Monoclonal antibody and paired monoclonal antibody of giardia duodenalis cwp2 protein and application thereof

Abstract

The application relates to a monoclonal antibody of duodenum giardia CWP2 protein, a paired monoclonal antibody and application. The application obtains five pairs of paired monoclonal antibodies with better immunization effects through immunoscreening of duodenum giardia CWP2 protein, wherein the immunization effect is best when YC301 is used as a capture antibody and YC201 is used as a detection antibody, and a double-antibody sandwich ELISA detection method established by using the paired antibodies is better in sensitivity, specificity and stability, thereby laying a solid foundation for development of a duodenum giardia CWP2 antigen detection kit.

Description

Technical Field

[0001] This invention relates to the field of immunoassay technology, specifically to monoclonal antibodies and paired monoclonal antibodies against Giardia lamblia CWP2 protein and their applications. Background Technology

[0002] duodenal giardiasis is a disease caused by Giardia (a type of parasite). Giardia Giardiasis is a zoonotic disease caused by intestinal protozoa, primarily transmitted via the fecal-oral route. After infection, hosts often experience various symptoms such as vomiting, diarrhea, dehydration, and weight loss, seriously endangering livestock farming and public health. Currently, there is no specific treatment for duodenal giardiasis, relying mainly on imidazole drugs. However, long-term use can easily lead to drug resistance in the parasite. Therefore, improving the diagnostic level of giardiasis is a crucial aspect of conducting epidemiological surveys and clinical testing.

[0003] The life cycle of Giardia lamblia consists of two distinct stages: trophozoites and cysts. During the infectious period, mature cysts enter the human body through contaminated food and water. They exuviae in the duodenum, forming two trophozoites. These trophozoites parasitize the small intestine, particularly the duodenum. Some trophozoites form cysts in the terminal ileum and are excreted in feces, contaminating food, water, and interfaces. The cysts are highly resistant to environmental changes and can survive for extended periods, enabling the continuous transmission of Giardiasis between hosts and increasing the risk of foodborne and waterborne transmission.

[0004] Currently, microscopic examination is the primary method for diagnosing Giardia lamblia infection in hosts. However, this method is highly subjective, depending on the observer's experience, and is time-consuming and labor-intensive, sometimes failing to detect the infection due to low infection rates. Therefore, an increasing number of biological detection methods are being applied. Among these, molecular biological detection methods, such as PCR amplification to detect Giardia lamblia DNA, while highly sensitive, have limited their widespread adoption at the grassroots level due to the high level of expertise required for equipment and personnel. In contrast, immunological diagnostic techniques offer advantages such as speed, simplicity, high sensitivity, and strong specificity, demonstrating promising application prospects. However, existing immunological diagnostic techniques for giardiasis still have some problems. First, for patients with mild infection or in the incubation period, current techniques may not be able to accurately detect Giardia antigens or antibodies, leading to missed diagnoses. Second, some advanced immunological diagnostic techniques require expensive reagents and equipment, and are cumbersome and time-consuming to operate, increasing the cost of testing, such as indirect immunofluorescence and immunoblotting. Third, due to the complexity of immune reactions and individual differences, misjudgments may occur. For example, some other pathogens with similar antigens to Giardia may lead to false positive results. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to overcome the problems of existing immunological diagnostic techniques for giardiasis, such as complex operation, high cost, low sensitivity and low specificity.

[0006] To address this technical problem, the present invention provides a pairing monoclonal antibody with high sensitivity and specificity for detecting Giardia lamblia CWP2 protein.

[0007] A monoclonal antibody YC201 for the Giardia lamblia CWP2 protein, wherein the monoclonal antibody YC201 has three nucleotide sequences encoding the heavy chain complementarity-determining region CDR3 as shown in SEQ ID NO: 17, the heavy chain complementarity-determining region CDR1 as shown in SEQ ID NO: 18, and the heavy chain complementarity-determining region CDR2 as shown in SEQ ID NO: 19; and three nucleotide sequences encoding the light chain complementarity-determining region CDR3 as shown in SEQ ID NO: 25, the light chain complementarity-determining region CDR1 as shown in SEQ ID NO: 26, and the light chain complementarity-determining region CDR2 as shown in SEQ ID NO: 27.

[0008] The monoclonal antibody YC201 includes a heavy chain variable region nucleotide sequence encoding the heavy chain variable region nucleotide sequence shown in SEQ ID NO: 20; and a light chain variable region nucleotide sequence shown in SEQ ID NO: 28.

[0009] A monoclonal antibody YC201 of Giardia lamblia CWP2 protein, wherein the monoclonal antibody YC201 has three amino acid sequences of the heavy chain complementarity-determining region CDR3 as shown in SEQ ID NO: 21, the heavy chain complementarity-determining region CDR1 as shown in SEQ ID NO: 22, and the heavy chain complementarity-determining region CDR2 as shown in SEQ ID NO: 23; and three amino acid sequences of the light chain complementarity-determining region CDR3 as shown in SEQ ID NO: 29, the light chain complementarity-determining region CDR1 as shown in SEQ ID NO: 30, and the light chain complementarity-determining region CDR2 as shown in SEQ ID NO: 31.

[0010] The monoclonal antibody YC201 includes the heavy chain variable region amino acid sequence as shown in SEQ ID NO: 24; and the light chain variable region amino acid sequence as shown in SEQ ID NO: 32.

