Monoclonal antibody 4H12 of duodenal giardia CP1 protein and application thereof

Abstract

This invention belongs to the field of biotechnology, and particularly relates to a monoclonal antibody 4H12 for Giardia duodenalis CP1 protein and its application. The monoclonal antibody 4H12 for Giardia duodenalis CP1 protein has a heavy chain complementarity-determining region (CDR1) as shown in SEQ ID NO:5, a heavy chain complementarity-determining region (CDR2) as shown in SEQ ID NO:6, and a heavy chain complementarity-determining region (CDR3) as shown in SEQ ID NO:7; the monoclonal antibody 4H12 also has a light chain complementarity-determining region (CDR1) as shown in SEQ ID NO:13, a light chain complementarity-determining region (CDR2) with a KVS amino acid sequence, and a light chain complementarity-determining region (CDR3) as shown in SEQ ID NO:15. A "polyclonal antibody capture-monoclonal antibody detection" sandwich system is constructed by combining a rabbit-derived polyclonal antibody with the monoclonal antibody 4H12, which ensures antigen binding efficiency, reduces non-specific interference, and improves the sensitivity and accuracy of the detection system.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, and in particular relates to a monoclonal antibody 4H12 against Giardia lamblia CP1 protein and its applications. Background Technology

[0002] Giardiasis is a globally distributed protozoan parasitic disease primarily transmitted via the fecal-oral route. It infects a wide range of hosts, including humans, and clinical symptoms include diarrhea, dehydration, vomiting, and weight loss. It poses a serious threat to both humans and animals and has been listed as a key protozoan disease for prevention and control by the World Health Organization. Currently, nitroimidazole drugs are commonly used for treatment; however, these drugs have safety concerns such as gastrointestinal adverse reactions, drug resistance in the parasite, and potential teratogenic and carcinogenic effects. Therefore, improving the diagnostic accuracy of giardiasis is a crucial aspect of conducting epidemiological surveys and carrying out clinical testing.

[0003] Currently, microscopic examination remains the "gold standard" for diagnosing Giardia infection. However, this method is time-consuming, subjective, has low sensitivity, and is prone to false negatives when the infection rate is low. Conventional ELISA immunoassay is more suitable for large-scale batch screening of samples. Existing commercial Giardia ELISA kits mostly target the CWP protein in the cyst wall. This protein is only expressed during the cyst formation stage of the parasite and is affected by intermittent cyst expulsion, making it unable to detect acute trophozoite infection.

[0004] Using the trophozoite-specific secreted CP1 protein as a target, which is secreted in large quantities into the intestinal lumen during the active infection phase, allows for early detection of trophozoite infection, solving the core problem of existing reagents. Patent No. CN 116626292 B discloses an indirect ELISA detection method and kit for Giardia duodenalis CP1 and CP2 genes, which uses recombinant protein-coated antigens to detect serum antibodies, showing good sensitivity and specificity. However, the indirect ELISA method detects host antibodies, which still cannot avoid the problem of distinguishing between current and past infections, and is greatly affected by the host's immune status. Summary of the Invention

[0005] To address the problems of existing Giardia detection methods, such as difficulty in distinguishing between current and past infections, low sensitivity, complex operation, and high cost, this invention provides a monoclonal antibody 4H12 against the Giardia duodenalis CP1 protein and its application.

[0006] The specific technical solution of the present invention is as follows: A monoclonal antibody 4H12 of Giardia duodenalis CP1 protein, wherein the monoclonal antibody 4H12 has a heavy chain complementarity-determining region (CDR1) as shown in SEQ ID NO: 5, a heavy chain complementarity-determining region (CDR2) as shown in SEQ ID NO: 6, and a heavy chain complementarity-determining region (CDR3) as shown in SEQ ID NO: 7; the monoclonal antibody 4H12 also has a light chain complementarity-determining region (CDR1) as shown in SEQ ID NO: 13, a light chain complementarity-determining region (CDR2) with a KVS amino acid sequence, and a light chain complementarity-determining region (CDR3) as shown in SEQ ID NO: 15.

[0007] The monoclonal antibody 4H12 has a heavy chain variable region with an amino acid sequence as shown in SEQ ID NO: 8; and a light chain variable region with an amino acid sequence as shown in SEQ ID NO: 16.

[0008] The polynucleotide encoding the monoclonal antibody 4H12 mentioned above.

[0009] The above-mentioned monoclonal antibody 4H12 is used in the preparation of products for detecting Giardia lamblia CP1 protein.

[0010] A double-antibody sandwich ELISA kit for detecting Giardia duodenalis, the kit comprising a rabbit polyclonal antibody against Giardia duodenalis CP1 protein and the monoclonal antibody 4H12 as described in claim 1 or claim 2; wherein the rabbit polyclonal antibody against Giardia duodenalis CP1 protein is a capture antibody and the monoclonal antibody 4H12 is a detection antibody.

[0011] The kit also includes an enzyme-labeled plate, blocking solution, diluent, washing solution, chromogenic agent, and stop solution.

[0012] Preferably, the blocking solution is a 2% PVP solution; the coating solution is a 0.05 mol / L pH 9.5 carbonate buffer; the washing solution is a PBST buffer; the chromogenic agent is a single-component TMB substrate chromogenic solution; and the stop solution is a 2 mol / L sulfuric acid stop solution.

