Monoclonal antibody of giardia duodenalis CWP2 protein, paired monoclonal antibody and application

Abstract

The invention relates to a monoclonal antibody of giardia duodenalis CWP2 protein, a paired monoclonal antibody and application. Five pairs of paired monoclonal antibodies are obtained through immune screening of the giardia duodenalis CWP2 protein, the immune effect is good, the immune effect is best when YC301 serves as a capture antibody and YC201 serves as a detection antibody, and a double-antibody sandwich ELISA detection method established by utilizing the paired antibodies is good in sensitivity, specificity and stability and has good application prospects. And a solid foundation is laid for the development of a giardia duodenalis CWP2 antigen detection kit.

Description

Technical Field

[0001] The invention relates to the technical field of immune detection, and in particular to a monoclonal antibody and a paired monoclonal antibody of Giardia duodenalis CWP2 protein and applications thereof. Background Art

[0002] Giardiasis is a disease caused by Giardia spp. Giardia ) is a zoonosis caused by intestinal protozoan infection, which is mainly transmitted through the fecal-oral route. After the host is infected with Giardia, vomiting, diarrhea, dehydration and weight loss often occur, which seriously endangers the livestock and poultry farming industry and public health safety. At present, there is no specific treatment for duodenal giardiasis, which mainly relies on imidazole drugs for treatment, but long-term use can easily make the parasite resistant. Therefore, improving the diagnosis level of giardiasis is a crucial link in conducting epidemiological surveys and clinical testing.

[0003] There are two different morphological stages in the life cycle of Giardia, namely trophozoites and cysts. During the infection period, mature cysts enter the human body through contaminated food and water, and exude in the duodenum to form two trophozoites. Trophozoites parasitize in the small intestine, especially in the duodenum. Some trophozoites form cysts in the terminal small intestine and are excreted with feces, contaminating food, water sources and interfaces. The cysts have strong environmental resistance and can survive in the environment for a long time, allowing giardiasis to continue to spread between hosts, increasing the risk of food-borne and water-borne transmission.

[0004] At present, microscopy is the main method for diagnosing whether a host is infected with Giardia, but this method is highly subjective, depends on the experience of the observer, is time-consuming and laborious, and sometimes cannot be detected due to low infection rates. Therefore, more and more biological detection methods are being used. Among them, molecular biological detection methods, such as the use of PCR amplification to detect the deoxyribonucleic acid of Giardia intestinalis, have high sensitivity, but due to the high professional requirements for instruments and operators, their promotion and popularization at the grassroots level are limited to a certain extent. In contrast, immunological diagnostic technology has the advantages of being fast, simple, highly sensitive, and highly specific, showing good application prospects. However, there are still some problems with the various existing immunological diagnostic technologies for giardiasis. First, for patients with mild infection or in the incubation period, existing technologies may not be able to accurately detect Giardia antigens or antibodies, resulting in missed diagnosis; second, some advanced immunological diagnostic technologies require expensive reagents and equipment, and the operation is cumbersome and time-consuming, which increases the cost of detection, such as indirect immunofluorescence and immunoblotting; third, due to the complexity of the immune response and individual differences, misjudgment may occur. For example, some other pathogens with similar antigens to Giardia may cause false positive results. Summary of the invention

[0005] The technical problem to be solved by the present invention is to overcome the problems of complex operation, high cost, low sensitivity and low specificity in the existing immunological diagnosis technology of giardiasis.

[0006] In order to solve this technical problem, the present invention provides a paired monoclonal antibody with high sensitivity and strong specificity for detecting Giardia duodenalis CWP2 protein.

[0007] A monoclonal antibody YC201 against Giardia duodenalis CWP2 protein, the monoclonal antibody YC201 having three nucleotide sequences encoding a heavy chain complementary determining region CDR1 as shown in SEQ ID NO: 17, a heavy chain complementary determining region CDR2 as shown in SEQ ID NO: 18, and a heavy chain complementary determining region CDR3 as shown in SEQ ID NO: 19; and three nucleotide sequences encoding a light chain complementary determining region CDR1 as shown in SEQ ID NO: 25, a light chain complementary determining region CDR2 as shown in SEQ ID NO: 26, and a light chain complementary determining region CDR3 as shown in SEQ ID NO: 27.

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

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

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

[0011] A paired monoclonal antibody for Giardia duodenalis CWP2 protein, comprising a capture antibody and a detection antibody; the capture antibody YC301 has three nucleotide sequences encoding a heavy chain complementary determining region CDR1 as shown in SEQ ID NO: 1, a heavy chain complementary determining region CDR2 as shown in SEQ ID NO: 2, and a heavy chain complementary determining region CDR3 as shown in SEQ ID NO: 3; and three nucleotide sequences encoding a light chain complementary determining region CDR1 as shown in SEQ ID NO: 13, a light chain complementary determining region CDR2 as shown in SEQ ID NO: 14, and a light chain complementary determining region CDR3 as shown in SEQ ID NO: 15; The detection antibody YC201 has three nucleotide sequences encoding the heavy chain complementary determining region CDR1 as shown in SEQ ID NO: 17, the heavy chain complementary determining region CDR2 as shown in SEQ ID NO: 18, and the heavy chain complementary determining region CDR3 as shown in SEQ ID NO: 19; and three nucleotide sequences encoding the light chain complementary determining region CDR1 as shown in SEQ ID NO: 25, the light chain complementary determining region CDR2 as shown in SEQ ID NO: 26, and the light chain complementary determining region CDR3 as shown in SEQ ID NO: 27.

