monoclonal antibody described in the first aspect.

[0034] Preferably, the nucleic acid sequence of the heavy chain variable region includes CDR1 shown in SEQ ID No. 13 or SEQ ID No. 14, CDR2 shown in SEQ ID No. 15 or SEQ ID No. 16, and CDR3 shown in SEQ ID No. 17 or SEQ ID No. 28;

[0035] The nucleic acid sequence of the light chain variable region includes CDR1 shown in SEQ ID No. 19 or SEQ ID No. 20, CDR2 shown in CTGGTGTCC or AAGGTGTCT, and CDR3 shown in SEQ ID No. 21 or SEQ ID No. 22.

[0036] SEQ ID No. 13: GGCTACACCTTCACCGAGTATACC.

[0037] SEQ ID No. 14: GGATACTCCATCACCTCTGGCTACTAT.

[0038] SEQ ID No. 15: TTTAATCCCAACAACGGCGGAACA.

[0039] SEQ ID No. 16: ATCTCCTACGACGGCTCCAAC.

[0040] SEQ ID No. 17: TCCAAGAACTTCGACGTG.

[0041] SEQ ID No. 18: GCCAGAATCTCCACCGGCGCCAAGGACTAC.

[0042] SEQ ID No. 19: AAGTCCGTGTCTACCTCTGGCTACTCCTAC.

[0043] SEQ ID No. 20: CAAAGCCTGGTGCATTCCAACGGCAACACCTAC.

[0044] SEQ ID No. 21: CAGCACATCAGAGAGCTGACCAGA.

[0045] SEQ ID No. 22: TCTCAGTCTACCCACATCCCTTGGACC.

[0046] Thirdly, the present invention provides an expression vector containing the nucleic acid molecule described in the second aspect; and after transfection, transduction or transformation of host cells, the expression vector causes the host cells to express the humanized Helicobacter pylori cytotoxin-associated protein A monoclonal antibody described in the first aspect.

[0047] Preferably, the expression vector further includes a kozak sequence and a signal peptide nucleic acid sequence.

[0048] Fourthly, the present invention provides the application of the humanized Helicobacter pylori cytotoxin-associated protein A monoclonal antibody according to the first aspect, the nucleic acid molecule according to the second aspect, or the expression vector according to the third aspect in the preparation of a reagent for detecting the content of cytotoxin-associated protein A.

[0049] Fifthly, the present invention provides an antibody pair for detecting cytotoxin-associated protein A, the antibody pair comprising humanized Helicobacter pylori cytotoxin-associated protein A monoclonal antibody 1 and humanized Helicobacter pylori cytotoxin-associated protein A monoclonal antibody 2.

[0050] Preferably, the amino acid sequence of the heavy chain variable region of the humanized Helicobacter pylori cytotoxin-associated protein A monoclonal antibody 1 includes CDR1 shown in SEQ ID No. 1, CDR2 shown in SEQ ID No. 3, and CDR3 shown in SEQ ID No. 5.

[0051] Preferably, the amino acid sequence of the light chain variable region of the humanized Helicobacter pylori cytotoxin-associated protein A monoclonal antibody 1 includes CDR1 shown in SEQ ID No. 7, CDR2 with the LVS sequence, and CDR3 shown in SEQ ID No. 9.

[0052] Preferably, the amino acid sequence of the heavy chain variable region of the humanized Helicobacter pylori cytotoxin-associated protein A monoclonal antibody 2 includes CDR1 shown in SEQ ID No. 2, CDR2 shown in SEQ ID No. 4, and CDR3 shown in SEQ ID No. 6.

[0053] Preferably, the amino acid sequence of the light chain variable region of the humanized Helicobacter pylori cytotoxin-associated protein A monoclonal antibody 2 includes CDR1 shown in SEQ ID No. 8, CDR2 with the sequence KVS, and CDR3 shown in SEQ ID No. 10.

[0054] In a sixth aspect, the present invention provides a method for detecting cytotoxin-associated protein A, the method comprising using the antibody for detecting cytotoxin-associated protein A as described in the fifth aspect of the invention.

[0055] Preferably, the method includes a double-anti-sandwich method.

[0056] Compared with the prior art, the present invention has at least the following beneficial effects:

[0057] 1. The antibodies screened in this invention have been verified to have excellent binding activity and pairing effects. A dual-antibody sandwich ELISA detection system established using these antibodies as the core exhibits a wide linear range, high precision, and good clinical relevance. This provides a reliable antibody source and core technical solution for developing domestically produced, high-performance Helicobacter pylori CagA genotyping kits, immunohistochemical reagents, or rapid test strips. The obtained highly specific antibodies are powerful molecular tools for studying the pathogenic mechanism of CagA protein in gastric diseases and intracellular signal transduction. Simultaneously, they also provide potential candidate molecules for future development of antibody-targeted therapies or drug conjugates based on CagA targets.

