Echinococcus granulosus monoclonal antibody, canine animal excrement detection kit and application of canine animal excrement detection kit

Abstract

The invention provides an echinococcus granulosus antibody, a kit for detecting echinococcus granulosus pathogen from excrement and application of the echinococcus granulosus antibody. Firstly, a monoclonal antibody 4E6 is provided, the monoclonal antibody 4E6 can stably and efficiently recognize soluble antigens of adult echinococcus granulosus, soluble antigens excreted and secreted by the echinococcus granulosus and soluble antigens of eggs of the echinococcus granulosus, and the monoclonal antibody 4E6 has high titer and good specificity on the various mixed antigens. Therefore, the antibody can be used for preparing a kit for detecting echinococcus granulosus eggs in canine animal excrement or environment. The preparation method of the monoclonal antibody 4E6 for resisting echinococcus granulosus provided by the invention is simple, the antibody purification process is simple, the efficiency is high, and the cost is low. The monoclonal antibody 4E6 provided by the invention is used for detecting polluted echinococcus granulosus in excrement of canine animals or environment, and has the advantages of rapidness, sensitivity, high specificity, simplicity in operation, low cost and the like.

Description

Technical Field

[0001] This invention relates to the field of bioimmunology, specifically to an antibody against Echinococcus granulosus, a kit for detecting Echinococcus granulosus pathogens from feces, and its applications. Background Technology

[0002] Echinococcosis, also known as cystic echinococcosis (CE), is caused by the tapeworm Echinococcus granulosus. Echinococcus granulosus , E. granulosus Echinococcosis is a zoonotic parasitic disease caused by the parasitic larvae of echinococcosis in the liver, lungs, and other organs of cattle, sheep, pigs, and humans. Humans become infected through ingestion of contaminated food, water, or soil containing parasite eggs, or through direct contact with the definitive host. The disease is widespread and distributed globally; the WHO classifies it as a Neglected Tropical Disease (NTD). Echinococcosis seriously endangers human health and livestock development. Due to its widespread prevalence, diverse hosts, and complex geographical ecology, different strains of *Echinococcus granulosus* may exist in my country. Because infection in livestock and dogs is asymptomatic, it is difficult to monitor cystic echinococcosis in animals, and the importance of monitoring this disease is not recognized. Current control measures are not fully implemented in practice; the large number of wild canids in pastoral areas means that missing even one animal can render all previous efforts futile. Therefore, accurate diagnosis and the development of vaccines are the best solutions for preventing echinococcosis.

[0003] Currently, diagnostic methods for canine feces mainly include fecal ova testing, necropsy, arecoline hydrobromide purging, fecal antigen ELISA, and fecal worm DNA testing. However, each method has its own shortcomings. For example, necropsy has a much higher positive detection rate than other methods, but it is relatively cumbersome and not very feasible in practice. Arecoline hydrobromide purging is the second most effective method after necropsy, but a considerable number of dogs are not sensitive to arecoline. Furthermore, it is difficult to perform, time-consuming, labor-intensive, carries the potential risk of infection for staff with ova, and poses a risk of environmental contamination. Fecal antigen ELISA and fecal worm DNA testing often require specialized technicians, instruments, or equipment, making rapid identification in the field difficult. Moreover, these methods are prone to contamination and false positives during operation, and the testing process is complex, consuming significant manpower and time.

[0004] At present, although there are various antibodies against Echinococcus granulosus, these antibodies often target outer membrane proteins or single antigens and do not react with other antigenic components, resulting in false negatives. Moreover, due to the complexity of fecal antigen components, false positive results often occur during testing, and the test results are often not very stable.

[0005] Therefore, there is currently a lack of methods and means for rapid screening of large numbers of samples in the field. Developing a rapid, safe, simple, highly specific, and sensitive detection method will provide effective support for the diagnosis and control of echinococcosis in canines. Summary of the Invention

[0006] To address the above problems, the present invention provides the following technical solution: First, this invention provides a method for preparing soluble antigens of adult Echinococcus granulosus. After purification, these antigens are used to prepare and screen monoclonal antibodies against soluble antigens of adult Echinococcus granulosus. After screening and four subcloning processes using an indirect ELISA method, eight single hybridoma cell lines resistant to soluble antigens of adult Echinococcus granulosus are obtained, named 1B4, 3C12, 2D2, 4E6, 3F7, 2G9, 5H5, and 5H10, respectively. Furthermore, the eight hybridoma cell lines were further screened using soluble antigens excreted by Echinococcus granulosus or soluble antigens from Echinococcus granulosus eggs as coating antigens. The ELISA method described above was used to identify the recognition ability of the eight hybridoma cell lines for different antigens. Finally, three hybridoma cell lines that showed positive reactions to all three antigens were selected: 3C12, 4E6, and 2G9. Analysis suggests that the same antigen protein with a relatively high expression level may exist in the three soluble mixed antigens.