[0012] The capture antibody YC301 comprises a nucleotide sequence encoding a heavy chain variable region as shown in SEQ ID NO: 4; and a nucleotide sequence encoding a light chain variable region as shown in SEQ ID NO: 12.

[0013] A paired monoclonal antibody for Giardia duodenalis CWP2 protein, comprising a capture antibody and a detection antibody; the capture antibody YC301 has three amino acid sequences of a heavy chain complementary determining region CDR1 as shown in SEQ ID NO: 5, a heavy chain complementary determining region CDR2 as shown in SEQ ID NO: 6, and a heavy chain complementary determining region CDR3 as shown in SEQ ID NO: 7; and three amino acid sequences of a light chain complementary determining region CDR1 as shown in SEQ ID NO: 13, a light chain complementary determining region CDR2 as shown in SEQ ID NO: 14, and a light chain complementary determining region CDR3 as shown in SEQ ID NO: 15; The detection antibody YC201 has three amino acid sequences of a heavy chain complementary determining region CDR1 as shown in SEQ ID NO: 21, a heavy chain complementary determining region CDR2 as shown in SEQ ID NO: 22, and a heavy chain complementary determining region CDR3 as shown in SEQ ID NO: 23; and three amino acid sequences of a light chain complementary determining region CDR1 as shown in SEQ ID NO: 29, a light chain complementary determining region CDR2 as shown in SEQ ID NO: 30, and a light chain complementary determining region CDR3 as shown in SEQ ID NO: 31.

[0014] The capture antibody YC301 includes a heavy chain variable region amino acid sequence as shown in SEQ ID NO: 8; and a light chain variable region amino acid sequence as shown in SEQ ID NO: 16.

[0015] A monoclonal antibody to Giardia duodenalis CWP2 protein, including monoclonal antibodies YC101, YC401, YC501 and the monoclonal paired antibodies YC301 and YC201, is prepared by the following method: (1) CWP2 protein immunization and antibody screening; 1 mg / mL of Giardia duodenalis CWP2 recombinant protein was mixed with an equal volume of Freund's adjuvant and immunized 5 BALB / c mice by subcutaneous injection at multiple points on the back. One week after the first immunization, booster immunization was performed every two weeks. After four immunizations, serum titer was tested and the titer reached 1:10. 5 Afterwards, the spleen of the immunized mouse was obtained, spleen cells were isolated, and fused with SP2 / 0 cells; The expressed CWP2 recombinant protein was used as the detection antigen, and the coating solution was diluted to 1 μg / mL and added to the ELISA plate, 100 μL per well, and placed at 4°C for overnight coating; washed 3 times with 0.05% PBS+Tween-20, added 5% skim milk solution, 200 μL per well, and blocked at 37°C for 2 h; added hybridoma cell culture medium, 100 μL per well, and blocked at 37°C for 1 h; washed 3 times with phosphate buffer PBST, added goat anti-mouse antibody labeled with horseradish peroxidase, and reacted at 37°C for 1 h; washed 5 times with phosphate buffer PBST, added TMB substrate solution for color development for 15 min, and read the absorbance at 450 nm on the ELISA reader. When the absorbance of the sample control was more than 3 times that of the control well, it was determined to be a positive clone well; then the hybridoma cells were cloned multiple times using the limiting dilution method, and five monoclonal cell lines were successfully screened.

[0016] (2) Preparation and purification of monoclonal antibody ascites; The mouse induction method was used. BALB / c mice were intraperitoneally injected with 500 μL of liquid paraffin oil. Nine days later, 1×10 6 The five hybridoma cells mentioned above can produce ascites 7 to 10 days after inoculation. The ascites of mice was collected with a 16-gauge needle on a clean bench. -1 Centrifuge for 10 min to remove cell components and other precipitates, and collect the supernatant as ascites; A Protein G column was used to affinity purify the ascites. The loaded rProtein G Beads gravity column was equilibrated with five times the volume of equilibration solution, so that the filler was in the same buffer system as the target protein, and the process was repeated 2-3 times. The collected ascites was added to the equilibrated gravity column, the sample was retained for 30 minutes, and the effluent was collected. The impurities were washed with 10-15 times the column volume of washing solution to remove non-specifically adsorbed impurities, and the washing solution was collected. The eluent was eluted with 5-10 times the column volume of elution solution, and the samples were collected in sections. One tube was collected for each column volume. The eluted components were immediately neutralized with 1 / 10 of the neutralization solution. The eluent was the purified monoclonal antibody, including YC101, YC201, YC301, YC401, and YC501. The purity of the monoclonal antibody was detected by SDS-PAGE gel electrophoresis. Among them, YC101, YC401, and YC501 are used as capture antibodies, and the YC201 is used as a detection antibody, constituting a paired monoclonal antibody for Giardia duodenalis CWP2 protein; Alternatively, the YC201 is used as a capture antibody and the YC501 is used as a detection antibody to form a paired monoclonal antibody for Giardia duodenalis CWP2 protein.

[0017] The monoclonal antibody or paired antibody is used in preparing a product for detecting Giardia duodenalis CWP2 protein.

[0018] The present invention achieves the following beneficial technical effects: The CWP2 protein selected in the present invention is one of the main cyst wall proteins of Giardia cyst wall, distributed in both trophozoites and cysts, and CWP2 is the main protein component in the cyst wall structure, with strong immunogenicity. Moreover, the CWP2 protein forms a stable complex with other CWP proteins within 5 minutes after synthesis, and therefore, can be used as a detection target for detecting Giardia duodenalis antigens.