[0058] 2. This invention successfully obtained the precise sequence information of the antibody's variable region and successfully completed the humanization of the antibody. This fundamentally avoids the human anti-mouse antibody reaction that occurs when traditional murine hybridoma antibodies are directly used in humans, greatly improving the safety and applicability of the antibody in diagnostic applications (especially in vivo imaging or treatment).

[0059] 3. This invention directly obtains antibody genes from single B cells of an immune host using flow cytometry sorting technology, fully preserving the natural pairing relationship between the antibody light and heavy chains formed in vivo. This is key to maintaining the optimal spatial conformation of the antibody and achieving high affinity and high specificity, fundamentally overcoming the affinity loss and screening difficulties caused by the random combination of light and heavy chains in technologies such as phage display. Compared to traditional hybridoma technology (which typically takes 3-6 months and involves cumbersome fusion and screening processes with unstable success rates), the single-cell isolation and PCR technology of this invention significantly shortens the cycle (antibody genes can be obtained within weeks), has a high degree of process standardization, significantly increases throughput, and can obtain multiple candidate antibody clones in parallel.

[0060] 4. This invention completely avoids the risk of antibody loss or reduced yield due to the instability of hybridoma cells during passage. The obtained antibody gene sequence is stable and can be produced on a large scale, stably, and reproducibly through a recombinant expression system, with uniform and controllable product quality. The method of this invention directly obtains the antibody DNA sequence, making subsequent humanization modification, affinity maturation, conversion to different expression systems (such as CHO, HEK293, etc.), and large-scale industrial production of antibodies direct, efficient, and purposeful. Attached Figure Description

[0061] Figure 1 This is a graph showing the results of flow cytometry analysis.

[0062] Figure 2 This is a graph showing the electrophoresis results of RT-PCR products.

[0063] Figure 3 This is a graph showing the results of antibody SDS-PAGE detection.

[0064] Figure 4 This is a graph showing the results of the correlation analysis.

[0065] Figure 5 To detect the linear range analysis graph.

[0066] Figure 6 The result of assigning values ​​to antibody #3.

[0067] Figure 7 The result of assigning values ​​to antibody #5. Detailed Implementation

[0068] The technical solution of the present invention will be further described below with reference to the accompanying drawings and specific embodiments. However, the following examples are merely simplified examples of the present invention and do not represent or limit the scope of protection of the present invention. The scope of protection of the present invention is determined by the claims.

[0069] Example 1

[0070] This embodiment describes the acquisition of the variable region gene of mouse anti-CagA antibody.

[0071] Mice were first immunized with prokaryotically expressed cytotoxin-associated protein A emulsified with Freund's complete adjuvant at a 1:1 ratio. Two weeks later, mice were immunized a second time with Freund's incomplete adjuvant, followed by a third immunization two weeks later with Freund's incomplete adjuvant. One week later, blood was collected from the orbital sinus to determine antibody titers. One week later, a shock immunization was performed using the antigen. Three days later, mice were sacrificed, and spleens were harvested. Spleen cell suspensions were prepared, and CagA protein was conjugated using PE to form fluorescent protein. These fluorescent proteins were then incubated with flow cytometry-fluorescent antibodies. The detection items and fluorescence carriers are shown in Table 1. Flow cytometry analysis was performed using a BD Ariafusion flow cytometer. To obtain highly specific antibodies, the flow cytometry analysis results are as follows: Figure 1 As shown, we will eventually use CD45R + F4 / 80-IgG + CagA + The top 10 cells were sorted into 96-well plates containing 10 μL of lysis buffer.

[0072] Table 1

[0073]

[0074] RNA was extracted using the ThermoFisher RNA Mini Kit following standardized procedures. cDNA was prepared using the TransScript® All-in-One First-Strand cDNA Synthesis SuperMix following standardized procedures. Using single B-cell cDNA as a template, pre-designed light and heavy chain mouse variable region amplification primers were added. The primer sequences are shown in Table 2 below. A nested PCR amplification system was prepared using TakaraTaq enzyme, with cycling steps of 98℃-10s, 55℃-30s, and 72℃-60s for 30 cycles. Mouse GAPDH was used as a positive control.

[0075] Table 2

[0076]

[0077] Electrophoresis was performed using a 2% agarose gel to determine the fragment size of the variable regions of the light and heavy chains after PCR amplification. Figure 2 As shown, a total of 10 successfully amplified PCR products were obtained, with the light chain being approximately 350 bp and the heavy chain fragment being slightly larger than the light chain.