[0013] Preferably, the rabbit polyclonal antibody against Giardia lamblia CP1 protein is prepared by immunizing New Zealand white rabbits with recombinant CP1 protein as an antigen according to an immunization program, and then detecting the titer as qualified by indirect ELISA.

[0014] A double-antibody sandwich ELISA method for detecting Giardia lamblia CP1 protein for non-diagnostic purposes includes the following steps: (1) Pretreatment of test samples: Lyse the test samples, centrifuge, transfer the supernatant to a new sterile centrifuge tube, add 10 μL of protease inhibitor, and obtain the test sample lysate; (2) Capture antibody coating: The rabbit polyclonal antibody against Giardia duodenalis CP1 protein was diluted to 3 μg / mL with coating buffer and added to the microplate, 100 μL per well. The plate was placed at 4°C overnight for coating. The coating buffer was a carbonate buffer with a concentration of 0.05 mol / L and a pH of 9.5. (3) Blocking: Wash 3 times with 1×PBST buffer, add 2% PVP solution, 200 μL per well, and block at room temperature for 1.5 h; (4) Incubation of the test sample: Remove the blocking solution PVP solution, add 100 μL of the lysis solution of the test sample obtained in step (1), and react at 37°C for 60 min; (5) Antibody detection: Wash 3 times with 1×PBST buffer, add 100 μL of the monoclonal antibody 4H12 as described in claim 1 or claim 2 diluted to 1:3200, and react at 37°C for 90 min; (6) Secondary antibody incubation: Wash 3 times with 1×PBST buffer, add 100μL of HRP-labeled goat anti-mouse IgG diluted to 1:10000, and react at 37℃ for 45min; (7) Color development: Wash 5 times with 1×PBST buffer, add single-component TMB substrate color development solution, and react at 37°C in the dark for 10 min; (8) Termination: Add 50 μL of 2 mol / L sulfuric acid stop solution, and read the OD value using an ELISA reader within 30 min. 450nm value; (9) Result calculation and judgment: OD 450nm A value >0.0617 indicates a positive result; OD 450nm A value <0.0578 is considered negative. Values ​​between these two values ​​are considered suspicious and require retesting.

[0015] Compared with the prior art, the beneficial effects of the present invention are as follows: This invention provides a monoclonal antibody 4H12 for Giardia duodenalis CP1 protein. A "polyclonal antibody capture-monoclonal antibody detection" sandwich system is constructed using a rabbit-derived polyclonal antibody paired with monoclonal antibody 4H12. This system ensures antigen binding efficiency while reducing non-specific interference, improving the sensitivity and accuracy of the detection system. By optimizing the double-antibody sandwich ELISA reaction conditions for Giardia duodenalis detection, this invention achieves a minimum detectable antigen concentration of 1 μg / mL, exhibiting excellent detection sensitivity and no cross-reactivity with common intestinal pathogens such as Toxoplasma gondii, Cryptosporidium, and Escherichia coli. The intra- and inter-batch coefficients of variation are both below 8. The overall concordance rate between 50 clinical fecal samples from dogs and sheep and double nested PCR is 98%. The paired antibodies can be assembled into commercially available ELISA kits for large-scale screening of Giardia duodenalis in livestock and companion animals. Furthermore, the variable region nucleic acid sequence of monoclonal antibody 4H12 can be used for the preparation of recombinant antibodies. Simultaneously, this invention provides key specific biomaterials for basic research on the pathogenic mechanism and invasion mechanism of Giardia duodenalis CP1 protein. Attached Figure Description

[0016] Figure 1 The results are for the indirect ELISA ascites titer determination of the purified monoclonal antibodies 1B7, 5D6, 1D8, 4B11, and 4H12 of this invention.

[0017] Figure 2 This is a Western blot verification result of the reaction between the monoclonal antibody 4H12, which has the best titer in this invention, and the natural protein CP1.

[0018] Figure 3 This is a Western blot verification result of the reaction between the monoclonal antibody 4H12 with the recombinant protein CP1, which has the best titer in this invention.

[0019] Figure 4 This is a diagram showing the results of indirect immunofluorescence localization verification of the monoclonal antibody 4H12, which has the highest titer in this invention, in Giardia trophozoites.

[0020] Figure 5 The figure shows the ELISA titer results of the rabbit polyclonal antibody against Giardia lamblia CP1 protein of this invention.

[0021] Figure 6 This is a Western blot verification result of the rabbit polyclonal antibody against Giardia lamblia CP1 protein of the present invention.

[0022] Figure 7 The figure shows the sensitivity test results of the double-antibody sandwich ELISA method for detecting Giardia lamblia CP1 protein established in this invention. Detailed Implementation

[0023] The present invention will be further described in detail below with reference to the embodiments and accompanying drawings, but the embodiments of the present invention are not limited thereto.

[0024] I. Sources of biological materials, reagents, and instruments (1) Species: The Giardia lamblia species in this embodiment is aggregate A, which was donated by Professor Zhang Xichen of Jilin University.

[0025] (2) CP1 recombinant protein: Using Giardia lamblia aggregate A trophozoite cDNA as a template, the complete coding gene of CP1 was amplified, digested with enzymes and ligated into the pET-32a(+) prokaryotic expression vector, transformed into BL21 competent engineered bacteria, expressed by IPTG, and purified by urea dissolution of inclusion bodies and nickel column affinity chromatography. The CP1 recombinant protein was independently constructed and purified in our laboratory.