[0019] The present invention obtains 5 pairs of paired monoclonal antibodies with good immune effects through immune screening of Giardia duodenalis CWP2 protein, among which the best immune effect is achieved when YC301 is used as a capture antibody and YC201 is used as a detection antibody. In addition, a double-antibody sandwich ELISA detection method established by using the paired antibodies is experimentally verified to have a minimum detectable concentration of CWP2 protein of 0.6 ug / ml, no cross-reaction with some common parasites, and good repeatability. Therefore, it performs well in sensitivity, specificity and stability. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 These are the results of SDS-PAGE after purification of 5 kinds of antibodies. Among them, M: protein marker, 1~5: respectively represent purified antibodies YC101, YC201, YC301, YC401, and YC501. 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.

[0021] Figure 2 These are the results of ELISA titer determination of the purified monoclonal antibodies YC101, YC201, YC301, YC401, and YC501.

[0022] Figure 3 The following are the Western blot results of 5 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.

[0023] Figure 4 It is the result of ELISA titer determination after enzyme labeling of YC101, YC201, YC301, YC401 and YC501 monoclonal antibodies.

[0024] Figure 5 This is a double antibody sandwich ELISA image using capture antibody YC301 and detection antibody YC201. DETAILED DESCRIPTION

[0025] 1. Biological Materials and Reagents Species: The species of Giardia duodenalis in this example is Aggregate A, which was donated by Professor Zhang Xichen of Jilin University.

[0026] Main reagents: agar powder, tryptone, yeast extract, kanamycin, IPTG, SDS, Tris, EDTA, Coomassie Brilliant Blue R-250, urea, Triton X-100, color pre-stained protein Marker, New Cyme 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 color development solution TMB, phosphate buffer PBST, HRP rapid labeling kit, etc.

[0027] Main instruments and equipment: desktop 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.; cell culture incubator purchased from Thermo; multifunctional microplate reader purchased from Tecan Trading AG; electrophoresis instrument purchased from Bio-Ra.

[0028] 2. CWP2 protein immunization and antibody screening 1 mg / mL of Giardia duodenalis CWP2 recombinant protein was mixed with an equal volume of Freund's adjuvant and immunized 5 BALB / c mice with multiple subcutaneous injections on the back. One week after the first immunization, booster immunization was performed every two weeks. After four immunizations, serum titer was tested and the titer reached 1:10. 5 Afterwards, the spleen of the immunized mouse was taken, spleen cells were separated and fused with SP2 / 0 cells.

[0029] The expressed CWP2 recombinant protein was used as the detection antigen, and the coating solution was diluted to 1 μg / mL and added to the ELISA plate, 100 μL per well, and placed at 4°C for overnight coating; washed 3 times with 0.05% PBS+Tween-20, added 5% skim milk solution, 200 μL per well, and blocked at 37°C for 2 h; added hybridoma cell culture medium, 100 μL per well, and blocked at 37°C for 1 h; washed 3 times with phosphate buffer PBST, added goat anti-mouse antibody labeled with horseradish peroxidase, and reacted at 37°C for 1 h; washed 5 times with phosphate buffer PBST, added TMB substrate solution for color development for 15 min, and read the absorbance at 450 nm on the ELISA reader. When the absorbance of the sample control was more than 3 times that of the control well, it was determined to be a positive clone well; then the hybridoma cells were cloned multiple times using the limiting dilution method, and five monoclonal cell lines were successfully screened.

[0030] 3. Preparation and purification of monoclonal antibody ascites The mouse induction method was used. BALB / c mice were intraperitoneally injected with 500 μL of liquid paraffin oil. Nine days later, 1×10 6 The five hybridoma cells mentioned above can produce ascites 7 to 10 days after inoculation. The ascites of mice was collected with a 16-gauge needle on a clean bench. -1 Centrifuge for 10 min to remove cell components and other precipitates, and collect the supernatant as ascites; Protein G column was used for affinity purification of ascites. The loaded rProtein G Beads gravity column was balanced with five times the volume of balance solution, so that the filler was in the same buffer system as the target protein, and repeated 2-3 times; the collected ascites was added to the balanced gravity column, the sample was retained for 30 minutes, and the effluent was collected; 10-15 times the column volume of washing solution was used for washing to remove non-specifically adsorbed impurities, and the washing solution was collected; 5-10 times the column volume of eluent was used for elution, and the segmented collection was carried out, and one tube was collected for each column volume. The eluted component volume was immediately neutralized with 1 / 10 of the neutralizing solution. The eluent was the purified monoclonal antibody, including YC101, YC201, YC301, YC401, and YC501; the purity of the monoclonal antibody was detected by SDS-PAGE gel electrophoresis ( Figure 1 ).

[0031] 4. Determination of the titer of monoclonal antibodies The purified monoclonal antibodies were titered. The purified monoclonal antibodies YC101, YC201, YC301, YC401, and YC501 were diluted to 1 μg / mL with the coating solution, added to the ELISA plate, 100 μL per well, and placed at 4°C for overnight coating; washed 3 times with phosphate buffered saline (PBST), added 5% skim milk solution, 200 μL per well, and blocked at 37°C for 2 h; the purified monoclonal antibodies were diluted 1:1000 to 1:512000, 100 μL per well, and blocked at 37°C for 1 h; washed 3 times with 0.05% PBS+Tween20, added goat anti-mouse antibody labeled with horseradish peroxidase, and reacted at 37°C for 1 h; washed 5 times with 0.05% PBS+Tween-20, added TMB substrate solution for color development for 15 min, and read the absorbance value at 450nm on the ELISA reader ( Figure 2 ), the titers of the five monoclonal antibodies all reached 1:512000.