[0007] Furthermore, the three hybridoma cell lines were further screened to obtain the monoclonal antibody 4E6 with the highest titer, and it was sequenced to obtain its heavy chain variable region sequence as shown in SEQ ID NO.1, wherein the sequences of HCDR1-3 are shown in SEQ ID NO.3-5, respectively, and the light chain variable region sequence is shown in SEQ ID NO.2, wherein the sequences of LCDR1-3 are shown in SEQ ID NO.6-8, respectively.

[0008] Furthermore, the present invention provides the application of the monoclonal antibody 4E6 in the preparation of a kit for detecting Echinococcus granulosus.

[0009] Furthermore, the present invention provides the application of the monoclonal antibody 4E6 in the preparation of a colloidal gold test strip for detecting Echinococcus granulosus.

[0010] Furthermore, the present invention provides a kit for detecting Echinococcus granulosus containing the monoclonal antibody 4E6.

[0011] Furthermore, the present invention provides a colloidal gold test strip containing the monoclonal antibody 4E6 for detecting Echinococcus granulosus.

[0012] The test samples for the above-mentioned kits or test strips include, but are not limited to: in vitro samples; water samples; feces; food; soil samples; and environmental samples.

[0013] Beneficial effects The monoclonal antibody 4E6 of this invention can stably and efficiently recognize soluble antigens from adult Echinococcus granulosus, soluble antigens from Echinococcus granulosus excretions, and soluble antigens from Echinococcus granulosus eggs. It also exhibits high titer and good specificity against various mixed antigens. Therefore, it can be used to prepare kits and colloidal gold test strips for detecting Echinococcus granulosus. The preparation method of the anti-Echinococcus granulosus monoclonal antibody 4E6 provided by this invention is simple, the antibody purification process is straightforward, efficient, and low-cost. Using the anti-Echinococcus granulosus monoclonal antibody 4E6 provided by this invention for detecting anti-Echinococcus granulosus is rapid, sensitive, highly specific, simple to operate, and low-cost. Attached Figure Description

[0014] Figure 1 Schematic diagram of SDS-PAGE of Echinococcus granulosus components, where M is Marker, 1 is soluble antigen of adult Echinococcus granulosus, 2 is soluble antigen of excretion and secretion of Echinococcus granulosus, and 3 is soluble antigen of Echinococcus granulosus eggs. Figure 2 SDS-PAGE results of purified hybridoma cells 3C12, 4E6, and 2G9, where M is the marker, and lanes 1-3 are the heavy and light chains of hybridoma cells 3C12, 4E6, and 2G9, respectively. Two bands of antibody heavy and light chains can be seen at molecular weights of 50 kDa and 25 kDa, respectively. Figure 3 In this embodiment of the invention, Western blotting was used to detect the interaction between monoclonal antibody 4E6 and Echinococcus granulosus cyst fluid antigen, scolex antigen, vesicular larvae antigen, Giardia canis antigen, and Ascaris canis antigen. In this study, A represents the target protein, B is a β-actin positive control, lane M is a marker, lanes 1-5 represent Echinococcus granulosus cyst fluid antigen, scolex antigen, vesicular larvae antigen, Giardia canis antigen, and Ascaris canis antigen, respectively, and NC is a negative control. The results showed that the antibody reacted only with Echinococcus granulosus components. Detailed Implementation

[0015] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0016] Example 1: Preparation of Echinococcus granulosus antigen Preparation of soluble antigens from adult Echinococcus granulosus tapeworms The adult E. granulocytic tapeworms preserved in liquid nitrogen were removed, rinsed with sterile water, and then placed on ice for ultrasonic disruption until no visible fragments remained. The disrupted solution was centrifuged at 13,000 rpm for 10 min at 4°C. The supernatant was the soluble E. granulocytic tapeworm antigen (AWA), which was stored at -80°C for later use.