[0078] Add pUCm-T vector, PCR product, ligation buffer, and T4 DNA Ligase, and ligate at 20℃ for 6 h to form a circular plasmid. Take 100 μL of competent E. coli DH5α, add 100 ng of plasmid DNA, incubate on ice for 30 min, perform precise heat shock at 42℃ for 90 s, and immediately incubate on ice for 2 min; add 900 μL of antibiotic-free LB medium, and incubate at 37℃ for 50 min (shaking at 150 rpm); pipette 100 μL of bacterial culture and spread evenly on an LB agar plate containing 0.5% ampicillin using an L-shaped spreader, and incubate upside down at 37℃ for 12 h. Select a single colony and send it to Sangon Biotech (Shanghai) Co., Ltd. for sequencing.

[0079] Example 2

[0080] This embodiment involves antibody expression.

[0081] The monoclonal antibody heavy and light chain variable region sequences obtained from the 10 cells in Example 1, as well as the human heavy and light chain constant region sequences downloaded from IMGT, were given to Sangon Biotech (Shanghai) Co., Ltd. to synthesize the nucleic acid sequence of the complete antibody. In addition, the kozak sequence and signal peptide sequence also need to be synthesized. Subsequently, the monoclonal antibody heavy chain, monoclonal antibody light chain, kozak sequence, and signal peptide sequence were inserted into the pcDNA3.4 plasmid to complete the construction of the recombinant plasmid.

[0082] Kozak sequence (SEQ ID No. 50): GCCACCATGG.

[0083] Signal peptide:

[0084] Heavy chain signal peptide (SEQ ID No. 51):

[0085] ATGGGCTGGAGCTGCATCATCCTGTTCCTGGTGGCAACCGCAACAGGAGTGCACTCT.

[0086] Light chain signal peptide (SEQ ID No. 52): ATGGAAACAGATACCCTGCTTCTCTGGGTCCTGCTGCTGTGGGTGCCCGGCAGCACAGGC.

[0087] Competent E. coli were collected, plasmid DNA was added, and the culture was incubated on ice for 30 minutes, followed by precise heat shock at 42°C for 90 seconds. The culture was then added to antibiotic-free LB medium and incubated at 37°C. The bacterial culture was then spread evenly on LB agar plates containing ampicillin using an L-shaped spreader and incubated upside down at 37°C for 12 hours. Single colonies were picked and incubated in 200 mL of liquid medium for 12 hours to harvest E. coli for plasmid extraction.

[0088] The monoclonal antibody was expressed in CHO cells at a mass ratio of PEI transfection reagent to recombinant plasmid of monoclonal antibody of 4:1. After culturing for 7 days, the cell culture medium was collected, sonicated, and then centrifuged at 9000 rpm for 15 min. The supernatant was collected and filtered through a 0.22 μm filter membrane.

[0089] Monoclonal antibodies were purified using Protein A gravity strain. IgG antibodies were eluted from the column using 0.1 M citric acid elution buffer, followed by dialyzing in PBS with a 34 mm, 3.5 kDa dialysis bag and magnetic stirrer for 8 h. 15 μL / well of each purified fraction was taken for SDS-PAGE electrophoresis, followed by Coomassie brilliant blue staining for 1 h, and destaining in 30% methanol and 10% acetic acid for 8 h. The light and heavy chain fragments of the target antibody were observed using a gel imaging system. The results are shown below. Figure 3 As shown, the light chain (approximately 25 kDa) and heavy chain (approximately 55 kDa) bands of the monoclonal antibody were single in the elution buffer, indicating that 10 monoclonal antibodies were successfully purified.

[0090] Example 3

[0091] This embodiment performs monoclonal antibody pair screening.

[0092] (1) Indirect ELISA detection

[0093] To verify antibody specificity, CagA protein was used to coat ELISA plates for 8 h, followed by blocking with 5% BSA for 2 h. Protein expression supernatant was added and incubated at 37°C for 1 h. The plates were washed three times with PBST, and HRP-labeled rabbit anti-human IgG monoclonal antibody (1:10000) was added and incubated at 37°C for 1 h. The plates were washed three times with PBST, and TMB chromogenic buffer was added for color development at room temperature in the dark for 30 min. The reaction was terminated by adding 0.5M H2SO4. 100 μL was added to each well, and the absorbance was measured at OD=450 nm using a microplate reader.