[0026] (3) Main experimental animals: 6-8 week old female BALB / c mice, purchased from Beijing Spefol Biotechnology Co., Ltd. 3-month-old SPF-grade female New Zealand white rabbits, purchased from Qingdao Kangda Biotechnology Co., Ltd.

[0027] (4) Control pathogens: Cryptosporidium microsporidium, Toxoplasma gondii, Coccidia, Escherichia coli, and Salmonella standard strains were long-term passaged and preserved by the Zoonotic Diseases Laboratory of Henan Agricultural University.

[0028] (5) Cells and strains: SP2 / 0 myeloma cells and pET-32a (+) prokaryotic expression vector were preserved by the Zoonotic Diseases Laboratory of Henan Agricultural University. DH5α competent engineered bacteria and BL21 competent engineered bacteria were purchased from Nanjing Novizan Biotechnology Co., Ltd.

[0029] (6) Main reagents: Agar powder, tryptone, yeast extract, ampicillin, kanamycin, IPTG, SDS, Tris, EDTA, urea, Triton X-100, HAT selective medium, HT medium, PEG1450 cell fusion agent, bovine serum albumin (BSA), polyvinylpyrrolidone (PVP), single-component TMB substrate chromogenic solution, 2 mol / L sulfuric acid stop solution, PBS buffer, PBST buffer, DAPI nuclear staining solution, RIPA protein lysis buffer, and 4% paraformaldehyde fixative were all purchased from Beijing Solarbio Science & Technology Co., Ltd. Coomassie Brilliant Blue R-250, color pre-stained protein molecular weight markers, and 10% PAGE gel preparation kits were purchased from Shanghai Yamei Biotechnology Co., Ltd. Freund's complete adjuvant and Freund's incomplete adjuvant were purchased from Beijing MecoWander Biotechnology Co., Ltd. rProtein G affinity chromatography packing material was purchased from Changzhou Tiandi Renhe Technology Co., Ltd. HRP rapid labeling kit, HRP-labeled goat anti-rabbit IgG, and fluorescently labeled goat anti-mouse IgG secondary antibody were purchased from Wuhan Sanying Biotechnology Co., Ltd.

[0030] (7) Major instruments and equipment: benchtop centrifuge purchased from Hunan Xiangyi Laboratory Instrument Development Co., Ltd.; pH meter purchased from Shanghai Yidian Scientific Instrument Co., Ltd.; constant temperature water bath purchased from Beijing Changfeng Instrument Co., Ltd.; CO2 cell culture incubator purchased from Thermo; multifunctional microplate reader purchased from Tecan Trading AG; protein electrophoresis apparatus and membrane transfer apparatus purchased from Bio-Rad; fluorescence inverted microscope purchased from Olympus; ultra-clean workbench purchased from Suzhou Sujing Antai; ultraviolet gel imaging system purchased from Analytick Jena, Germany; spectrophotometer purchased from Shimadzu; horizontal shaker purchased from Shanghai Huake Experimental Equipment Co., Ltd.; electronic balance purchased from Mettler-Toledo Group; ice maker purchased from Beijing Changfeng Instrument Co., Ltd.; ultrasonic cell disruptor purchased from Ningbo Xinzhi Biotechnology Co., Ltd.

[0031] II. Animal Immunization (1) Take 1 mg / mL of recombinant protein CP1 and an equal volume of Freund's adjuvant and fully emulsify it. Immunize 5 6-8 week old SPF grade BALB / c mice subcutaneously at multiple points. After the first immunization, boost the immunization twice every 14 days. (2) Seven days after the last immunization, collect blood from the tail vein to measure the serum indirect ELISA titer. If the titer reaches the target, inject pure CP1 protein into the peritoneum for shock immunization 3 days before fusion. (3) Aseptically remove the mouse spleen, grind it to prepare spleen cells, fuse it with SP2 / 0 myeloma cells in a certain proportion, and plate them into a 96-well cell culture plate containing feeder cells. Culture in HAT selective medium.

[0032] III. Screening of Hybridoma Cells (1) Dilute recombinant protein CP1 to 1 μg / mL with coating buffer, which is 0.05 mol / L carbonate buffer with pH 9.5, 100 μL per well, and coat overnight at 4℃; wash 3 times with 0.05% PBST buffer, add 200 μL of 5% skim milk powder to each well, and block at 37℃ for 2 h; (2) Discard the blocking solution, add 100 μL of hybridoma cell supernatant to each well, and incubate at 37℃ for 1 h; wash 4 times with PBST buffer, add 1:5000 diluted HRP-goat anti-mouse IgG, and incubate at 37℃ for 1 h; (3) After thorough washing, add single-component TMB substrate chromogenic solution and develop at room temperature in the dark for 10~15 min, stop with 2 mol / L sulfuric acid stop solution, and read OD using an ELISA reader. 450nm ; Sample well OD 450nm / Negative pore OD 450nm ≥2.1 The wells were determined to be positive hybridoma cells; (4) The positive hybridoma cells were plated with limited dilution and three rounds of monoclonal subcloning were carried out. After each round of subcloning, the indirect ELISA was used to retest until all monoclonal wells were consistently positive; five hybridoma cell lines secreting specific monoclonal antibodies were obtained.