[0032] 5. Identification of the reactivity of monoclonal antibodies with specific antigens The purified monoclonal antibodies YC101, YC201, YC301, YC401, and YC501 were subjected to Western blot analysis. According to the instructions of the New Cyme PAGE gel kit, a 1.0 mm thick, 10% SDS-PAGE protein gel was prepared; the extracted Giardia trophozoite total protein and 5× protein loading buffer were added to a centrifuge tube at a ratio of 4:1, mixed evenly by blowing and pipetting, boiled at 100°C for 10 min, loaded in each well with 20 μL, the upper gel voltage was 100 V, and the lower gel voltage was 120 V; after the electrophoresis, the PVDF membrane was activated by methanol solution for 1 min and arranged in the order of "sponge-filter paper-gel-PVDF membrane-filter paper-sponge", with the gel close to the negative electrode and the PVDF membrane close to the positive electrode. The protein moved from the negative electrode to the positive electrode under a constant voltage of 100 V, thereby wet-transferring the protein to the PVDF membrane; PBST was prepared into 5% skim milk and blocked at 37°C for 2 h; after blocking, the membrane was washed 4 times in PBST solution for 5 min each time; monoclonal antibodies diluted in PBST solution were added and incubated on a shaker at room temperature for 2 h; discard the primary antibody, wash the membrane with PBST solution 4 times; 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 with PBST solution 5 times; ECL chemiluminescence color development, observe the size and luminescence of the target protein ( Figure 3 ), all five monoclonal antibodies could react with the total protein of Giardia trophozoites, and the bands were obvious without other mixed bands.

[0033] 6. Monoclonal Antibody Horseradish Peroxidase (HRP) Labeling The monoclonal antibody was labeled with HRP enzyme using HRP rapid labeling kit. The monoclonal antibody was fully dialyzed in 0.01M CB buffer to remove interfering substances; the HRP enzyme labeling kit was taken out from the 4℃ refrigerator to mix all the components thoroughly; 250 ul (about 0.5 mg) of one of the above purified monoclonal antibodies was added to 50 uL pre-activated HRP (0.5 mg) liquid, mixed well, and then 54ul of HRP labeling starter solution was added, and continued to mix several times to avoid bubbles; then, protected from light, the coupling reaction was carried out in a 30℃-37℃ incubator for 2-3 h; after the coupling was completed, 1 mL of deionized water was added to the reaction termination solution powder tube and mixed well, 25 uL was added to the above reaction solution, mixed well, and placed at room temperature for 1 h; after the termination was completed, an equal volume of the marker preservation solution was directly added, mixed well, and stored at -20℃; the titer of the monoclonal antibody after HRP labeling was detected by indirect ELISA method ( Figure 4 ), the titers of the five enzyme-labeled antibodies all reached 1:64000.

[0034] 7. Screening of paired antibodies The five monoclonal antibodies were paired in pairs to screen the best paired antibodies. The purified five monoclonal antibodies were diluted to 1 μg / mL as capture antibodies and coated at 37℃ for 2 h; washed 3 times with phosphate buffer PBST, added 5% skim milk solution, 200 μL per well, and sealed at 37℃ for 2 h; added diluted Giardia antigen, 100 μL per well, incubated at 37℃ for 1 h; added HRP enzyme-labeled monoclonal antibodies, incubated at 37℃ for 1 h; washed 5 times with phosphate buffer PBST, added TMB substrate solution for color development for 15 min, and read the absorbance value at 450 nm on the microplate reader. The pairing results are shown in Table 1. According to the color development results, the signal is the strongest when CWP2 monoclonal antibody YC301 is used as the capture antibody and HRP-labeled monoclonal antibody YC201-HRP is used as the detection antibody; the signal is stronger when CWP2 monoclonal antibody YC501, YC101, and YC401 are used as the capture antibody and HRP-labeled monoclonal antibody YC201-HRP is used as the detection antibody, and when CWP2 monoclonal antibody YC201 is used as the capture antibody and HRP-labeled monoclonal antibody YC501-HRP is used as the detection antibody.

[0035] Table 1 Antibody pairing test results 8. Sequencing of the best paired antibodies The hybridoma cells corresponding to the best paired antibodies YC301 and YC201 were sequenced and analyzed. The hybridoma cells were lysed to extract total RNA; the RNA was reverse transcribed into cDNA using the rapid amplification of cDNA ends (RACE) technique; the heavy chain and light chain variable region sequences were obtained by PCR amplification; the target fragments were connected to the vector pcDNA3.4 using ligase, and the ligation products were transformed into Escherichia coli competent cells DH5α, and then single clones were picked for sequencing. The core sequence of the paired antibodies (the variable regions of the heavy chain and light chain) was included.

[0036] Among them, the capture antibody YC301 has three amino acid sequences of the heavy chain complementary determining region CDR1 as shown in SEQ ID NO: 5, the heavy chain complementary determining region CDR2 as shown in SEQ ID NO: 6, and the heavy chain complementary determining region CDR3 as shown in SEQ ID NO: 7; and three amino acid sequences of the light chain complementary determining region CDR1 as shown in SEQ ID NO: 13, the light chain complementary determining region CDR2 as shown in SEQ ID NO: 14, and the light chain complementary determining region CDR3 as shown in SEQ ID NO: 15.