[0094] Judgment: S / N = Sample OD value / Negative control OD value. If S / N > 2.0, it is judged as positive. The results are shown in Table 3. The S / N value of the monoclonal antibody is greater than 2.0, and it is judged as a positive sample, indicating that the monoclonal antibody prepared in this invention can bind to CagA.

[0095] Table 3

[0096]

[0097] (2) BLI detection of CagA antibody affinity

[0098] To evaluate the affinity between the prepared CagA monoclonal antibody and the CagA standard protein, this embodiment establishes a detection procedure for the affinity between the CagA monoclonal antibody and the standard protein using biomembrane interference (BLI) technology. The antibody is immobilized on the tip of a biosensor, immersed in a buffer to establish a baseline, and then exposed to an antigen solution for binding reaction, with the binding signal monitored in real time. Subsequently, it is transferred to a buffer for dissociation, and kinetic data (such as binding rate kon, dissociation rate koff, and equilibrium dissociation constant KD) are obtained.

[0099] The experimental procedure for analyzing the binding kinetics of CagA monoclonal antibody and CagA antigen using the Fortebio biomolecular interaction assay instrument is as follows: First, seven AMC sensors, moistened with SD buffer, were placed in the designated positions on the sensor holder. SD buffer, 25 μg / mL CagA monoclonal antibody working solution, 1000 pg / mL CagA protein solution, blank solution, regeneration buffer, and neutralization SD buffer were added to each well in a preset order in a 96-well black sample plate. The sample volume for each well was 250 μL. Then, in the Data Acquisition 12 software, the kinetic detection module was selected, the sample plate temperature was set to 30℃, and the data acquisition frequency was set to 2Hz. The program was run to sequentially complete the real-time detection of the sensor baseline equilibration, antibody loading, secondary equilibration, CagA antibody binding, and dissociation phases. Finally, the sensors were eluted with glycine regeneration buffer and neutralized to obtain binding kinetic data. The test conditions are shown in Table 4.

[0100] Table 4

[0101]

[0102] Data was analyzed using Data Analysis 12.0 software. Blank wells without bound samples were used as controls to subtract background. Steady-state fitting was performed with concentration as the x-axis and binding value of each concentration sample as the y-axis to calculate the affinity (KD) between CagA monoclonal antibody and CagA protein. The results are shown in Table 5.

[0103] Table 5

[0104]

[0105] (3) Double-antibody sandwich method for screening antibody pairs

[0106] Ten antibodies were paired, and a double-antibody sandwich assay was used to screen for suitable antibody pairs. The ten antibodies were then used as capture and detection antibodies, respectively. The experimental steps included: coating 96-well ELISA plates with CagA monoclonal antibodies 1#, 2#, 3#, 4#, 5#, 6#, 7#, 8#, 9#, and 10# at a concentration of 5.0 μg / mL and incubating overnight at 4°C; washing the ELISA plate three times with PBST, blocking with 2% BSA at 37°C for 1 h, discarding the blocking solution, and then washing the ELISA plate three more times with PBST; diluting the CagA protein with 1×ELISA diluent to concentrations of 4000 pg / mL, 2000 pg / mL, 1000 pg / mL, and 0 pg / mL, adding 100 μL to each well of the ELISA plate, mixing well, and incubating at 37°C for 30 hours. min; after discarding the liquid in the wells, wash the plate three times with PBST; add biotin-labeled diluted CagA monoclonal antibodies (1:4000 dilution, 100 μL per well), mix well, and incubate at 37°C for 30 min. After discarding the liquid in the wells, wash the plate three times with PBST; add TMB chromogenic solution to the plate (100 μL per well) and incubate at room temperature for 15 min; add 0.5 M H2SO4 to the plate to stop the reaction (100 μL per well); measure the absorbance at OD=450 nm using an ELISA reader. The detection results of the antibody ELISA combination screening of the coated antibodies are shown in Tables 6, 7, and 8.

[0107] Table 6

[0108]

[0109] Table 7

[0110]

[0111] Table 8

[0112]

[0113] The results showed that the best experimental results were obtained when using 5# as the capture antibody and 3# as the detection antibody. The amino acid sequence of the heavy chain variable region of antibody 3# is CDR1 shown in SEQ ID No. 1, CDR2 shown in SEQ ID No. 3, and CDR3 shown in SEQ ID No. 5; the amino acid sequence of the light chain variable region is CDR1 shown in SEQ ID No. 7, CDR2 with LVS sequence, and CDR3 shown in SEQ ID No. 9.

[0114] The amino acid sequences of the heavy chain variable region of antibody #5 are CDR1 shown in SEQ ID No. 2, CDR2 shown in SEQ ID No. 4, and CDR3 shown in SEQ ID No. 6; the amino acid sequences of the light chain variable region include CDR1 shown in SEQ ID No. 8, CDR2 with the KVS sequence, and CDR3 shown in SEQ ID No. 10.