[0033] IV. Preparation and Purification of Monoclonal Antibody Ascites Fluid (1) Intraperitoneally inject 500 μL of sterile liquid paraffin into female BALB / c mice for pretreatment for 7–14 days; (2) Resuscitate the above 5 hybridoma cell lines, culture them in T25 flasks until confluence reaches 70%, collect the cells, resuspend them in sterile PBS buffer, and inject approximately 1 × 10⁻⁶ cells into each mouse intraperitoneally. 6 (3) After the abdomen of mice is enlarged 7-14 days after inoculation, ascites is extracted with a sterile syringe, centrifuged at 4500r / min for 5 min to remove red blood cells and fat precipitates, and the ascites supernatant is collected; (4) The rProtein G affinity chromatography column is equilibrated with 5 column volumes of equilibration buffer 2-3 times, and the ascites sample is loaded onto the column at room temperature for 30 min; (5) 10-15 column volumes of washing buffer are used to wash away non-specific proteins; 5-10 column volumes of elution buffer are used to elute the antibody, and 1 / 10 volume of neutralization buffer is added to the elution buffer immediately to neutralize it, and purified monoclonal antibodies are obtained, including 1B7, 5D6, 1D8, 4B11, and 4H12.

[0034] V. Monoclonal Antibody Titer Determination (1) Dilute recombinant protein CP1 to 1 μg / mL with coating buffer, which is 0.05 mol / L carbonate buffer with pH 9.5, 100 μL per well, and incubate overnight at 4°C; (2) Wash 3 times with 1×PBST buffer, add 5% skim milk powder, 200 μL per well, and block at room temperature for 2 h; (3) Dilute the purified monoclonal antibodies 1B7, 5D6, 1D8, 4B11, and 4H12 sequentially from 1:100000 to 1:72600000, 100 μL per well, and react at 37°C for 60 min; (4) After thorough washing with PBST buffer, add 1:5000 HRP-labeled goat anti-mouse IgG, and incubate at 37°C for 1 h; (5) After washing, develop the color with single-component TMB substrate in the dark for 10-15 min, terminate with 2 mol / L sulfuric acid ELISA stop solution, and read the OD. 450nm The positive criterion is P / N ≥ 2.1; Figure 1 As shown, the OD of monoclonal antibody 4H12 at the same dilution gradient 450nm The P / N ratio of 4H12 was significantly higher than that of the other four plants. At the dilution gradient of 1:24,300,000, the highest P / N ratio of 4H12 reached 2.6, while the P / N ratios of the other four plants were all below 2.1 at the same dilution gradient. Only at the highest dilution gradient of 1:72,600,000 did 4H12 still meet the positive criteria. Therefore, 4H12 was determined to be the plant with the best potency.

[0035] VI. Identification of the reactivity of monoclonal antibodies with specific antigens (a) Western blot identification After screening five hybridoma ascites titers by indirect ELISA, the monoclonal antibody 4H12 with the highest titer was selected for Western blot analysis. The specific steps are as follows: (1) Prepare an SDS-PAGE protein gel with a thickness of 1.0 mm and a concentration of 10%; (2) Add recombinant protein CP1 and total protein extracted from Giardia duodenalis trophozoites to a centrifuge tube at a ratio of 4:1, mix thoroughly by blowing and aspiration, boil at 100℃ for 10 min, and load 15 μL per well. The upper gel voltage is 90 V and the lower gel voltage is 120 V; (3) After electrophoresis, the PVDF membrane is activated with methanol solution for 5 min and arranged in the order of "sponge-filter paper-gel-PVDF membrane-filter paper-sponge". The gel is close to the negative electrode and the PVDF membrane is close to the positive electrode. The protein is transferred at a constant current of 400 mA for 25 min. The transfer is then completed. Then, transfer the protein wet onto a PVDF membrane; (4) prepare 5% skim milk powder with PBST buffer and block at room temperature for 2 hours; (5) discard the blocking solution after blocking and wash simply with PBST buffer; (6) add monoclonal antibody diluted with PBST buffer and incubate slowly on a shaker at room temperature for 2 hours; (7) discard the primary antibody and wash the membrane 4 times with PBST buffer, 5 minutes each time; (8) add goat anti-mouse IgG diluted with PBST buffer and incubate on a shaker at room temperature in the dark for 1 hour; (9) discard the secondary antibody and wash the membrane 5 times with PBST buffer, 5 minutes each time; (10) perform ECL chemiluminescence staining and observe the size and luminescence of the target protein, such as Figure 2 , 3 As shown, the monoclonal antibody 4H12 can specifically recognize both natural and recombinant proteins and has good reactivity.

[0036] (ii) Indirect immunofluorescence identification Indirect immunofluorescence analysis was performed on the monoclonal antibody 4H12 with the highest titer: (1) Giardia lamblia trophozoites in the logarithmic growth phase were inoculated into 12-well culture plates and incubated at 37°C and 5% CO2 for 6 h to allow the worms to adhere to the plates; (2) the plates were washed twice with PBS buffer, and each well was fixed with 4% paraformaldehyde fixative at room temperature for 5 min. The plates were then washed with PBST buffer containing 0.1 M glycine to neutralize any residual fixative; (3) the plates were incubated with PBST permeabilization buffer containing 0.1% Triton X-100 at room temperature for 30 min, and then washed with PBST buffer for 3 minutes. (4) Block with 3% BSA blocking solution at room temperature for 1 h, discard the blocking solution and wash briefly with PBST buffer; (5) Use diluted 4H12 monoclonal antibody as primary antibody, 1 mL per well, incubate at 37℃ in the dark for 2 h, and wash 3 times with PBST buffer; (6) Add fluorescently labeled goat anti-mouse IgG secondary antibody, incubate at room temperature in the dark for 1 h, and wash 3 times with PBST buffer; (7) Stain nuclei with DAPI nuclear staining solution in the dark for 15 min, and wash twice with PBST buffer; (8) Observe and photograph under a fluorescent inverted microscope, such as Figure 4 As shown, the monoclonal antibody 4H12 can produce a specific green fluorescent signal with Giardia duodenalis trophozoites, while the negative control group does not produce specific fluorescence, proving that it can specifically recognize the CP1 protein in the worm.