[0011] A paired monoclonal antibody against Giardia lamblia CWP2 protein comprises a capture antibody and a detection antibody; the capture antibody YC301 has three nucleotide sequences encoding the heavy chain complementarity-determining region CDR3 as shown in SEQ ID NO: 1, the heavy chain complementarity-determining region CDR1 as shown in SEQ ID NO: 2, and the heavy chain complementarity-determining region CDR2 as shown in SEQ ID NO: 3; and three nucleotide sequences encoding the light chain complementarity-determining region CDR3 as shown in SEQ ID NO: 9, the light chain complementarity-determining region CDR1 as shown in SEQ ID NO: 10, and the light chain complementarity-determining region CDR2 as shown in SEQ ID NO: 11.

[0012] The detection antibody YC201 has three nucleotide sequences encoding the heavy chain complementarity-determining region (CDR3) as shown in SEQ ID NO: 17, the heavy chain complementarity-determining region (CDR1) as shown in SEQ ID NO: 18, and the heavy chain complementarity-determining region (CDR2) as shown in SEQ ID NO: 19; and the light chain complementarity-determining region (CDR3) as shown in SEQ ID NO: 25, the light chain complementarity-determining region (CDR1) as shown in SEQ ID NO: 26, and the light chain complementarity-determining region (CDR2) as shown in SEQ ID NO: 27.

[0013] The capture antibody YC301 includes a heavy chain variable region nucleotide sequence encoding the heavy chain variable region nucleotide sequence shown in SEQ ID NO: 4; and a light chain variable region nucleotide sequence shown in SEQ ID NO: 12.

[0014] A paired monoclonal antibody against Giardia lamblia CWP2 protein comprises a capture antibody and a detection antibody; the capture antibody YC301 has three amino acid sequences of the heavy chain complementarity-determining region CDR3 as shown in SEQ ID NO: 5, the heavy chain complementarity-determining region CDR1 as shown in SEQ ID NO: 6, and the heavy chain complementarity-determining region CDR2 as shown in SEQ ID NO: 7; and three amino acid sequences of the light chain complementarity-determining region CDR3 as shown in SEQ ID NO: 13, the light chain complementarity-determining region CDR1 as shown in SEQ ID NO: 14, and the light chain complementarity-determining region CDR2 as shown in SEQ ID NO: 15;

[0015] The detection antibody YC201 has three amino acid sequences as shown in SEQ ID NO: 21 (heavy chain complementarity-determining region CDR3), SEQ ID NO: 22 (heavy chain complementarity-determining region CDR1), and SEQ ID NO: 23 (heavy chain complementarity-determining region CDR2); and three amino acid sequences as shown in SEQ ID NO: 29 (light chain complementarity-determining region CDR3), SEQ ID NO: 30 (light chain complementarity-determining region CDR1), and SEQ ID NO: 31 (light chain complementarity-determining region CDR2).

[0016] The capture antibody YC301 includes the heavy chain variable region amino acid sequence as shown in SEQ ID NO: 8; and the light chain variable region amino acid sequence as shown in SEQ ID NO: 16.

[0017] A monoclonal antibody against Giardia lamblia CWP2 protein, comprising monoclonal antibodies YC101, YC401, YC501 and said monoclonal paired antibodies YC301 and YC201, is prepared by the following method:

[0018] (1) CWP2 protein immunization and antibody screening;

[0019] Five BALB / c mice were immunized with a mixture of 1 mg / mL Giardia lamblia CWP2 recombinant protein and an equal volume of Freund's adjuvant via subcutaneous injection at multiple sites on the back. Boosted immunizations were administered every two weeks after the initial immunization, and serum titers were measured after four immunizations, with a titer reaching 1:10. 5 Subsequently, the spleen of the immunized mice was taken, spleen cells were separated, and fused with SP2 / 0 cells;

[0020] Using the expressed CWP2 recombinant protein as the detection antigen, the coating buffer was diluted to 1 μg / mL and added to each well of the ELISA plate at 100 μL. The plate was incubated overnight at 4°C. After washing three times with 0.05% PBS + Tween-20, 200 μL of 5% skim milk solution was added to each well, and the plate was blocked at 37°C for 2 h. Hybridoma cell culture medium was added to each well at 100 μL, and the plate was blocked at 37°C for 1 h. The plate was then washed three times with phosphate-buffered saline (PBST), and horseradish peroxidase-labeled goat anti-mouse antibody was added. The plate was incubated at 37°C for 1 h. After washing five times with PBST, TMB substrate solution was added for color development for 15 min. The absorbance was read at 450 nm using an ELISA reader. A positive clone well was defined as one whose absorbance was more than three times that of the control well. The hybridoma cells were then subjected to multiple clonal screenings using the limiting dilution method, successfully identifying five monoclonal cell lines.

[0021] (2) Preparation and purification of monoclonal antibody ascites fluid;

[0022] Using an in vivo induction method in mice, BALB / c mice were intraperitoneally injected with 500 μL of liquid paraffin oil, followed by an intraperitoneal injection of 1×10⁻⁶ mol / L of liquid paraffin oil 9 days later. 6 The above five hybridoma cell lines produced ascites 7-10 days after inoculation; ascites was collected from mice using a 16-gauge needle in a clean bench; and subjected to 8000 r·min -1 Centrifuge for 10 minutes to remove cellular components and other precipitates, and collect the supernatant as ascites.