[0037] The detection antibody YC201 has three amino acid sequences of the heavy chain complementary determining region CDR1 as shown in SEQ ID NO: 21, the heavy chain complementary determining region CDR2 as shown in SEQ ID NO: 22, and the heavy chain complementary determining region CDR3 as shown in SEQ ID NO: 23; and three amino acid sequences of the light chain complementary determining region CDR1 as shown in SEQ ID NO: 29, the light chain complementary determining region CDR2 as shown in SEQ ID NO: 30, and the light chain complementary determining region CDR3 as shown in SEQ ID NO: 31.

[0038] The capture antibody YC301 has three nucleotide sequences encoding the heavy chain complementary determining region CDR1 as shown in SEQ ID NO: 1, the heavy chain complementary determining region CDR2 as shown in SEQ ID NO: 2, and the heavy chain complementary determining region CDR3 as shown in SEQ ID NO: 3; and three nucleotide sequences encoding the light chain complementary determining region CDR1 as shown in SEQ ID NO: 13, the light chain complementary determining region CDR2 as shown in SEQ ID NO: 14, and the light chain complementary determining region CDR3 as shown in SEQ ID NO: 15.

[0039] The detection antibody YC201 has three nucleotide sequences encoding the heavy chain complementary determining region CDR1 as shown in SEQ ID NO: 17, the heavy chain complementary determining region CDR2 as shown in SEQ ID NO: 18, and the heavy chain complementary determining region CDR3 as shown in SEQ ID NO: 19; and three nucleotide sequences encoding the light chain complementary determining region CDR1 as shown in SEQ ID NO: 25, the light chain complementary determining region CDR2 as shown in SEQ ID NO: 26, and the light chain complementary determining region CDR3 as shown in SEQ ID NO: 27.

[0040] The capture antibody YC301 includes the heavy chain variable region amino acid sequence shown in SEQ ID NO: 8; and the light chain variable region amino acid sequence shown in SEQ ID NO: 16.

[0041] The detection antibody YC201 includes a heavy chain variable region amino acid sequence as shown in SEQ ID NO: 24; and a light chain variable region amino acid sequence as shown in SEQ ID NO: 32.

[0042] The capture antibody YC301 includes a nucleotide sequence encoding a heavy chain variable region as shown in SEQ ID NO: 4; and a nucleotide sequence encoding a light chain variable region as shown in SEQ ID NO: 12.

[0043] The detection antibody YC201 comprises a nucleotide sequence encoding a heavy chain variable region as shown in SEQ ID NO: 20; and a nucleotide sequence encoding a light chain variable region as shown in SEQ ID NO: 28.