[0037] VII. Sequencing of Monoclonal Antibodies Hybridoma cell lines corresponding to the monoclonal antibody 4H12 with the best titer and specific binding were screened for sequencing analysis. Total RNA was extracted from hybridoma cells after lysis; cDNA was synthesized by reverse transcription using Oligo (dT) primers; PCR amplification was performed to obtain the heavy and light chain variable region sequences; the target fragment was ligated into a T-cloning vector using ligase, and the ligation product was transformed into DH5α competent engineered bacteria. Single clones were then selected for bidirectional sequencing. The sequencing results included the core sequence of the 4H12 antibody (the nucleic acid and amino acid sequences of the heavy chain variable region, light chain variable region, and each CDR region).

[0038] The monoclonal antibody 4H12 has a heavy chain complementarity-determining region (CDR1) as shown in SEQ ID NO: 5, a heavy chain complementarity-determining region (CDR2) as shown in SEQ ID NO: 6, and a heavy chain complementarity-determining region (CDR3) as shown in SEQ ID NO: 7; the monoclonal antibody 4H12 also has a light chain complementarity-determining region (CDR1) as shown in SEQ ID NO: 13, a light chain complementarity-determining region (CDR2) as shown in SEQ ID NO: 14, and a light chain complementarity-determining region (CDR3) as shown in SEQ ID NO: 15.

[0039] The monoclonal antibody 4H12 includes a heavy chain variable region with an amino acid sequence as shown in SEQ ID NO: 8, and a light chain variable region with an amino acid sequence as shown in SEQ ID NO: 16.

[0040] The nucleotide sequence of the heavy chain complementarity-determining region (CDR1) encoding monoclonal antibody 4H12 is shown in SEQ ID NO: 1, the nucleotide sequence of the heavy chain complementarity-determining region (CDR2) encoding monoclonal antibody 4H12 is shown in SEQ ID NO: 2, and the nucleotide sequence of the heavy chain complementarity-determining region (CDR3) encoding monoclonal antibody 4H12 is shown in SEQ ID NO: 3.

[0041] The nucleotide sequence of the light chain complementarity-determining region CDR1 encoding monoclonal antibody 4H12 is shown in SEQ ID NO: 9, the nucleotide sequence of the light chain complementarity-determining region CDR2 encoding monoclonal antibody 4H12 is shown in SEQ ID NO: 10, and the nucleotide sequence of the light chain complementarity-determining region CDR3 encoding monoclonal antibody 4H12 is shown in SEQ ID NO: 11.

[0042] The nucleotide sequence encoding the heavy chain variable region of monoclonal antibody 4H12 is shown in SEQ ID NO: 4, and the nucleotide sequence encoding the light chain variable region of monoclonal antibody 4H12 is shown in SEQ ID NO: 12.