[0023] Ascites fluid was purified using a Protein G column. The packed rProtein G Beads gravity column was equilibrated with five volumes of equilibration buffer to ensure the packing material was in the same buffer system as the target protein. This process was repeated 2-3 times. The collected ascites fluid was added to the equilibrated gravity column, and the sample was retained for 30 minutes. The eluent was collected. The column was then washed with 10-15 column volumes of washing buffer to remove non-specifically adsorbed proteins, and the washing buffer was collected. Elution was performed with 5-10 column volumes of elution buffer, collecting fractions (one column volume per tube). The eluted fraction was immediately neutralized with 1 / 10 of the neutralization buffer. The eluent was the purified monoclonal antibody, including YC101, YC201, YC301, YC401, and YC501. The purity of the monoclonal antibodies was determined by SDS-PAGE gel electrophoresis.

[0024] Among them, YC101, YC401, and YC501 are used as capture antibodies, and YC201 is used as a detection antibody, which constitutes a pairwise monoclonal antibody against Giardia lamblia CWP2 protein.

[0025] Alternatively, YC201 can be used as a capture antibody and YC501 as a detection antibody to form a paired monoclonal antibody against Giardia lamblia CWP2 protein.

[0026] The application of the monoclonal antibody or paired antibody in the preparation of products for detecting Giardia lamblia CWP2 protein.

[0027] The present invention achieves the following beneficial technical effects:

[0028] The CWP2 protein selected in this invention is one of the main cyst wall proteins of Giardia lamblia, distributed in both trophozoites and cysts. CWP2 is a major protein component of the cyst wall structure and possesses strong immunogenicity. Furthermore, CWP2 protein forms a stable complex with other CWP proteins within 5 minutes of synthesis, thus it can serve as a detection target for Giardia lamblia antigens.

[0029] This invention obtained five pairs of paired monoclonal antibodies with good immunogenicity through immunoscreening of Giardia lamblia CWP2 protein. Among them, YC301 as the capture antibody and YC201 as the detection antibody showed the best immunogenicity. Furthermore, the double-antibody sandwich ELISA detection method established using these paired antibodies has been experimentally verified to detect CWP2 protein at a minimum concentration of 0.6 ug / ml. It also showed no cross-reactivity with some common parasites and good reproducibility. Therefore, it exhibited good sensitivity, specificity, and stability. Attached Figure Description

[0030] Figure 1 These are the SDS-PAGE results of five purified antibodies. M: Protein Marker; 1-5: Representing purified antibodies YC101, YC201, YC301, YC401, and YC501, respectively. A: SDS-PAGE analysis of purified antibody YC101; B: SDS-PAGE analysis of purified antibody YC201; C: SDS-PAGE analysis of purified antibody YC301; D: SDS-PAGE analysis of purified antibody YC401; E: SDS-PAGE analysis of purified antibody YC501.

[0031] Figure 2 These are the ELISA titer results after purification of monoclonal antibodies YC101, YC201, YC301, YC401, and YC501.

[0032] Figure 3 These are the Western blot results for five monoclonal antibodies. M: Protein Marker; 1: Giardia trophozoite protein. A: Western blot analysis of YC101 monoclonal antibody; B: Western blot analysis of YC201 monoclonal antibody; C: Western blot analysis of YC301 monoclonal antibody; D: Western blot analysis of YC401 monoclonal antibody; E: Western blot analysis of YC501 monoclonal antibody.

[0033] Figure 4 These are the results of ELISA titer assays of YC101, YC201, YC301, YC401, and YC501 monoclonal antibodies after enzyme labeling.

[0034] Figure 5 This is a sandwich ELISA image using a double antibody sandwich ELISA with capture antibody YC301 and detection antibody YC201. Detailed Implementation

[0035] I. Biological materials and reagents

[0036] Species: The Giardia lamblia species used in this embodiment is aggregate A, which was donated by Professor Zhang Xichen of Jilin University.

[0037] Main reagents: agar powder, tryptone, yeast extract, kanamycin, IPTG, SDS, Tris, EDTA, Coomassie Brilliant Blue R-250, urea, Triton X-100, color prestained protein marker, 10% PAGE gel preparation kit, Freund's complete adjuvant, Freund's incomplete adjuvant, hybridoma culture medium, HRP rapid labeling kit, bovine serum albumin (BSA), substrate chromogenic solution TMB, phosphate buffer PBST, HRP rapid labeling kit, etc.

[0038] Major instruments and equipment: benchtop centrifuge purchased from Hunan Xiangyi Laboratory Instrument Development Co., Ltd.; pH meter purchased from Shanghai Instrument & Electronics Scientific Instrument Co., Ltd.; constant temperature water bath purchased from Beijing Changfeng Instrument Co., Ltd.; cell culture incubator purchased from Thermo; multi-functional microplate reader purchased from Tecan Trading AG; electrophoresis apparatus purchased from Bio-Ra.

[0039] II. CWP2 protein immunization and antibody screening

[0040] Five BALB / c mice were immunized with a mixture of 1 mg / mL Giardia lamblia CWP2 recombinant protein and an equal volume of Freund's adjuvant via subcutaneous injection at multiple sites on the back. Boosted immunizations were administered every two weeks after the initial immunization, and serum titers were measured after four immunizations, with a titer reaching 1:10. 5 Subsequently, the spleen of the immunized mice was taken, spleen cells were separated, and fused with SP2 / 0 cells.