[0017] Preparation of soluble antigens excreted and secreted by Echinococcus granulosus: The adult Echinococcus granulosus tapeworms preserved in liquid nitrogen were removed and gently shaken in sterile physiological saline to mix. After the tapeworms settled naturally, the saline was removed, and the mixture was rinsed repeatedly 3-5 times. The tapeworms were cultured at a density of approximately 500 worms / mL in RPMI 1640 medium containing 2% glucose, 100 U / mL penicillin G, and 100 ng / mL streptomycin at 37°C for 24 h. The tapeworms and supernatant were separated by centrifugation at 1500 rpm for 3 min. The supernatant was collected, filtered through a 0.22 µm filter membrane, and transferred to a 3 kDa ultrafiltration tube. The culture was concentrated by centrifugation at 5000 rpm for approximately 1 h at 4°C. The concentrated liquid is the excretory antigen (ES) of Echinococcus granulosus and stored at -80°C for later use.

[0018] Preparation of soluble antigens from Echinococcus granulosus eggs: Beagle dogs were infected orally with 150,000 purified Echinococcus granulosus protocercariae (isolated from the liver of sheep naturally infected with echinococcosis in a slaughterhouse in Tibet). Starting on day 45 post-infection, daily tests were performed using a transparent tape method around the anus until eggs were detected under a microscope. Subsequently, the eggs were isolated from the dog's feces.

[0019] Take a certain amount of dog feces, add sterile 0.85% NaCl solution, stir and dilute, then filter through a 100-120 mesh sieve. After centrifugation, resuspend the filtrate in sterile 0.85% NaCl solution, and repeat centrifugation 2-3 times to obtain a fecal suspension containing Echinococcus granulosus eggs.

[0020] Add 9 mL of saturated sucrose solution (an aqueous solution containing 67.1% sucrose) to a 15 mL sterile centrifuge tube. Using a pipette, slowly add 3 mL of fecal egg suspension to the surface of the liquid. Then, insert the pipette tip 2-3 cm below the boundary between the sample and the sucrose solution, and continuously and uniformly rotate and stir along the tube wall to allow the fecal suspension to diffuse fully into the saturated sucrose solution, forming a gradient. Carefully aspirate the egg-rich layer at the interface (avoid aspirating fecal samples adhering to the tube wall, as this can lead to impure egg separation later). Add 3 times the volume of sterile 0.85% NaCl solution and mix well. Centrifuge at 1000×g for 5 min, discard the supernatant, and retain the precipitate, which is the purified Echinococcus canis eggs.

[0021] Resuspend the precipitate of eggs in sterile 0.85% NaCl solution. Add 10 µL of penicillin-streptomycin (100×) per mL of suspension to a final concentration of 1%, and simultaneously add 10 µL of nystatin solution (10 mg / mL) to a final concentration of 10 mg / L. The purified egg suspension is then stored at 4°C. Homogenize the collected eggs repeatedly in an ice bath, then incubate at 4°C for 48 h. Centrifuge at 10,000×g for 1 h, collect the supernatant, aliquot, and store. This is the soluble egg antigen (SEA).

[0022] The above three soluble antigens were concentrated and confirmed to be correct by SDS-PAGE (see [link to SDS-PAGE]). Figure 1 The concentration was determined using the Amersham Biosciences 2D Quant Kit protein quantification kit.

[0023] Example 2: Preparation and Screening of Monoclonal Antibodies 1. Mouse immunization Four 40-day-old female BALB / c mice were selected, with three as the immunization group and one as the negative control. The mice were immunized with the soluble antigen of the adult Echinococcus granulosus obtained in Example 1. Each mouse received four immunizations, with 200 μL of the emulsion injected subcutaneously at multiple sites each time. The specific immunization procedure was as follows: the first immunization was performed on day 0, with multiple subcutaneous injections of an emulsion containing 50 μg of antigen (mixed 1:1 with Freund's complete adjuvant); the second and third immunizations were performed on days 14 and 28, respectively, with multiple subcutaneous injections of 100 μg of antigen (mixed 1:1 with Freund's incomplete adjuvant); the fourth immunization was performed on day 35, with a direct intraperitoneal injection of 100 μg of antigen (without adjuvant). The negative control mice received only the same volume of either Freund's complete or incomplete adjuvant.