[0043] The specific sequence information is as follows: SEQ ID NO: 1 (nucleotide sequence encoding the CDR1 complementarity-determining region of the heavy chain of the monoclonal antibody 4H12) GGGTATACCTTCACAGACTATGGA SEQ ID NO: 2 (nucleotide sequence encoding the CDR2 complementarity-determining region of the heavy chain of the monoclonal antibody 4H12) ATAAACACCAACACTGGAGAGCCA SEQ ID NO: 3 (nucleotide sequence encoding the complementarity-determining region CDR3 of the heavy chain of the monoclonal antibody 4H12) ACAAGATCTAGCATTTCGGCAGACTATGCTATGGACTAC SEQ ID NO: 4 (nucleotide sequence encoding the variable region of the 4H12 heavy chain of the monoclonal antibody) CAGATCCAGTTGGTGCAGTCTGGACCTGAGCTGAGGAAGCCTGGAGAGACAGTCAAGATTTCCTGCAAGGCTTCTGGGTATACCTTCACAGACTATGGAATGAACTGGGTGAAGCAGACTCCAGGAAAGGGTTTAAAGTGGATGGGCTGGATAAACACCAACACTGGAGAGCCAACATAT GCTGAAGAGTTCAAGGGACGGTTTGCCTTCCTTTGGCAACCTCTGCCCGCACTGCCTTTTTGCAGATCAACAACCTCCAAAATGAGGACACGGCTACATATTTCTGTACAAGATCTAGCATTTCGGCAGACTATGCTATGGACTACTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCA SEQ ID NO: 5 (Amino acid sequence of CDR1 in the heavy chain complementarity-determining region of monoclonal antibody 4H12) GYTFTDYG SEQ ID NO: 6 (Amino acid sequence of CDR2 in the complementarity-determining region of the 4H12 heavy chain of monoclonal antibody) INTNTGEP SEQ ID NO: 7 (Amino acid sequence of CDR3 in the complementarity-determining region of the 4H12 heavy chain of monoclonal antibody) TRSSISADYAMDY SEQ ID NO: 8 (Amino acid sequence of the variable region of the 4H12 heavy chain of monoclonal antibody) QIQLVQSGPELRKPGETVKISCKASGYTFTDYGMNWVKQTPGKGLKWMGWINTNTGEPTYAEEFKGRFAFSLATSARTAFLQINNLQNEDTATYFCTRSSISADYAMDYWGQGTSVTVSS SEQ ID NO: 9 (nucleotide sequence encoding the complementarity-determining region CDR1 of the light chain of the monoclonal antibody 4H12) CAGAGCATTGTACATAGTAATGGAAACACCTAT SEQ ID NO: 10 (nucleotide sequence encoding the complementarity-determining region CDR2 of the light chain of monoclonal antibody 4H12) AAAGTTTCC SEQ ID NO: 11 (nucleotide sequence encoding the complementarity-determining region CDR3 of the light chain of monoclonal antibody 4H12) TTTCAAGGTTCATATGTTCCTCCGACG SEQ ID NO: 12 (nucleotide sequence encoding the variable region of the light chain of monoclonal antibody 4H12) GATATTTTGATGACCCAAACTCCACTCTCCCTGCCTGTCAGTCTTGGAGATCAAGCCTCCATCTCTTGCAAATCTAGTCAGAGCATTGTACATAGTAATGGAAACACCTATTTAGAATGGTACCTGCAGAAACCAGGCCAGTCTCCAAAGCTCCTGATCTACAAAGTT TCCAACCGATTTTCTGGGGTCCCAGACAGGTTCAGTGGCAGTGGATCAGGGACAGATTTCACACTCAAGATCAGCAGATGGAGGCTGAGGATCTGGGAGTTTATTACTGCTTTCAAGGTTCATATGTTCCTCCGACGTTCGGTGGAGGCACCAAGCTGGAAATCAAA SEQ ID NO: 13 (Amino acid sequence of CDR1 complementarity-determining region of 4H12 light chain of monoclonal antibody) QSIVHSNGNTY SEQ ID NO: 14 (Amino acid sequence of CDR2 in the complementarity-determining region of the 4H12 light chain of monoclonal antibody) KVS SEQ ID NO: 15 (Amino acid sequence of CDR3 in the complementarity-determining region of the 4H12 light chain of monoclonal antibody) FQGSYVPPT SEQ ID NO: 16 (Amino acid sequence of the variable region of the 4H12 light chain of monoclonal antibody) DILMTQTPLSLPVSLGDQASISCKSSQSIVHSNGNTYLEWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQGSYVPPTFGGGTKLEIK.

[0044] VIII. Preparation and Identification of Polyclonal Antibodies (1) 1 mg / mL recombinant protein CP1 was thoroughly emulsified with an equal volume of Freund's adjuvant and injected into the back of two healthy 3-month-old SPF-grade female New Zealand white rabbits. After the first immunization, booster immunizations were performed every 14 days for a total of 3 immunizations. After the last immunization, blood was collected for indirect ELISA titer detection. After the titer reached the target, all rabbit blood was collected by cardiac puncture. (2) The blood was allowed to stand at room temperature to separate into layers. The serum was separated by centrifugation and purified using an rProtein G affinity chromatography column to obtain a high-purity rabbit polyclonal antibody against Giardia duodenalis CP1 protein.

[0045] Using indirect ELISA to detect the titer, the rabbit-derived polyclonal antibody against Giardia lamblia CP1 protein achieved a titer exceeding 10.24 million times. The results are as follows: Figure 5 As shown.

[0046] A rabbit polyclonal antibody against Giardia lamblia CP1 protein was used as the primary antibody to validate the recombinant CP1 protein using Western blot. The results are as follows: Figure 6 As shown, the rabbit-derived polyclonal antibody against Giardia lamblia CP1 protein prepared has good reactivity against recombinant protein CP1.

[0047] IX. Establishment of a double-antibody sandwich ELISA method for detecting Giardia lamblia CP1 protein. Using the polyclonal antibody prepared above as the capture antibody and the prepared monoclonal antibody 4H12 as the detection antibody, a sandwich ELISA method was established to detect CP1 protein. The optimal capture antibody coating concentration, blocking solution, blocking time, antigen incubation time, secondary antibody dilution factor, and secondary antibody incubation time were explored and optimized.