[0041] Using the expressed recombinant CWP2 protein as the detection antigen, the coating buffer was diluted to 1 μg / mL and added to each well of the ELISA plate (100 μL per well). The plate was incubated overnight at 4°C. After washing three times with 0.05% PBS + Tween-20, 200 μL of 5% skim milk solution was added to each well, and the plate was blocked at 37°C for 2 h. Hybridoma cell culture medium was added to each well (100 μL per well), and the plate was blocked at 37°C for 1 h. The plate was then washed three times with phosphate-buffered saline (PBST), and horseradish peroxidase-labeled goat anti-mouse antibody was added. The plate was reacted at 37°C for 1 h. After washing five times with PBST, TMB substrate was added for color development for 15 min. The absorbance was read at 450 nm using an ELISA reader. A positive clone well was defined as one whose absorbance was more than three times that of the control well. The hybridoma cells were then subjected to multiple clonal screenings using a limiting dilution method, successfully identifying five monoclonal cell lines.

[0042] III. Preparation and Purification of Monoclonal Antibody Ascites Fluid

[0043] Using an in vivo induction method in mice, BALB / c mice were intraperitoneally injected with 500 μL of liquid paraffin oil, followed by an intraperitoneal injection of 1×10⁻⁶ mol / L of liquid paraffin oil 9 days later. 6 The above five hybridoma cell lines produced ascites 7-10 days after inoculation; ascites was collected from mice using a 16-gauge needle in a clean bench; and subjected to 8000 r·min -1 Centrifuge for 10 minutes to remove cellular components and other precipitates, and collect the supernatant as ascites.

[0044] Ascites fluid was purified using a Protein G column. The packed rProtein G Beads gravity column was equilibrated with five volumes of equilibration buffer to ensure the packing material was in the same buffer system as the target protein. This process was repeated 2-3 times. The collected ascites fluid was added to the equilibrated gravity column, and the sample was retained for 30 minutes. The eluent was collected. The column was then washed with 10-15 column volumes of washing buffer to remove non-specifically adsorbed proteins, and the washing buffer was collected. Elution was performed with 5-10 column volumes of elution buffer, collected in fractions (one column volume per tube). The eluted fraction was immediately neutralized with 1 / 10 of the neutralization buffer. The eluent was the purified monoclonal antibody, including YC101, YC201, YC301, YC401, and YC501. The purity of the monoclonal antibodies was determined by SDS-PAGE gel electrophoresis. Figure 1 ).

[0045] IV. Monoclonal Antibody Titer Determination

[0046] The titer of the purified monoclonal antibodies was determined. Purified monoclonal antibodies YC101, YC201, YC301, YC401, and YC501 were diluted to 1 μg / mL with coating buffer and added to 100 μL per well of an ELISA plate. The plate was incubated overnight at 4°C. The plates were washed three times with phosphate-buffered saline (PBST), and 200 μL of 5% skim milk solution was added to each well. The plates were then blocked at 37°C for 2 h. The purified monoclonal antibodies were serially diluted 1:1000 to 1:512000, and 100 μL of the solution was added to each well. The plates were then blocked at 37°C for 1 h. The plates were washed three times with 0.05% PBS + Tween-20, and horseradish peroxidase-labeled goat anti-mouse antibody was added. The reaction was carried out at 37°C for 1 h. The plates were washed five times with 0.05% PBS + Tween-20, and TMB substrate solution was added for color development for 15 min. The absorbance was read at 450 nm using an ELISA reader. Figure 2 The titers of all five monoclonal antibodies reached 1:512000.

[0047] V. Identification of the reactivity of monoclonal antibodies with specific antigens

[0048] The purified monoclonal antibodies YC101, YC201, YC301, YC401, and YC501 were analyzed by Western blot. Following the instructions of the NewSemi PAGE gel kit, prepare a 1.0 mm thick, 10% SDS-PAGE protein gel. Add the extracted Giardia trophozoite total protein and 5× protein loading buffer at a 4:1 ratio to centrifuge tubes, mix thoroughly by pipetting, and boil at 100°C for 10 min. Load 20 μL per well. Set the upper gel voltage to 100 V and the lower gel voltage to 120 V. After electrophoresis, activate the PVDF membrane with methanol solution for 1 min and arrange it in the following order: sponge-filter paper-gel-PVDF membrane-filter paper-sponge, with the gel near the negative electrode and the PVDF membrane near the positive electrode. Under constant voltage (100 V), the protein moves from the negative electrode to the positive electrode, thus transferring the protein wet to the PVDF membrane. Prepare a 5% skim milk solution with PBST and block at 37°C for 2 h. After blocking, wash the membrane four times in PBST solution for 5 min each time. Add monoclonal antibody diluted in PBST solution and incubate at room temperature on a shaker for 2 hours. h; Discard the primary antibody, wash the membrane 4 times with PBST solution; Add goat anti-mouse IgG diluted with PBST solution and incubate on a shaker at room temperature for 2 h; Discard the secondary antibody, wash the membrane 5 times with PBST solution; Perform ECL chemiluminescence staining and observe the size and luminescence of the target protein. Figure 3 All five monoclonal antibodies reacted with total protein from Giardia trophozoites, and the bands were clear with no other contaminants.