[0024] 2. Detect serum antibody titer An indirect ELISA method for screening hybridoma cells was established using serum from mice after the fourth immunization. The specific steps are as follows: (1) Dilute the soluble antigen of adult Echinococcus granulosus to 0.5 μg / mL with antigen coating solution (0.05 mol / L, pH 9.6±0.2 carbonate buffer), add 100 μL to each well of the ELISA plate, coat overnight at 4°C (about 8 h), and wash 3 times with PBST.

[0025] (2) Add 200 μL of 5% skim milk solution to each well, block at 37℃ for 2 h, and wash 3 times with PBST.

[0026] (3) The mouse serum samples were serially diluted from 1:200 to 1:25,600. 100 μL was added to each well and each sample was in 3 replicates. The samples were incubated at 37°C for 1 h and washed 3 times with PBST.

[0027] (4) Dilute the HRP-labeled goat anti-mouse IgG (H+L) antibody with 2.5% skim milk at a ratio of 1:5000, add 100 μL to each well, incubate at 37°C for 1 h, and wash 3 times with PBST.

[0028] (5) Add 100 μL of TMB colorimetric solution to each well and let stand at room temperature in the dark for 10 min.

[0029] (6) Add 50 μL of stop solution (1 M HCl) to each well to stop the color development, and measure the OD within 5 min afterward. 450 absorbance value in nm.

[0030] (7) The ratio of the absorbance value of the serum of the immunized group mice to that of the negative control mice (P / N value) was calculated as the judgment criterion. When P / N>2, it was judged as positive (indicating that the immunized group mice successfully produced antibodies against the soluble antigens of the parasite), otherwise it was judged as negative. The mice with the highest antibody titer were selected for cell fusion experiments to prepare monoclonal antibodies.

[0031] 3. Cell fusion and screening SP2 / 0 cells were resuscitated 30 days prior to fusion, passaged to the logarithmic growth phase, and cells in good condition were collected for use. Culture supernatant was also collected as a negative control. Feeder cells were prepared 24 hours prior to fusion by diluting peritoneal cells from healthy BALB / c mice to approximately 1 × 10⁻⁶ cells. 5 Spleen cells were cultured at a density of [number] cells / mL in 96-well plates. Spleen cells for fusion were obtained from BALB / c mice on day 3 after booster immunization. Spleen cells were aseptically obtained after anesthesia and blood collection, and a splenocyte suspension was prepared for later use. Splenocytes were mixed with SP2 / 0 cells at a 2:1 ratio and fusion was induced using 50% PEG1450. After fusion, the cells were slowly resuspended and seeded into 96-well plates containing feeder cells, and cultured at 37°C in a 5% CO2 incubator. Cell growth was observed on day 4 post-fusion, and antibody activity in the supernatant was detected by ELISA on day 7 to screen for positive clones. HAT medium was used initially, with half the medium changed every 3-4 days; HT medium was used between the second and third rounds of subcloning, and finally DMEM medium was used to screen and amplify stable hybridoma cell lines.

[0032] Following the indirect ELISA method described in step "2. Detection of serum antibody titer", soluble antigens from adult Echinococcus granulosus were used as coating antigens to screen hybridoma cell culture supernatants. Coating conditions were optimized using a checkerboard titration method, with the highest positive / negative absorbance ratio (P / N value) and the highest OD value of positive serum.450 A P / N ratio close to 1.0 was considered optimal, and a P / N ratio > 2 was considered positive. Indirect ELISA results showed that the P / N ratio was highest at an antigen coating concentration of 0.5 μg / mL. Using this concentration as a standard, an indirect ELISA method was established to detect the monoclonal antibody supernatant secreted by hybridoma cells and screen for positive monoclonal cells. Hybridoma cells that tested positive in both tests were subjected to limiting dilution subcloning, and after 2-4 cloning processes, cell lines stably secreting monoclonal antibodies were obtained. After screening using the indirect ELISA method and four subcloning processes, eight single hybridoma cell lines resistant to soluble antigens from adult Echinococcus granulosus were obtained, named 1B4, 3C12, 2D2, 4E6, 3F7, 2G9, 5H5, and 5H10.

[0033] Using soluble antigens from the excretion and secretion of Echinococcus granulosus and soluble antigens from Echinococcus granulosus eggs as coating antigens, the above-mentioned indirect ELISA method was used to detect the recognition ability of eight hybridoma cell lines for different antigens. Finally, three hybridoma cell lines that showed positive reactions to all three antigens were selected, namely 3C12, 4E6, and 2G9. It was analyzed that there may be a common antigen protein with a relatively high expression level in the above three soluble mixed antigens.