[0044] The specific sequence information is as follows: SEQ ID NO: 1 (capture antibody YC301 heavy chain complementary determining region CDR1 nucleotide sequence) GCAAGAAGGGGAAGGTACTACGGTAGTAGCGTTGGGGACTAC SEQ ID NO: 2 (capture antibody YC301 heavy chain complementary determining region CDR2 nucleotide sequence) GGCTACACCTTCACCAGCTATTGG SEQ ID NO: 3 (capture antibody YC301 heavy chain complementary determining region CDR3 nucleotide sequence) ATTGACCCTAGTAACGGTCGTACT SEQ ID NO: 4 (capture antibody YC301 heavy chain variable region nucleotide sequence) CAGGTCCAACTGCAGCAGCCTGGGGCTGAACTGGTGAAGCCTGGGGCTTCAGAGAAGCTGTCCTGCAAGGCTTCTGGCTACACCTTCACCAGCTATTGGATGCACTGGATGAAGCAGAGGCCTGGACAAGGCCTTGAGTGGATTGGAGAGATTGACCCTAGTAACGGTCGTACTAACTACA ATGAGAAGTTCAGGAGCAAGGCCACACTGACTGTAGACAAATCCTCCAACACAGCCTACATGCAACTCAGCAGCCTGACATCTGAGGACTCTGCGGTCTATTACTGTGCAAGAAGGGGAAGGTACTACGGTAGTAGCGTTGGGGACTACTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCA SEQ ID NO: 5 (capture antibody YC301 heavy chain complementary determining region CDR1 amino acid sequence) ARRGRYYGSSVGDY SEQ ID NO: 6 (capture antibody YC301 heavy chain complementary determining region CDR2 amino acid sequence) GYTFTSYW SEQ ID NO: 7 (capture antibody YC301 heavy chain complementary determining region CDR3 amino acid sequence) IDPSNGRT SEQ ID NO: 8 (capture antibody YC301 heavy chain variable region amino acid sequence) QVQLQQSGAELAKPGASVKMSCKASGYTFTSYWMHWVKQRPGQGLEWIGYINPSTGYTEYNQKFKDKATLTADKSSSTAYMQLSSLTSEDSAVYSCARRVFYAYFDVWGAGTTVTVSS SEQ ID NO: 9 (capture antibody YC301 light chain complementary determining region CDR1 nucleotide sequence) CAACAGGTTAATAAGTTTCCGCTCACG SEQ ID NO: 10 (capture antibody YC301 light chain complementary determining region CDR2 nucleotide sequence) CAGGACGTTAACAATTAT SEQ ID NO: 11 (capture antibody YC301 light chain complementary determining region CDR3 nucleotide sequence) TACACTTCA SEQ ID NO: 12 (capture antibody YC301 light chain variable region nucleotide sequence) TATATCCAGATGACACAGACAACATCCTCCCTGTCTGCCTCTCTGGGAGACAGAGTCACCATCAGTTGCAGGGCAAGTCAGGACGTTAACAATTATTTAAACTGGTATCAGCAGAAACCAGATGGAACTGTTAAACTCCTGATCTACTACACTTCAAGAT TACATTCAGGAGTCCCATCAAGGTTCAGTGGCAGTGGGTCTGGAACAGATTATTCTCTCACCATTAGCAACCTGGACCAAGAAGATATTGCCACTTACTTTTGCCAACAGGTTAATAAGTTTCCGCTCACGTTCGGTGCTGGGACCAAGCTGGAGCTGAAA SEQ ID NO: 13 (capture antibody YC301 light chain complementary determining region CDR1 amino acid sequence) QQVNKFPLT SEQ ID NO: 14 (capture antibody YC301 light chain complementary determining region CDR2 amino acid sequence) QDV SEQ ID NO: 15 (capture antibody YC301 light chain complementary determining region CDR3 amino acid sequence) YTS SEQ ID NO: 16 (capture antibody YC301 light chain variable region amino acid sequence) YIQMTQTTSSLSASLGDRVTISCRASQDVNNYLNWYQQKPDGTVKLLIYYTSRLHSGVPSRFSGSGSGTDYSLTISNLDQEDIATYFCQQVNKFPLTFGAGTKLELK SEQ ID NO: 17 (detection antibody YC201 heavy chain complementary determining region CDR1 nucleotide sequence) GCAATATCTAGTGGTAACTACTAC SEQ ID NO: 18 (detection antibody YC201 heavy chain complementary determining region CDR2 nucleotide sequence) GGCTACACCTTCACTAAATACTGG SEQ ID NO: 19 (detection antibody YC201 heavy chain complementary determining region CDR3 nucleotide sequence) ATTAATCCTAGCACTGATTATACT SEQ ID NO: 20 (nucleotide sequence of the heavy chain variable region of detection antibody YC201) CAGGTCCAACTTCAGCAGTCTGGGGCTGAACTGGCAAAACCTGGGGCCTCAGTGAAGATGTCCTGCAAGGCTTCTGGCTACACCTTCACTAAATACTGGATACACTGGGTAAAACAGAGGCCTGGACAGGGTCTGGAATGGATTGGATACATTAATCCTAGCACTGATTATA CTGAGTACAATCAGAACTTCAAGGACAAGGCCACATTGACTGCAGACAAATCCTCCAGCACAGCCTACATGCAACTGAGCAGCCTGACATCTGAAGACTCTGCAGTCTTTTACTGTGCAATATCTAGTGGTAACTACTGGGGCCAAGGCGCCACTCTCACAGTCTCCTCA SEQ ID NO: 21 (detection antibody YC201 heavy chain complementary determining region CDR1 amino acid sequence) AISSGNYY SEQ ID NO: 22 (detection antibody YC201 heavy chain complementary determining region CDR2 amino acid sequence) GYTFTKYW SEQ ID NO: 23 (detection antibody YC201 heavy chain complementary determining region CDR3 amino acid sequence) INPSTDYT SEQ ID NO: 24 (amino acid sequence of the heavy chain variable region of detection antibody YC201) QVQLQQSGAELAKPGASVKMSCKASGYTFTKYWIHWVKQRPGQGLEWIGYINPSTDYTEYNQNFKDKATLTADKSSSTAYMQLSSLTSEDSAVFYCAISSGNYYWGQGATLTVSS SEQ ID NO: 25 (detection antibody YC201 light chain complementary determining region CDR1 nucleotide sequence) AAGCAATCTTATAATCTTCCGTGGACG SEQ ID NO: 26 (detection antibody YC201 light chain complementary determining region CDR2 nucleotide sequence) CAGAGTCTGCTCATCAGTAGAACCCGAAAGAGTTAC SEQ ID NO: 27 (detection antibody YC201 light chain complementary determining region CDR3 nucleotide sequence) TGGGCATCC SEQ ID NO: 28 (detection antibody YC201 light chain variable region nucleotide sequence) GACATTGTGATGTCACAGTCTCCTTCCTCCCTGGCTGTGTCAACAGGAGAGAAGGTCACTATGAGCTGCAAATCCAGTCAGAGTCTGCTCATCAGTAGAACCCGAAAGAGTTACTTGGCTTGGTACCAGCAGAAACCAGGGCAGTCTCCTAAACTGCTGATCTACTGGG CATCCACTAGGGAATCTGGGGTCCCTGATCGCTTCACAGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTGTGCAGGCTGAAGACCTGGCAGTTTTTTACTGCAAGCAATCTTATAATCTTCCGTGGACGTTCGGTGGAGGCACCAAGCTGGAAATCAAA SEQ ID NO: 29 (amino acid sequence of the light chain complementary determining region CDR1 of the detection antibody YC201) KQSYNLPWT SEQ ID NO: 30 (detection antibody YC201 light chain complementary determining region CDR2 amino acid sequence) QSLLISRTRKSY SEQ ID NO: 31 (detection antibody YC201 light chain complementary determining region CDR3 amino acid sequence) WAS SEQ ID NO: 32 (amino acid sequence of the light chain variable region of detection antibody YC201) DIVMSQSPSLAVSTGEKVTMSCKSSQSLLISRTRKSYLAWYQQKPGQSPKLLIYWASTRESGVPDRFTGSGSGTDFTLTISSVQAEDLAVFYCKQSYNLPWTFGGGTKLEIK IX. Double Antibody Sandwich ELISA for Detection of Giardia duodenalis CWP2 Protein Take the above-purified monoclonal antibody YC301, dilute the coating solution to 4 μg / mL, add it to the ELISA plate, 100 μL per well, and leave it at 4°C for overnight coating; wash 3 times with 1×PBST, add 5% BSA solution, 200 μL per well, and block at room temperature for 2 hours; remove the blocking solution, add 100 μL Giardia duodenalis whole worm protein, and react at 37°C for 90 minutes; wash 3 times with 1×PBST, add 100 μL of the above-mentioned horseradish peroxidase-labeled monoclonal antibody YC201 (diluted to 1:2000), and react at 37°C for 1 hour; wash 5 times with 1×PBST, add the luminescent substrate solution, react at room temperature for 10 minutes, and read the OD with an ELISA reader. 450nm value( Figure 5 ).