[0049] VI. Monoclonal antibody horseradish peroxidase (HRP) labeling

[0050] HRP rapid labeling kit was used to label monoclonal antibodies with HRP enzyme. The monoclonal antibody was dialyzed thoroughly in 0.01M CB buffer to remove interfering substances. The HRP labeling kit was removed from the 4°C freezer and all components were thoroughly mixed. 250 μL (approximately 0.5 mg) of one of the purified monoclonal antibodies was added to 50 μL of pre-activated HRP (0.5 mg), mixed well, and then 54 μL of HRP labeling initiation solution was added. Mixing was repeated several times to avoid air bubbles. The mixture was then incubated in the dark at 30°C-37°C for 2-3 h. After coupling, 1 mL of deionized water was added to the reaction termination solution powder tube and mixed well. 25 μL of this solution was added to the above reaction solution, mixed thoroughly, and incubated at room temperature for 1 h. After termination, an equal volume of labeling preservation solution was added directly, mixed thoroughly, and stored at -20°C. The titer of the HRP-labeled monoclonal antibody was detected using an indirect ELISA method. Figure 4 The titers of all five enzyme-labeled antibodies reached 1:64000.

[0051] VII. Screening for paired antibodies

[0052] Five monoclonal antibodies were paired to select the optimal pairing antibody. The purified five monoclonal antibodies were diluted to 1 μg / mL as capture antibodies and coated at 37℃ for 2 h. After washing three times with phosphate-buffered saline (PBST), 200 μL of 5% skim milk solution was added to each well, and the mixture was incubated at 37℃ for 2 h. Diluted Giardia antigen was added to each well, and the mixture was incubated at 37℃ for 1 h. HRP-labeled monoclonal antibody was added, and the mixture was incubated at 37℃ for 1 h. After washing five times with PBST, TMB substrate solution was added for color development for 15 min, and the absorbance was read at 450 nm using a microplate reader. The pairing results are shown in Table 1. According to the colorimetric results, the strongest signal was obtained when CWP2 monoclonal antibody YC301 was used as the capture antibody and HRP-labeled monoclonal antibody YC201-HRP was used as the detection antibody; the signals were also relatively strong when CWP2 monoclonal antibodies YC501, YC101, and YC401 were used as the capture antibodies and HRP-labeled monoclonal antibody YC201-HRP was used as the detection antibody, as well as when CWP2 monoclonal antibody YC201 was used as the capture antibody and HRP-labeled monoclonal antibody YC501-HRP was used as the detection antibody.

[0053] Table 1 Results of antibody pairing test

[0054]

[0055] 8. Sequencing of the best paired antibodies

[0056] Hybridoma cells corresponding to the selected optimal paired antibodies YC301 and YC201 were sequenced and analyzed. Total RNA was extracted from the hybridoma cells after lysis; cDNA was synthesized by reverse transcription using rapid cDNA end amplification (RACE); the variable regions of the heavy and light chains were obtained by PCR amplification; the target fragments were ligated into the vector pcDNA3.4 using ligase, and the ligation product was transformed into *E. coli* competent cells DH5α. Single clones were then selected for sequencing. The sequence included the core sequences of the paired antibodies (variable regions of the heavy and light chains).

[0057] The capture antibody YC301 has three amino acid sequences as shown in SEQ ID NO: 5 (heavy chain complementarity-determining region CDR3), SEQ ID NO: 6 (heavy chain complementarity-determining region CDR1), and SEQ ID NO: 7 (heavy chain complementarity-determining region CDR2); and three amino acid sequences as shown in SEQ ID NO: 13 (light chain complementarity-determining region CDR3), SEQ ID NO: 14 (light chain complementarity-determining region CDR1), and SEQ ID NO: 15 (light chain complementarity-determining region CDR2).

[0058] The detection antibody YC201 has three amino acid sequences as shown in SEQ ID NO: 21 (heavy chain complementarity-determining region CDR3), SEQ ID NO: 22 (heavy chain complementarity-determining region CDR1), and SEQ ID NO: 23 (heavy chain complementarity-determining region CDR2); and three amino acid sequences as shown in SEQ ID NO: 29 (light chain complementarity-determining region CDR3), SEQ ID NO: 30 (light chain complementarity-determining region CDR1), and SEQ ID NO: 31 (light chain complementarity-determining region CDR2).

[0059] The capture antibody YC301 has three nucleotide sequences encoding the heavy chain complementarity-determining region CDR3 as shown in SEQ ID NO: 1, the heavy chain complementarity-determining region CDR1 as shown in SEQ ID NO: 2, and the heavy chain complementarity-determining region CDR2 as shown in SEQ ID NO: 3; and the light chain complementarity-determining region CDR3 as shown in SEQ ID NO: 9, the light chain complementarity-determining region CDR1 as shown in SEQ ID NO: 10, and the light chain complementarity-determining region CDR2 as shown in SEQ ID NO: 11.

[0060] The detection antibody YC201 has three nucleotide sequences encoding the heavy chain complementarity-determining region (CDR3) as shown in SEQ ID NO: 17, the heavy chain complementarity-determining region (CDR1) as shown in SEQ ID NO: 18, and the heavy chain complementarity-determining region (CDR2) as shown in SEQ ID NO: 19; and the light chain complementarity-determining region (CDR3) as shown in SEQ ID NO: 25, the light chain complementarity-determining region (CDR1) as shown in SEQ ID NO: 26, and the light chain complementarity-determining region (CDR2) as shown in SEQ ID NO: 27.