[0034] 4. Monoclonal antibody preparation and purification The selected hybridoma cell lines were cultured at 5 × 10⁻⁶. 5 Inoculate T175 culture flasks at a density of 1 / mL, add 30 mL of DMEM medium containing 10% fetal bovine serum for expansion culture, and collect the supernatant after the medium turns yellow. Centrifuge at 1000 rpm for 10 min, transfer the supernatant and freeze for later use. Antibody purification is performed using the caprylic acid-ammonium sulfate method. Mix the supernatant with acetate buffer at a ratio of 1:2, add caprylic acid (33 μL / mL), stir at room temperature for 30 min, and incubate at 4℃ for 4 h. Then add ammonium sulfate to a final concentration of 45%, incubate at 4℃ for 4 h, and centrifuge to precipitate. Resuspend the precipitate with PBS, centrifuge at 12,000 rpm for 5 min to remove impurities, ultrafilter to remove ammonium sulfate and change the medium 3 times to obtain purified monoclonal antibodies for each hybridoma cell. Figure 2 Antibody concentration was determined using the BCA method.

[0035] 5. Monoclonal antibody titer determination An ELISA plate was coated with soluble antigen from adult Echinococcus granulosus, and HRP-labeled goat anti-mouse IgG antibody was used as the secondary antibody. The purified monoclonal antibodies secreted by various hybridoma cells were serially diluted and detected according to the ELISA method in step "2. Detection of Serum Antibody Titer". The P / N value was calculated (P is the OD of the sample). 450 nm value, N is the negative control OD 450(nm value; negative control was DMEM medium). The monoclonal antibody with the highest dilution factor (P / N greater than 2) was selected for subsequent experiments. Results showed that monoclonal antibody 4E6 had the highest titer, with a detection limit of 1:51,200, while monoclonal antibodies 3C12 and 2G9 had titers of 1:25,600 (Table 1). The above experiments were repeated using soluble antigens excreted by *Echinococcus granulosus* or soluble antigens from *Echinococcus granulosus* eggs as coating antigens. Similar results were observed: monoclonal antibody 4E6 had the highest titer, with a detection limit of 1:51,200, while monoclonal antibodies 3C12 and 2G9 had titers of 1:25,600 and 1:12,800, respectively. Finally, monoclonal antibody 4E6 was selected for subsequent experiments.

[0036] Table 1. Determination of Monoclonal Antibody Titer

[0037] 6. Stability determination of monoclonal antibodies The obtained hybridoma cell line 4E6 was cryopreserved for one month and then thawed. It was then passaged 10 times in vitro, and the cell supernatant was collected. The stability of the antibody secreted by the hybridoma cell line was determined using an established indirect ELISA method. The results showed that after 10 passages, the 4E6 cell line could still stably secrete monoclonal antibodies, exhibiting good stability.

[0038] 7. Specificity assay of monoclonal antibodies Echinococcus granulosus cyst fluid antigen, scolex antigen, Echinococcus multilocularis somatic antigen, Giardia canis flagellate antigen, and Ascaris lumbricoides antigen were prepared (preparation method as described in Example 1). 4E6 monoclonal antibody (1:2,000 dilution) was used as the primary antibody, and HRP-labeled goat anti-mouse IgG (H+L) antibody (1:5,000 dilution) was used as the secondary antibody. Western blot analysis was performed to determine the specificity of the monoclonal antibodies. The results showed that 4E6 monoclonal antibody could recognize Echinococcus granulosus cyst fluid antigen and scolex antigen, but did not react with Echinococcus multilocularis somatic antigen, Giardia canis flagellate antigen, or Ascaris lumbricoides antigen. Figure 3 ).

[0039] 8. Monoclonal antibody sequencing After the 4E6 monoclonal antibody was expanded and cultured, it was sent to a gene company for sequencing. The results showed that: The heavy chain variable region sequence of 4E6 is as follows: PSQALGLVKPSQSTVTGYSITSDAGWWNWIRAFPNKLLEWMGFIYSAIGSTSYSYALKSRLSITRATDTSKNQFFLQLNSVTAEDTATTEDTYCATFANYPFDFWGAGCALTVSS (SEQ ID NO. 1); HCDR1 is: SDAGWWN (SEQ ID NO.3); HCDR2 is: FIYSAIGSTSYSYALKS (SEQ ID NO.4); HCDR3 is: FANYPFDF (SEQ ID NO.5). The light chain variable region sequence is as follows: QEKVTMTSCRSSYLVHSAYWYLDWYLQKPGQSPTPLLINDSSSHRLAASSGVPPARSGSGSSGTSYATLKIREAEEAATYFCSQSADPPPTFGGTAKLEIK (SEQ ID NO. 2); LCDR1 is: RSSYLVHSAYWYLD (SEQ ID NO.6); LCDR2 is: NDSSSHRLAASS (SEQ ID NO.7); LCDR3 is: SQSADPPPT (SEQ ID NO.8).