[0045] 10. Evaluation of Detection Methods (1) Specificity test According to the established double antibody sandwich ELISA method, Cryptosporidium parvum, Toxoplasma gondii, Blastocystis, Leishmania, and Coccidia were detected, while Giardia was set as a positive control and PBS was set as a negative control to evaluate the specificity of the double antibody sandwich ELISA detection method established with the paired antibodies YC301 and YC201 of the present invention.

[0046] The results showed that the reaction with Giardia trophozoites was positive, but there was no cross reaction with other parasites, indicating that the double antibody sandwich ELISA detection method established with the paired antibodies YC301 and YC201 of the present invention has good specificity.

[0047] (2) Sensitivity test The sensitivity of the double antibody sandwich ELISA detection method established with the paired antibodies YC301 and YC201 of the present invention was determined by using whole giardia trophozoite protein of whole giardia trophozoite with 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 as the test antigen and PBS as the negative control.

[0048] The sensitivity of the method was detected by Giardia trophozoites, and the P / N value was >2.1, which was judged as 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 of the method for detecting Giardia trophozoite lysate was 0.6 μg / mL (Table 2). This shows that the double antibody sandwich ELISA detection method established with the paired antibodies YC301 and YC201 in the present invention has good sensitivity.

[0049] Table 2 Sensitivity test results (3) Repeatability test The double antibody sandwich ELISA method established above was subjected to inter-batch repeatability and intra-batch repeatability experiments, and the test samples were 5 positive samples for detection, and each sample was repeated 3 times. The mean value, 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 in the present invention.

[0050] The results showed that the coefficient of variation of each group was 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 the present invention has good repeatability.

[0051] Table 3 Repeatability test results The preferred specific embodiments of the present invention are described in detail above. It should be understood that ordinary technicians in the field can make many modifications and changes based on the concept of the present invention without creative work. Therefore, all technical solutions that can be obtained by technicians in the technical field based on the concept of the present invention through logical analysis, reasoning or limited experiments on the basis of the prior art should be within the scope of protection determined by the claims.

Claims

1. A monoclonal antibody YC201 against Giardia duodenalis CWP2 protein, characterized in that: The monoclonal antibody YC201 has three nucleotide sequences encoding the heavy chain complementary determining region CDR1 as shown in SEQ ID NO: 17, the heavy chain complementary determining region CDR2 as shown in SEQ ID NO: 18, and the heavy chain complementary determining region CDR3 as shown in SEQ ID NO: 19; and three nucleotide sequences encoding the light chain complementary determining region CDR1 as shown in SEQ ID NO: 25, the light chain complementary determining region CDR2 as shown in SEQ ID NO: 26, and the light chain complementary determining region CDR3 as shown in SEQ ID NO:

27.

2. The monoclonal antibody YC201 according to claim 1, characterized in that The monoclonal antibody YC201 comprises a heavy chain variable region nucleotide sequence as shown in SEQ ID NO: 20; and a light chain variable region nucleotide sequence as shown in SEQ ID NO:

28.

3. A monoclonal antibody YC201 against Giardia duodenalis CWP2 protein, characterized in that: The monoclonal antibody YC201 has three amino acid sequences of a heavy chain complementary determining region CDR1 as shown in SEQ ID NO: 21, a heavy chain complementary determining region CDR2 as shown in SEQ ID NO: 22, and a heavy chain complementary determining region CDR3 as shown in SEQ ID NO: 23; and three amino acid sequences of a light chain complementary determining region CDR1 as shown in SEQ ID NO: 29, a light chain complementary determining region CDR2 as shown in SEQ ID NO: 30, and a light chain complementary determining region CDR3 as shown in SEQ ID NO:

31.

4. The monoclonal antibody YC201 according to claim 3, characterized in that The monoclonal antibody YC201 comprises a heavy chain variable region amino acid sequence as shown in SEQ ID NO: 24; and a light chain variable region amino acid sequence as shown in SEQ ID NO:

32.

5. A paired monoclonal antibody against Giardia duodenalis CWP2 protein, characterized in that: It comprises a capture antibody and a detection antibody; the capture antibody YC301 has three nucleotide sequences encoding a heavy chain complementary determining region CDR1 as shown in SEQ ID NO: 1, a heavy chain complementary determining region CDR2 as shown in SEQ ID NO: 2, and a heavy chain complementary determining region CDR3 as shown in SEQ ID NO: 3; and three nucleotide sequences encoding a light chain complementary determining region CDR1 as shown in SEQ ID NO: 13, a light chain complementary determining region CDR2 as shown in SEQ ID NO: 14, and a light chain complementary determining region CDR3 as shown in SEQ ID NO: 15; The detection antibody YC201 has three nucleotide sequences encoding the heavy chain complementary determining region CDR1 as shown in SEQ ID NO: 17, the heavy chain complementary determining region CDR2 as shown in SEQ ID NO: 18, and the heavy chain complementary determining region CDR3 as shown in SEQ ID NO: 19; and three nucleotide sequences encoding the light chain complementary determining region CDR1 as shown in SEQ ID NO: 25, the light chain complementary determining region CDR2 as shown in SEQ ID NO: 26, and the light chain complementary determining region CDR3 as shown in SEQ ID NO:

27.