[0115] Example 4

[0116] Monoclonal antibody specificity and cross-reactivity verification

[0117] To verify the detection specificity of antibody #5 as the capture antibody and antibody #3 as the detection antibody, a cross-reactivity assay was performed using a double-antibody sandwich ELISA to examine the cross-reactivity of the antibodies with other virulence factors of Helicobacter pylori, common gastrointestinal pathogens, and human interfering proteins.

[0118] Target antigen: Recombinant Helicobacter pylori CagA protein. Cross antigens: VacA, UreA, UreB, NapA, HpaA, Escherichia coli, Salmonella, Staphylococcus aureus, human serum albumin, Klebsiella pneumoniae, Proteus, human IgG, and total protein from human gastric mucosa.

[0119] Results judgment criteria: Cross-reactivity rate (%) = (CagA antigen OD value - Blank OD value) / (CagA antigen OD value - Blank OD value) × 100%. A cross-reactivity rate ≤ 1.0% is considered as no cross-reactivity. Specific results are shown in Table 9.

[0120] Table 9

[0121]

[0122] Example 5

[0123] This embodiment performs application scenario detection using monoclonal antibodies.

[0124] The results of the double-antibody sandwich ELISA kit for CagA detection manufactured by Company A were compared with those of antibody pairs #3 and #5. A total of 45 samples were collected, including 34 CagA-positive sera and 11 CagA-negative sera from patients with gastric ulcers whose gastric mucosa cultures were positive for Helicobacter pylori. The kit was operated according to the instructions. The results are shown in Tables 10 and 11. The detection steps for the antibody pairs were consistent with the double-antibody sandwich method described in Example 3. The absorbance values ​​at OD=450nm on the microplate reader are shown in Table 9. The concordance rate with the purchased kit results is shown in Table 12.

[0125] Compared with Company A's kit, the antibody pair in this invention can achieve a higher positive concordance rate and a negative concordance rate of 100%.

[0126] A scatter plot was drawn with CagA protein concentration as the ordinate and the measured CagA protein concentration as the abscissa, and correlation analysis was performed. The detection results are as follows: Figure 4 As shown, the detection results were subjected to linear regression analysis (y = 0.9254x + 99.896, R²). 2 =0.9182), indicating a good correlation between the two detection results.

[0127] Table 10

[0128]

[0129] Table 11

[0130]

[0131] Table 12

[0132]

[0133] Example 6

[0134] This embodiment performs a performance evaluation of the ELISA system.

[0135] (1) Test of the analytical range of the standard curve

[0136] CagA protein was used as the detection standard and serially diluted with PBS to prepare concentrations of 5500 pg / mL, 4000 pg / mL, 2000 pg / mL, 1000 pg / mL, 500 pg / mL, 250 pg / mL, 125 pg / mL, 62.5 pg / mL, 31.25 pg / mL, 15.625 pg / mL, and 0.20 pg / mL. The constructed ELISA system was tested using antibody #5 as the capture antibody and antibody #3 as the detection antibody. A standard curve was plotted with protein concentration on the x-axis and the corresponding OD=450 nm on the y-axis. The linear range analysis results of the CagA protein ELISA detection system are shown below. Figure 5 As shown, regression analysis results indicate that CagA protein concentrations between 0 and 5500 pg / mL exhibit good detection linearity: y = 0.0006x + 0.132, R0. 2 =0.9933.

[0137] (2) Precision analysis

[0138] CagA protein was diluted with PBS to 1000 pg / mL, 500 pg / mL, and 250 pg / mL as quality control samples. The constructed ELISA system was used to measure the above samples in 4 batches, with 8 replicates per batch. Intra-batch precision and inter-batch precision were calculated. The results are shown in Table 13. The results showed that the intra-batch coefficient of variation (CV) (CV = standard deviation / mean × 100%) was 3.67–4.42%, which was less than the standard value of 10%, and the inter-batch coefficient of variation was 5.41–7.40%, which was less than the standard value of 15%. The results indicate that the precision of CagA protein detection is good.

[0139] Table 13

[0140]

[0141] (3) Time stability test

[0142] Antibodies #3 and #5 were aliquoted into 50 μL portions at a concentration of 1 mg / mL and stored at -20°C. Every 6 months, one unopened antibody sample was taken and the hydrodynamic radius (rh), polydispersity index (PDI), and melting temperature (Tm) of the monoclonal antibodies #3 and #5 were determined using a Prometheus Panta multi-functional protein stability analyzer. Each sample was tested three times, and the average value was calculated. The absolute value of the relative deviation between the rh and Tm values ​​and the original sample test results should be ≤5%, and the PDI value should be less than 0.15.