[0061] The capture antibody YC301 includes the heavy chain variable region amino acid sequence as shown in SEQ ID NO: 8; and the light chain variable region amino acid sequence as shown in SEQ ID NO: 16.

[0062] The detection antibody YC201 includes the heavy chain variable region amino acid sequence as shown in SEQ ID NO: 24; and the light chain variable region amino acid sequence as shown in SEQ ID NO: 32.

[0063] The capture antibody YC301 includes a heavy chain variable region nucleotide sequence encoding the heavy chain variable region nucleotide sequence shown in SEQ ID NO: 4; and a light chain variable region nucleotide sequence shown in SEQ ID NO: 12.

[0064] The detection antibody YC201 includes a heavy chain variable region nucleotide sequence encoding the heavy chain variable region nucleotide sequence shown in SEQ ID NO: 20; and a light chain variable region nucleotide sequence shown in SEQ ID NO: 28.

[0065] The specific sequence information is as follows:

[0066] SEQ ID NO: 1 (nucleotide sequence of CDR3 heavy chain complementarity-determining region of capture antibody YC301)

[0067] GCAAGAAGGGGAAGGTACTACGGTAGTAGCGTTGGGGACTAC

[0068] SEQ ID NO: 2 (nucleotide sequence of CDR1 heavy chain complementarity-determining region of capture antibody YC301)

[0069] GGCTACACCTTCACCAGCTATTGG

[0070] SEQ ID NO: 3 (nucleotide sequence of CDR2 in the heavy chain complementarity-determining region of capture antibody YC301)

[0071] ATTGACCCTAGTAACGGTCGTACT

[0072] SEQ ID NO: 4 (nucleotide sequence of the variable region of the heavy chain of capture antibody YC301)

[0073] CAGGTCCAACTGCAGCAGCCTGGGGCTGAACTGGTGAAGCCTGGGGCTTCAGAGAAGCTGTCCTGCAAGGCTTCTGGCTACACCTTCACCAGCTATTGGATGCACTGGATGAAGCAGAGGCCTGGACAAGGCCTTGAGTGGATTGGAGAGATTGACCCTAGTAACGGTCGTACTAACTACA ATGAGAAGTTCAGGAGCAAGGCCACACTGACTGTAGACAAATCCTCCAACACAGCCTACATGCAACTCAGCAGCCTGACATCTGAGGACTCTGCGGTCTATTACTGTGCAAGAAGGGGAAGGTACTACGGTAGTAGCGTTGGGGACTACTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCA

[0074] SEQ ID NO: 5 (Amino acid sequence of CDR3 in the heavy chain complementarity-determining region of capture antibody YC301)

[0075] ARRGRYYGSSVGDY

[0076] SEQ ID NO: 6 (Amino acid sequence of CDR1 in the heavy chain complementarity-determining region of capture antibody YC301)

[0077] GYTFTSYW

[0078] SEQ ID NO: 7 (Amino acid sequence of CDR2 in the heavy chain complementarity-determining region of capture antibody YC301)

[0079] IDPSNGRT

[0080] SEQ ID NO: 8 (Amino acid sequence of the variable region of the heavy chain of capture antibody YC301)

[0081] QVQLQQPGAELVKPGASEKLSCKASGYTFTSYWMHWMKQRPGQGLEWIGEIDPSNGRTNYNEKFRSKATLVDKSSNTAYMQLSSLTSEDSAVYYCARRGRYYGSSVGDYWGQGTSVTVSS

[0082] SEQ ID NO: 9 (nucleotide sequence of CDR3 light chain complementarity-determining region of capture antibody YC301)

[0083] CAACAGGTTAATAAGTTTCCGCTCACG

[0084] SEQ ID NO: 10 (nucleotide sequence of CDR1, light chain complementarity-determining region, of capture antibody YC301)

[0085] CAGGACGTTAACAATTAT

[0086] SEQ ID NO: 11 (nucleotide sequence of CDR2, light chain complementarity-determining region, of capture antibody YC301)

[0087] TACACTTCA

[0088] SEQ ID NO: 12 (nucleotide sequence of the variable region of the light chain of capture antibody YC301)

[0089] TATATCCAGATGACACAGACAACATCCTCCCTGTCTGCCTCTCTGGGAGACAGAGTCACCATCAGTTGCAGGGCAAGTCAGGACGTTAACAATTATTTAAACTGGTATCAGCAGAAACCAGATGGAACTGTTAAACTCCTGATCTACTACACTTCAAGAT TACATTCAGGAGTCCCATCAAGGTTCAGTGGCAGTGGGTCTGGAACAGATTATTCTCTCACCATTAGCAACCTGGACCAAGAAGATATTGCCACTTACTTTTGCCAACAGGTTAATAAGTTTCCGCTCACGTTCGGTGCTGGGACCAAGCTGGAGCTGAAA

[0090] SEQ ID NO: 13 (Amino acid sequence of CDR3 in the light chain complementarity-determining region of capture antibody YC301)

[0091] QQVNKFPLT

[0092] SEQ ID NO: 14 (Amino acid sequence of CDR1 in the light chain complementarity-determining region of capture antibody YC301)