[0048] (I) Optimization of Capture Antibody Coating Concentration: Rabbit polyclonal antibodies against Giardia duodenalis CP1 protein were diluted with coating buffer to six gradients: 0.25 μg / mL, 0.5 μg / mL, 1 μg / mL, 2 μg / mL, 3 μg / mL, and 4 μg / mL. The coating buffer was a carbonate buffer with a concentration of 0.05 mol / L and a pH of 9.5. Monoclonal antibody 4H12 was diluted sequentially at dilution gradients of 1:200, 1:400, 1:800, 1:1600, 1:3200, 1:6400, 1:12800, and 1:25600. The reaction was combined using a checkerboard titration method, and the OD was read after incubation. 450nm The P / N ratio was calculated, and the highest P / N ratio was found when a rabbit polyclonal antibody at 3 μg / mL against Giardia duodenalis CP1 protein was combined with a 1:3200 monoclonal antibody, which was determined to be the optimal working antibody concentration. (II) Optimization of sealing conditions: Three sealing solutions were prepared, namely 5% skim milk powder, 1% BSA and 2% PVP, and four sealing times were set: 0.5h, 1h, 1.5h and 2h. The other reaction conditions were kept constant. The positive absorbance value and negative background value of each group were compared. The negative background value of 2% PVP at room temperature for 1.5h was the lowest and was determined to be the optimal sealing system. (III) Optimization of antigen incubation time: With other optimal conditions fixed, four antigen incubation times were set: 0.5h, 1h, 1.5h, and 2h. OD was measured after incubation at 37℃. 450nm The P / N ratio was optimal in the 1-hour incubation group, thus determining the incubation time for the standard antigen. (iv) Optimization of antibody incubation time: Keeping other conditions unchanged, set gradient incubation times of 0.5h, 1h, 1.5h and 2h, and take readings after reaction at 37℃. The 1.5h group showed the strongest positive signal and was used as the standard incubation time for antibody detection. (V) Optimization of enzyme-labeled secondary antibody conditions: HRP-goat anti-mouse IgG was diluted at 1:5000, 1:10000, 1:15000, and 1:20000, with incubation times of 15 min, 30 min, 45 min, and 60 min, respectively. After checkerboard titration, the optimal overall effect was determined to be a secondary antibody diluted at 1:10000 and incubated at 37℃ for 45 min. (vi) Criterion determination: A rabbit-derived polyclonal antibody against Giardia duodenalis CP1 protein was used as the capture antibody, and the monoclonal antibody 4H12 was used as the detection antibody for sandwich ELISA. Twenty Giardia duodenalis-negative fecal supernatants were used for sandwich ELISA. The OD values ​​of the 20 Giardia duodenalis-negative proteins were determined. 450nmThe values ​​were: 0.053, 0.045, 0.054, 0.056, 0.046, 0.047, 0.053, 0.053, 0.047, 0.045, 0.053, 0.058, 0.046, 0.047, 0.050, 0.051, 0.045, 0.046, 0.046, and 0.049. The positive and negative cutoff values ​​for ELISA were determined using the cut-off formula, and the OD of the recombinant protein CP1 was... 450nm A value >0.0617 is considered positive; OD 450nm A value <0.0578 is considered negative. Values ​​between these two values ​​are considered suspicious and require retesting.

[0049] Based on the optimization of the above conditions, a double-antibody sandwich ELISA method for detecting Giardia lamblia CP1 protein includes the following steps: (1) Sample pretreatment: Lyse the sample, centrifuge, transfer the supernatant to a new sterile centrifuge tube, add 10 μL of protease inhibitor to obtain the sample lysate; (2) Capture antibody coating: Dilute the rabbit polyclonal antibody against Giardia duodenalis CP1 protein to 3 μg / mL with coating buffer, add 100 μL to each well of the microplate, and place at 4℃ overnight for coating. The coating buffer is carbonate buffer with a concentration of 0.05 mol / L and a pH of 9.5; (3) Blocking: Wash 3 times with 1×PBST buffer, add 2% PVP solution to each well, and block at room temperature for 1.5 h; (4) Sample incubation: Remove the blocking PVP solution, add 100 μL of the sample lysate obtained in step (1), and react at 37℃ for 60 min; (5) Detection antibody incubation: Wash 3 times with 1×PBST buffer, add monoclonal antibody 4H12 diluted to 1:3200. 100 μL, react at 37℃ for 90 min; (6) Secondary antibody incubation: wash 3 times with 1×PBST buffer, add 100 μL of HRP-labeled goat anti-mouse IgG diluted to 1:10000, react at 37℃ for 45 min; (7) Color development: wash 5 times with 1×PBST buffer, add single-component TMB substrate color development solution, react at 37℃ in the dark for 10 min; (8) Termination: add 50 μL of 2 mol / L sulfuric acid ELISA termination solution, read OD of the microplate reader within 30 min. 450nm Value; (9) Result calculation and judgment: OD 450nm A value >0.0617 indicates a positive result; OD 450nm A value <0.0578 is considered negative. Values ​​between these two values ​​are considered suspicious and require retesting.

[0050] 10. Evaluation of Detection Methods (a) Specificity test Following the established double-antibody sandwich ELISA method, protein samples were prepared for Toxoplasma gondii, Cryptosporidium microsporidium, Escherichia coli, Salmonella, Coccidia, and Giardia duodenalis. Giardia duodenalis was used as a positive control, and blank lysis buffer was used as a negative control for simultaneous detection. The results showed that only the Giardia duodenalis sample showed a high OD value. 450nm The results showed that the positive threshold was higher than the negative threshold, while all other pathogens were below the negative threshold, with no cross-reaction. This indicates that the double-antibody sandwich ELISA detection method established in this invention, using rabbit-derived polyclonal antibody against Giardia duodenalis CP1 protein and monoclonal antibody 4H12 as paired antibodies, has good specificity.

[0051] (ii) Sensitivity test The total protein concentration of Giardia trophozoites was serially diluted to 1 mg / mL, 500 μg / mL, 100 μg / mL, 50 μg / mL, 10 μg / mL, 5 μg / mL, 1 μg / mL, 500 ng / mL, 100 ng / mL, 50 ng / mL, 10 ng / mL, and 0 ng / mL; a P / N ratio > 2.1 was used as the positive criterion; the results showed (e.g. Figure 7 As shown in the figure, the lowest detectable antigen concentration is 1 μg / mL, and the detection sensitivity is excellent.