[0143] Results Analysis: After being stored at -20℃ for 6, 12, 18, 24, 30, and 36 months, the changes in rh and Tm values ​​of monoclonal antibodies #3 and #5 were all within the allowable deviation range, and the PDI was less than 0.15. Visually, both antibodies were clear, transparent liquids without precipitation, flocculent matter, or suspended matter, maintaining a stable appearance. The affinity of antibody #3 changed between -1.81% and -0.21%, while that of antibody #5 changed between -1.21% and -0.12%. The OD value deviation of the results detected by the double-antibody sandwich ELISA was between -2.49% and 0.37%. This indicates that monoclonal antibodies #3 and #5 maintain good performance and strong stability even after being stored at -20℃ for 3 years.

[0144] (4) Freeze-thaw stability test

[0145] Antibodies #3 and #5 were stored at -20°C for 12 months, followed by seven freeze-thaw cycles in a 37°C water bath. Using the Prometheus Panta multi-functional protein stability analyzer, rh, PDI, and Tm values ​​were measured. Each sample was tested three times, and the average value was calculated. The absolute value of the relative deviation between the rh and Tm values ​​and the original sample test results should be ≤5%, and the PDI value should be less than 0.15.

[0146] For samples stored at -20℃ for 12 months, seven freeze-thaw cycles were performed. The changes in rh and Tm values ​​of both monoclonal antibodies #3 and #5 were within the allowable deviation range, and the PDI was less than 0.15. Both antibodies maintained good appearance after seven freeze-thaw cycles, remaining clear and transparent liquids without precipitation, flocculent matter, or suspended matter, exhibiting stable appearance. The affinity of antibody #3 changed between -2.26% and -0.25%, while that of antibody #5 changed between -1.79% and -0.20%. The OD value deviation of the results obtained using a double-antibody sandwich ELISA was between -4.01% and -0.37%. This indicates that monoclonal antibodies #3 and #5 maintain good performance and strong stability even after seven freeze-thaw cycles.

[0147] (5) Uniformity test

[0148] Fifteen monoclonal antibodies, numbered 3 and 5, were randomly selected and their concentrations were calibrated using an immunoglobulin G1 assay kit (chemiluminescence method). The average deviation rate of antibody 3 was 0.51%, and that of antibody 5 was 0.33%, indicating that both antibodies have good accuracy.

[0149] (6) Recovery test

[0150] Fetal bovine serum was used as the matrix solution to dilute the CagA protein detection standard to prepare test samples with mass concentrations of 4000 pg / mL, 2000 pg / mL, 1000 pg / mL, and 500 pg / mL. The samples were tested according to the ELISA system described above, with eight replicates for each sample. The recovery rate was calculated by taking the average value (recovery rate = detected value / theoretical value × 100%). In the recovery experiment, the recovery rates of CagA detection at different concentrations ranged from 98.01% to 106.04%, with an average recovery rate of 103.41%, indicating good accuracy for CagA detection.

[0151] Example 7

[0152] Application as a calibrator

[0153] (1) Calibration material configuration

[0154] Prepare a series of CagA antibody calibrators (numbered 3#1-4, 5#1-4) with concentrations of 50 ng / mL, 250 ng / mL, 450 ng / mL, and 650 ng / mL using the sample diluent for antibodies 3# and 5# of the present invention. Aliquot the calibrators of these two antibodies into 1 mL packages and store them at 2℃~8℃ or below -18℃ for long-term storage, avoiding repeated freeze-thaw cycles.

[0155] (2) Assignment verification scheme

[0156] Using the immunoglobulin G1 assay kit, antibody calibrators #3 and #5 were independently and repeatedly tested, with each concentration measured three times, and the concentrations of antibody calibrators #3 and #5 were calculated.

[0157] Acceptance criteria: The relative deviation (Bias) between the average test result and the theoretical concentration is ≤10%. Relative deviation is calculated as follows: Detection deviation (%) = (Average test result - Theoretical value) / Theoretical value × 100%.

[0158] (3) Calibrator value assignment test results

[0159] The test results and deviation calculations for antibody calibrators #3 and #5 are shown in Tables 14 and 15, respectively.