[0093] QDVNNY

[0094] SEQ ID NO: 15 (Amino acid sequence of CDR2 in the light chain complementarity-determining region of capture antibody YC301)

[0095] YTS

[0096] SEQ ID NO: 16 (Amino acid sequence of the variable region of the light chain of capture antibody YC301)

[0097] YIQMTQTTSSLSASLGDRVTISCRASQDVNNYLNWYQQKPDGTVKLLIYYTSRLHSGVPSRFSGSGSGTDYSLTISNLDQEDIATYFCQQVNKFPLTFGAGTKLELK

[0098] SEQ ID NO: 17 (nucleotide sequence of CDR3 in the heavy chain complementarity-determining region of antibody YC201)

[0099] GCAATATCTAGTGGTAACTACTAC

[0100] SEQ ID NO: 18 (nucleotide sequence of CDR1 heavy chain complementarity-determining region of antibody YC201)

[0101] GGCTACACCTTCACTAAATACTGG

[0102] SEQ ID NO: 19 (nucleotide sequence of CDR2 heavy chain complementarity-determining region of antibody YC201)

[0103] ATTAATCCTAGCACTGATTATACT

[0104] SEQ ID NO: 20 (nucleotide sequence of the variable region of the heavy chain of antibody YC201)

[0105] CAGGTCCAACTTCAGCAGTCTGGGGCTGAACTGGCAAAACCTGGGGCCTCAGTGAAGATGTCCTGCAAGGCTTCTGGCTACACCTTCACTAAATACTGGATACACTGGGTAAAACAGAGGCCTGGACAGGGTCTGGAATGGATTGGATACATTAATCCTAGCACTGATTATA CTGAGTACAATCAGAACTTCAAGGACAAGGCCACATTGACTGCAGACAAATCCTCCAGCACAGCCTACATGCAACTGAGCAGCCTGACATCTGAAGACTCTGCAGTCTTTTACTGTGCAATATCTAGTGGTAACTACTGGGGCCAAGGCGCCACTCTCACAGTCTCCTCA

[0106] SEQ ID NO: 21 (Amino acid sequence of CDR3 in the heavy chain complementarity-determining region of antibody YC201)

[0107] AISSGNYY

[0108] SEQ ID NO: 22 (Amino acid sequence of CDR1 heavy chain complementarity-determining region of antibody YC201)

[0109] GYTFTKYW

[0110] SEQ ID NO: 23 (Amino acid sequence of CDR2 in the heavy chain complementarity-determining region of antibody YC201)

[0111] INPSTDYT

[0112] SEQ ID NO: 24 (Amino acid sequence of the variable region of the heavy chain of antibody YC201)

[0113] QVQLQQSGAELAKPGASVKMSCKASGYTFTKYWIHWVKQRPGQGLEWIGYINPSTDYTEYNQNFKDKATLTADKSSSTAYMQLSSLTSEDSAVFYCAISSGNYYWGQGATLTVSS

[0114] SEQ ID NO: 25 (nucleotide sequence of CDR3 in the light chain complementarity-determining region of antibody YC201)

[0115] AAGCAATCTTATAATCTTCCGTGGACG

[0116] SEQ ID NO: 26 (nucleotide sequence of CDR1 light chain complementarity-determining region of antibody YC201)

[0117] CAGAGTCTGCTCATCAGTAGAACCCGAAAGAGTTAC

[0118] SEQ ID NO: 27 (nucleotide sequence of CDR2 light chain complementarity-determining region of antibody YC201)

[0119] TGGGCATCC

[0120] SEQ ID NO: 28 (nucleotide sequence of the variable region of the light chain of antibody YC201)

[0121] GACATTGTGATGTCACAGTCTCCTTCCTCCCTGGCTGTGTCAACAGGAGAGAAGGTCACTATGAGCTGCAAATCCAGTCAGAGTCTGCTCATCAGTAGAACCCGAAAGAGTTACTTGGCTTGGTACCAGCAGAAACCAGGGCAGTCTCCTAAACTGCTGATCTACTGGG CATCCACTAGGGAATCTGGGGTCCCTGATCGCTTCACAGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTGTGCAGGCTGAAGACCTGGCAGTTTTTTACTGCAAGCAATCTTATAATCTTCCGTGGACGTTCGGTGGAGGCACCAAGCTGGAAATCAAA

[0122] SEQ ID NO: 29 (Amino acid sequence of CDR3 in the light chain complementarity-determining region of antibody YC201)

[0123] KQSYNLPWT

[0124] SEQ ID NO: 30 (Amino acid sequence of CDR1 in the light chain complementarity-determining region of antibody YC201)

[0125] QSLLISRTRKSY

[0126] SEQ ID NO: 31 (Amino acid sequence of CDR2 in the light chain complementarity-determining region of antibody YC201)

[0127] WAS

[0128] SEQ ID NO: 32 (Amino acid sequence of the variable region of the light chain of antibody YC201)

[0129] DIVMSQSPSLAVSTGEKVTMSCKSSQSLLISRTRKSYLAWYQQKPGQSPKLLIYWASTRESGVPDRFTGSGSGTDFTLTISSVQAEDLAVFYCKQSYNLPWTFGGGTKLEIK

[0130] IX. Detection of Giardia lamblia CWP2 protein using a double-antibody sandwich ELISA method

[0131] Take the purified monoclonal antibody YC301, dilute the coating buffer to 4 μg / mL, add 100 μL to each well of an ELISA plate, and incubate overnight at 4°C. Wash three times with 1×PBST, add 200 μL of 5% BSA solution to each well, and block at room temperature for 2 h. Remove the blocking buffer, add 100 μL of Giardia lamblia whole protein, and react at 37°C for 90 min. Wash three times with 1×PBST, add 100 μL of the horseradish peroxidase-labeled monoclonal antibody YC201 (diluted to 1:2000), and react at 37°C for 1 h. Wash five times with 1×PBST, add the luminescent substrate solution, react at room temperature for 10 min, and read the OD using an ELISA reader. 450nm value( Figure 5 ).