[0052] (iii) Repeatability test The established double-antibody sandwich ELISA method was tested for intra-batch and inter-batch repeatability. Ten positive fecal lysate supernatants were selected, with three control groups for each sample. The mean and coefficient of variation were calculated. Inter-batch tests were performed using two batches of protein coating assays, with 10 positive samples tested in each batch. The mean and coefficient of variation were then calculated.

[0053] Rabbit polyclonal antibody against Giardia lamblia CP1 protein was used as the capture antibody, and monoclonal antibody 4H12 was used as the detection antibody. The intra-assay coefficient of variation (CV) was 2.02%–7.76%, and the inter-assay coefficient of variation (CV) was 1.49%–6.55%. The CVs were all less than 8%, indicating that the method had good reproducibility. The results are shown in Table 1.

[0054] Table 1 Results of Repeatability Tests

[0055] (iv) Testing of clinical samples The established double-antibody sandwich ELISA method was used to detect 50 canine and sheep fecal samples (24 positive and 26 negative), and the results are shown in Table 2. The double-antibody sandwich ELISA method established in this application has high sensitivity and high concordance rate, and is suitable for detection.

[0056] Table 2. Detection results of clinical samples

[0057] The preferred embodiments of the present invention have been described in detail above. It should be understood that those skilled in the art can make numerous modifications and variations based on the concept of the present invention without creative effort. Therefore, all technical solutions that can be obtained by those skilled in the art based on the concept of the present invention through logical analysis, reasoning, or limited experimentation on the basis of existing technology should be within the scope of protection defined by the claims.

Claims

1. A monoclonal antibody 4H12 against Giardia duodenalis CP1 protein, characterized in that, The monoclonal antibody 4H12 has a heavy chain complementarity-determining region CDR1 as shown in SEQ ID NO: 5, a heavy chain complementarity-determining region CDR2 as shown in SEQ ID NO: 6, and a heavy chain complementarity-determining region CDR3 as shown in SEQ ID NO:

7. The monoclonal antibody 4H12 has a light chain complementarity-determining region CDR1 as shown in SEQ ID NO: 13, a light chain complementarity-determining region CDR2 with the amino acid sequence KVS, and a light chain complementarity-determining region CDR3 as shown in SEQ ID NO:

15.

2. The monoclonal antibody 4H12 against Giardia duodenalis CP1 protein according to claim 1, characterized in that, The monoclonal antibody 4H12 has a heavy chain variable region with an amino acid sequence as shown in SEQ ID NO: 8; and a light chain variable region with an amino acid sequence as shown in SEQ ID NO:

16.

3. A polynucleotide encoding the monoclonal antibody 4H12 as described in claim 1 or claim 2.

4. The use of the monoclonal antibody 4H12 according to claim 1 or claim 2 in the preparation of a product for detecting Giardia duodenalis CP1 protein.

5. A double-antibody sandwich ELISA kit for detecting Giardia lamblia, characterized in that, The kit comprises a rabbit polyclonal antibody against Giardia duodenalis CP1 protein and the monoclonal antibody 4H12 as described in claim 1 or claim 2; the rabbit polyclonal antibody against Giardia duodenalis CP1 protein is a capture antibody, and the monoclonal antibody 4H12 is a detection antibody.

6. The double-antibody sandwich ELISA kit for detecting Giardia lamblia according to claim 5, characterized in that, The kit also includes an enzyme-labeled plate, blocking solution, diluent, washing solution, chromogenic agent, and stop solution.

7. A double-antibody sandwich ELISA method for detecting Giardia lamblia CP1 protein for non-diagnostic purposes, characterized in that, Includes the following steps: (1) Pretreatment of test samples: Lyse the test samples, centrifuge, transfer the supernatant to a new sterile centrifuge tube, add 10 μL of protease inhibitor, and obtain the test sample lysate; (2) Capture antibody coating: The rabbit polyclonal antibody against Giardia duodenalis CP1 protein was diluted to 3 μg / mL with coating buffer and added to the microplate, 100 μL per well. The plate was placed at 4°C overnight for coating. The coating buffer was a carbonate buffer with a concentration of 0.05 mol / L and a pH of 9.

5. (3) Blocking: Wash 3 times with 1×PBST buffer, add 2% PVP solution, 200 μL per well, and block at room temperature for 1.5 h; (4) Incubation of the test sample: Remove the blocking solution PVP solution, add 100 μL of the lysis solution of the test sample obtained in step (1), and react at 37°C for 60 min; (5) Antibody detection: Wash 3 times with 1×PBST buffer, add 100 μL of the monoclonal antibody 4H12 as described in claim 1 or claim 2 diluted to 1:3200, and react at 37°C for 90 min; (6) Secondary antibody incubation: Wash 3 times with 1×PBST buffer, add 100μL of HRP-labeled goat anti-mouse IgG diluted to 1:10000, and react at 37℃ for 45min; (7) Color development: Wash 5 times with 1×PBST buffer, add single-component TMB substrate color development solution, and react at 37°C in the dark for 10 min; (8) Termination: Add 50 μL of 2 mol / L sulfuric acid stop solution, and read the OD value using an ELISA reader within 30 min. 450nm value; (9) Result calculation and judgment: OD 450nm A value >0.0617 indicates a positive result; OD 450nm A value <0.0578 is considered negative. Values ​​between these two values ​​are considered suspicious and require retesting.

Images