[0160] Table 14

[0161]

[0162] Table 15

[0163]

[0164] Results analysis showed that the relative deviations between the mean detection values ​​of the #3 CagA antibody at four concentration levels and the theoretical values ​​were 0.37%-0.76%, and the relative deviations between the mean detection values ​​of the #5 CagA antibody at four concentration levels and the theoretical values ​​were 0.29%-1.01%. The relative deviations for both antibodies were less than 10%, and the linear relationship was as follows: Figure 6 , Figure 7 As shown, antibodies #3 and #5 exhibited good linearity in the analytical method within a concentration range of 50 ng / mL to 650 ng / mL, with R² values ​​of 0.9985 and 0.9997, respectively. These results indicate that the calibrator concentrations are accurate and the linear relationship is good, meeting the requirements for use as a calibrator in the kit.

[0165] Example 8

[0166] Application as a quality control product

[0167] (1) Configuration of quality control materials

[0168] CagA antibody-negative artificial serum matrix was selected. Known concentrations of CagA antibody #3 were diluted to (50±10%) ng / mL and (100±10%) ng / mL using the artificial serum matrix; CagA antibody #5 was diluted to (50±10%) ng / mL and (100±10%) ng / mL. Two concentrations of each antibody were used as dual-level quality controls, numbered 3#50, 3#100, 5#50, and 5#100. These quality controls were aliquoted and stored for use as dual-level quality controls.

[0169] (2) Assignment scheme

[0170] The quality control samples were measured using an immunoglobulin G1 assay kit calibrated with calibrators. The assays were performed continuously for 5 days, with one batch tested each day and each batch tested four times. A total of 20 data points were obtained for each antibody at each concentration level.

[0171] Data processing and acceptance criteria: Outliers are removed from the test results according to the Grubbs criteria. The number of outliers removed should not exceed 5%. If the number of outliers exceeds 5%, the stability of the experimental system or the standardization of operation should be investigated and the results re-evaluated.

[0172] Accuracy and precision: The deviation between the detected value and the target value (theoretical mean) of all valid data is ≤10%.

[0173] Quality control limits are determined by calculating the arithmetic mean (xˉ) and standard deviation (s) after removing outliers, and setting the quality control range as xˉ±2s.

[0174] (3) Quality control product value assignment test results

[0175] The statistical analysis results of antibody quality control product #3 are shown in Tables 16 and 17.

[0176] Table 16

[0177]

[0178] Table 17

[0179]

[0180] Results Analysis: Experimental data show that when the 3#CagA antibody of this invention is used as a quality control at 50 ng / mL, the intra-assay coefficient of variation is 0.08%-0.48%, the inter-assay coefficient of variation is 1.30%, and the target value range is 48.98±2.3 ng / mL; when used as a quality control at 100 ng / mL, the intra-assay coefficient of variation is 0.16%-0.8%, the inter-assay coefficient of variation is 2.34%, and the target value range is 99.18±2.6 ng / mL. The detected values ​​are uniformly distributed within the set quality control limits and meet the quality control requirements.

[0181] The statistical analysis results of antibody quality control product #5 are shown in Tables 18 and 19.

[0182] Table 18

[0183]

[0184] Table 19

[0185]

[0186] Results Analysis: Experimental data show that when the 5#CagA antibody of this invention is used as a quality control at 50 ng / mL, the intra-assay coefficient of variation is 0.49%-1.02%, the inter-assay coefficient of variation is 2.34%, and the target value range is 49.16±2.30 ng / mL; when used as a quality control at 100 ng / mL, the intra-assay coefficient of variation is 0.25%-0.75%, the inter-assay coefficient of variation is 1.42%, and the target value range is 99.27±2.83 ng / mL. The detected values ​​are uniformly distributed within the set quality control limits and meet the quality control requirements.

[0187] The above results demonstrate that using the two antibodies of this invention as quality control materials exhibits good homogeneity, precision, and accuracy in value determination, making them suitable for quality control of the kits of this invention.

[0188] In summary, the anti-Helicobacter pylori CagA monoclonal antibodies prepared and screened by this invention exhibit good specificity and high affinity for antibodies #3 and #5, and can be used as detection antibodies and capture antibodies, respectively. They are effectively applied in ELISA methods, such as the ELISA double-antibody sandwich detection method, and possess good detection linearity, precision, stability, and uniformity, as well as good correlation with clinical serum values, meeting the requirements for CagA detection. In addition, these two antibodies also have good applications as calibrators and quality control materials.

[0189] The applicant declares that the above description is only a specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Those skilled in the art should understand that any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention fall within the protection and disclosure scope of the present invention.