6. The capture antibody YC301 according to claim 5, characterized in that The capture antibody YC301 comprises a nucleotide sequence encoding a heavy chain variable region as shown in SEQ ID NO: 4; and a nucleotide sequence encoding a light chain variable region as shown in SEQ ID NO:

12.

7. A paired monoclonal antibody against Giardia duodenalis CWP2 protein, characterized in that: Comprising a capture antibody and a detection antibody; the capture antibody YC301 has three amino acid sequences of a heavy chain complementary determining region CDR1 as shown in SEQ ID NO: 5, a heavy chain complementary determining region CDR2 as shown in SEQ ID NO: 6, and a heavy chain complementary determining region CDR3 as shown in SEQ ID NO: 7; and three amino acid sequences of a light chain complementary determining region CDR1 as shown in SEQ ID NO: 13, a light chain complementary determining region CDR2 as shown in SEQ ID NO: 14, and a light chain complementary determining region CDR3 as shown in SEQ ID NO: 15; The detection antibody YC201 has three amino acid sequences of a heavy chain complementary determining region CDR1 as shown in SEQ ID NO: 21, a heavy chain complementary determining region CDR2 as shown in SEQ ID NO: 22, and a heavy chain complementary determining region CDR3 as shown in SEQ ID NO: 23; and three amino acid sequences of a light chain complementary determining region CDR1 as shown in SEQ ID NO: 29, a light chain complementary determining region CDR2 as shown in SEQ ID NO: 30, and a light chain complementary determining region CDR3 as shown in SEQ ID NO:

31.

8. The capture antibody YC301 according to claim 7, characterized in that The capture antibody YC301 includes a heavy chain variable region amino acid sequence as shown in SEQ ID NO: 8; and a light chain variable region amino acid sequence as shown in SEQ ID NO:

16.

9. A monoclonal antibody against Giardia duodenalis CWP2 protein, comprising monoclonal antibodies YC101, YC401, YC501 and the monoclonal paired antibodies YC301 and YC201 according to claims 1 to 8, characterized in that: Prepared by the following method: (1) CWP2 protein immunization and antibody screening; 1 mg / mL of Giardia duodenalis CWP2 recombinant protein was mixed with an equal volume of Freund's adjuvant and immunized 5 BALB / c mice with multiple subcutaneous injections on the back. One week after the first immunization, booster immunization was performed every two weeks. After four immunizations, serum titer was tested and the titer reached 1:

10. 5 Afterwards, the spleen of the immunized mouse was obtained, spleen cells were isolated, and fused with SP2 / 0 cells; The expressed CWP2 recombinant protein was used as the detection antigen, and the coating solution was diluted to 1 μg / mL and added to the ELISA plate, 100 μL per well, and placed at 4°C for overnight coating; washed 3 times with 0.05% PBS+Tween-20, added 5% skim milk solution, 200 μL per well, and blocked at 37°C for 2 h; added hybridoma cell culture medium, 100 μL per well, and blocked at 37°C for 1 h; washed 3 times with phosphate buffer PBST, added goat anti-mouse antibody labeled with horseradish peroxidase, and reacted at 37°C for 1 h; washed 5 times with phosphate buffer PBST, added TMB substrate solution for color development for 15 min, and read the absorbance value at a wavelength of 450 nm on the ELISA reader. When the absorbance value of the sample control was more than 3 times that of the control well, it was determined to be a positive clone well; then the hybridoma cells were cloned multiple times using the limiting dilution method, and five monoclonal cell lines were successfully screened; (2) Preparation and purification of monoclonal antibody ascites; The mouse induction method was used. BALB / c mice were intraperitoneally injected with 500 μL of liquid paraffin oil. Nine days later, 1×10 6 The five hybridoma cells mentioned above can produce ascites 7 to 10 days after inoculation. The ascites of mice was collected with a 16-gauge needle on a clean bench. -1 Centrifuge for 10 min to remove cell components and other precipitates, and collect the supernatant as ascites; A Protein G column was used to affinity purify the ascites. The loaded rProtein G Beads gravity column was equilibrated with five times the volume of equilibration solution, so that the filler was in the same buffer system as the target protein, and the process was repeated 2-3 times. The collected ascites was added to the equilibrated gravity column, the sample was retained for 30 minutes, and the effluent was collected. The impurities were washed with 10-15 times the column volume of washing solution to remove non-specifically adsorbed impurities, and the washing solution was collected. The eluent was eluted with 5-10 times the column volume of elution solution, and the samples were collected in sections. One tube was collected for each column volume. The eluted components were immediately neutralized with 1 / 10 of the neutralization solution. The eluent was the purified monoclonal antibody, including YC101, YC201, YC301, YC401, and YC501. The purity of the monoclonal antibody was detected by SDS-PAGE gel electrophoresis. Among them, YC101, YC401, and YC501 are used as capture antibodies, and YC201 described in any one of claims 1 to 4 is used as a detection antibody, constituting a paired monoclonal antibody against Giardia duodenalis CWP2 protein; Or the YC201 described in any one of claims 1 to 4 is used as a capture antibody, and YC501 is used as a detection antibody to form a paired monoclonal antibody against the CWP2 protein of Giardia duodenalis.

10. Use of the monoclonal antibody or paired antibody according to any one of claims 1 to 9 in the preparation of a product for detecting CWP2 protein of Giardia duodenalis.

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