[0132] 10. Evaluation of Detection Methods

[0133] (1) Specificity test

[0134] The double-antibody sandwich ELISA method was used to detect Cryptosporidium microsporum, Toxoplasma gondii, Bacillus cystis, Leishmania, and Coccidia. Giardia was used as a positive control and PBS was used as a negative control. The specificity of the double-antibody sandwich ELISA method established with the paired antibodies YC301 and YC201 of this invention was evaluated.

[0135] The results showed a positive reaction with Giardia trophozoites, but no cross-reaction with other parasites, indicating that the double-antibody sandwich ELISA detection method established with the paired antibodies YC301 and YC201 in this invention has good specificity.

[0136] (2) Sensitivity test

[0137] Giardia trophozoite whole-cell protein at antigen concentrations of 0.08 mg / mL, 0.04 mg / mL, 0.02 mg / mL, 0.01 mg / mL, 0.005 mg / mL, 0.0025 mg / mL, 0.0012 mg / mL, 0.0006 mg / mL, and 0.0003 mg / mL were used as the test antigens, with PBS as the negative control. The sensitivity of the double-antibody sandwich ELISA detection method established with the paired antibodies YC301 and YC201 of this invention was determined.

[0138] The sensitivity of this method was tested using Giardia trophozoites; a P / N value > 2.1 was considered positive. The results showed that when the antigen concentration was 0.6 μg / mL, the P / N value was 4.55, indicating that the detection limit for Giardia trophozoite lysates was 0.6 μg / mL (Table 2). This demonstrates that the double-antibody sandwich ELISA method established using the paired antibodies YC301 and YC201 of this invention has good sensitivity.

[0139] Table 2 Sensitivity Test Results

[0140]

[0141] (3) Repeatability test

[0142] The established double-antibody sandwich ELISA method was subjected to inter-assay and intra-assay repeatability experiments. Five positive samples were tested, and each sample was tested in triplicate. The mean, standard deviation, and coefficient of variation of each group were calculated to verify the repeatability of the double-antibody sandwich ELISA detection method established with the paired antibodies YC301 and YC201 of this invention.

[0143] The results showed that the coefficients of variation for each group were between 0.30% and 3.84% (<5%, see Table 3), indicating that the double-antibody sandwich ELISA detection method established by the paired antibodies YC301 and YC201 in this invention has good reproducibility.

[0144] Table 3 Repeatability Test Results

[0145]

[0146] 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 YC201 against Giardia lamblia CWP2 protein, characterized in that, The monoclonal antibody YC201 has a heavy chain complementarity-determining region (CDR1) as shown in SEQ ID NO: 22, a heavy chain complementarity-determining region (CDR2) as shown in SEQ ID NO: 23, and a heavy chain complementarity-determining region (CDR3) as shown in SEQ ID NO: 21; and a light chain complementarity-determining region (CDR1) as shown in SEQ ID NO: 30, a light chain complementarity-determining region (CDR2) with the amino acid sequence WAS, and a light chain complementarity-determining region (CDR3) as shown in SEQ ID NO:

29.

2. The monoclonal antibody YC201 according to claim 1, characterized in that, The monoclonal antibody YC201 includes a heavy chain variable region with an amino acid sequence as shown in SEQ ID NO: 24; and a light chain variable region with an amino acid sequence as shown in SEQ ID NO:

32.

3. A polynucleotide encoding the monoclonal antibody YC201 as described in claim 1 or claim 2.

4. A pairing monoclonal antibody against Giardia lamblia CWP2 protein, characterized in that, The invention includes a capture antibody and a detection antibody; the capture antibody is a monoclonal antibody YC301, which has a heavy chain complementarity-determining region CDR1 as shown in SEQ ID NO: 6, a heavy chain complementarity-determining region CDR2 as shown in SEQ ID NO: 7, and a heavy chain complementarity-determining region CDR3 as shown in SEQ ID NO: 5; and a light chain complementarity-determining region CDR1 as shown in SEQ ID NO: 14, a light chain complementarity-determining region CDR2 with the amino acid sequence YTS, and a light chain complementarity-determining region CDR3 as shown in SEQ ID NO: 13; The detection antibody is the monoclonal antibody YC201 as described in claim 1 or claim 2.

5. The paired monoclonal antibody according to claim 4, characterized in that, The monoclonal antibody YC301 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.

6. A polynucleotide encoding the monoclonal antibody YC301 as described in claim 4 or claim 5.

7. The use of the paired monoclonal antibody according to claim 4 or claim 5 in the preparation of a product for detecting Giardia lamblia CWP2 protein.

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