Claims

1. A humanized Helicobacter pylori cytotoxin-associated protein A monoclonal antibody, characterized in that, The humanized Helicobacter pylori cytotoxin-associated protein A monoclonal antibody includes a heavy chain variable region and a light chain variable region; The amino acid sequence of the heavy chain variable region includes CDR1 shown in SEQ ID No. 1 or SEQ ID No. 2, CDR2 shown in SEQ ID No. 3 or SEQ ID No. 4, and CDR3 shown in SEQ ID No. 5 or SEQ ID No. 6; The amino acid sequence of the light chain variable region includes CDR1 as shown in SEQ ID No. 7 or SEQ ID No. 8, CDR2 as LVS or KVS, and CDR3 as shown in SEQ ID No. 9 or SEQ ID No.

10.

2. The humanized Helicobacter pylori cytotoxin-associated protein A monoclonal antibody according to claim 1, characterized in that, The amino acid sequence of the heavy chain variable region includes CDR1 shown in SEQ ID No. 1, CDR2 shown in SEQ ID No. 3, and CDR3 shown in SEQ ID No. 5; Preferably, the amino acid sequence of the light chain variable region includes CDR1 shown in SEQ ID No. 7, CDR2 with the LVS sequence, and CDR3 shown in SEQ ID No.

9.

3. The humanized Helicobacter pylori cytotoxin-associated protein A monoclonal antibody according to claim 1, characterized in that, The amino acid sequence of the heavy chain variable region includes CDR1 shown in SEQ ID No. 2, CDR2 shown in SEQ ID No. 4, and CDR3 shown in SEQ ID No. 6; Preferably, the amino acid sequence of the light chain variable region includes CDR1 shown in SEQ ID No. 8, CDR2 with the KVS sequence, and CDR3 shown in SEQ ID No.

10.

4. The humanized Helicobacter pylori cytotoxin-associated protein A monoclonal antibody according to any one of claims 1-3, characterized in that, The humanized Helicobacter pylori cytotoxin-associated protein A monoclonal antibody also includes a heavy chain constant region and a light chain constant region.

5. The humanized Helicobacter pylori cytotoxin-associated protein A monoclonal antibody according to claim 4, characterized in that, The amino acid sequence of the heavy chain constant region is as shown in SEQ ID No. 11; Preferably, the amino acid sequence of the light chain constant region is as shown in SEQ ID No.

12.

6. A nucleic acid molecule, characterized in that, The nucleic acid molecule encodes a humanized Helicobacter pylori cytotoxin-associated protein A monoclonal antibody as described in any one of claims 1-5.

7. An expression carrier, characterized in that, The expression vector contains the nucleic acid molecule of claim 6; and after transfection, transduction or transformation of host cells, the expression vector causes the host cells to express the humanized Helicobacter pylori cytotoxin-associated protein A monoclonal antibody of any one of claims 1-5. Preferably, the expression vector further includes a kozak sequence and a signal peptide nucleic acid sequence.

8. The use of the humanized Helicobacter pylori cytotoxin-associated protein A monoclonal antibody according to any one of claims 1-5, the nucleic acid molecule according to claim 6, or the expression vector according to claim 7 in the preparation of a reagent for detecting the content of cytotoxin-associated protein A.

9. An antibody pair for detecting cytotoxin-associated protein A, characterized in that, The antibody pair includes humanized Helicobacter pylori cytotoxin-associated protein A monoclonal antibody 1 and humanized Helicobacter pylori cytotoxin-associated protein A monoclonal antibody 2; Preferably, the amino acid sequence of the heavy chain variable region of the humanized Helicobacter pylori cytotoxin-associated protein A monoclonal antibody 1 includes CDR1 shown in SEQ ID No. 1, CDR2 shown in SEQ ID No. 3, and CDR3 shown in SEQ ID No. 5; Preferably, the amino acid sequence of the light chain variable region of the humanized Helicobacter pylori cytotoxin-associated protein A monoclonal antibody 1 includes CDR1 shown in SEQ ID No. 7, CDR2 with the LVS sequence, and CDR3 shown in SEQ ID No. 9; Preferably, the amino acid sequence of the heavy chain variable region of the humanized Helicobacter pylori cytotoxin-associated protein A monoclonal antibody 2 includes CDR1 shown in SEQ ID No. 2, CDR2 shown in SEQ ID No. 4, and CDR3 shown in SEQ ID No. 6; Preferably, the amino acid sequence of the light chain variable region of the humanized Helicobacter pylori cytotoxin-associated protein A monoclonal antibody 2 includes CDR1 shown in SEQ ID No. 8, CDR2 with the sequence KVS, and CDR3 shown in SEQ ID No.

10.

10. A method for detecting cytotoxin-associated protein A, characterized in that, The method includes detection using the antibody pair for detecting cytotoxin-associated protein A as described in claim 9.

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