[0040] Example 3: Clinical Sample Analysis Sixty clinical canine fecal samples were randomly selected from samples with known backgrounds submitted from various regions of Tibet (15 positive and 45 negative).

[0041] The above samples were added to 1 mL of PBST buffer solution at a rate of 1 g of fecal sample. The fecal sample with added buffer solution was shaken well and incubated overnight at 4°C. The overnight sample was then centrifuged at 4,000 rpm for 10 min at room temperature, and the supernatant was collected for later use.

[0042] The above samples were tested using the monoclonal antibody 4E6 via ELISA. The procedure was briefly described as follows: The antibody-coated plate was thawed and gently patted on a clean gauze to remove moisture. Well A1 was designated as a blank well, and 100 μL each of the control positive sample, control negative sample, and test sample were added to each well. A PVC membrane was attached, and the plate was incubated at 37°C in the dark for 30 min. The PVC membrane was then removed, and the solution in the wells was discarded. 300 μL of PBST solution was added to each well, and after standing for 30 s, the liquid was discarded. The plate was washed three times and then thoroughly patted dry on absorbent paper. Except for well A1, 100 μL of monoclonal antibody solution was added to each well, and a PVC membrane was attached. The plate was incubated at 37°C for 30 min. The PVC membrane was then removed, and the solution in the wells was discarded. 300 μL of PBST solution was added to each well, and after standing for 30 s, the liquid was discarded. The plate was washed three times and then thoroughly patted dry on absorbent paper. Except for well A1, 100 μL of enzyme-labeled secondary antibody solution was added to each well. μL of PBST solution was added to each well, covered with a PVC membrane, and incubated at 37°C for 30 min. The PVC membrane was then removed, the solution in the wells was discarded, and 300 μL of PBST solution was added to each well. After standing for 30 s, the liquid was discarded, and the solution was washed 5 times and patted dry on absorbent paper. 100 μL of chromogenic solution was added to each well, the PVC membrane was covered, and the solution was incubated at 37°C for 10 min. The PVC membrane was discarded, and 50 μL of the prepared stop solution was added to each well. The OD value of each well was measured at 450 nm using a microplate reader. ELISA results showed that the positive detection rate of the 4E6 monoclonal antibody was 93.3% (14 / 15), the negative detection rate was 100% (45 / 45), and the concordance rate was 98.3% (59 / 60), with only one false negative result. This indicates that the monoclonal antibody 4E6 screened in this application can achieve rapid detection of Echinococcus granulosus in canine feces and can be applied to the clinical detection of Echinococcus granulosus in dogs. This study lays the foundation for the rapid diagnosis of canine Echinococcus granulosus.

[0043] The above description of the embodiments is intended to enable those skilled in the art to understand and use the present invention. It will be apparent to those skilled in the art that various modifications can be made to these embodiments, and the general principles described herein can be applied to other embodiments without inventive effort. Therefore, the present invention is not limited to the above embodiments. Improvements and modifications made by those skilled in the art based on the principles of the present invention, without departing from the scope of the invention, should be within the protection scope of the present invention.

Claims

1. A monoclonal antibody 4E6 which specifically recognizes a component of Echinococcus granulosus, characterized in that The heavy chain variable region sequence of the antibody is shown as SEQ ID NO. 1, wherein the sequences of HCDR1-3 are shown as SEQ ID NO. 3-5 respectively, and the light chain variable region sequence is shown as SEQ ID NO. 2, wherein the sequences of LCDR1-3 are shown as SEQ ID NO. 6-8 respectively.

2. Use of the monoclonal antibody 4E6 of claim 1 in the preparation of a kit for detecting E. granulosus.

3. Use of the monoclonal antibody 4E6 of claim 1 in the preparation of a colloidal gold test strip for detecting E. granulosus.

4. A kit for detecting E. granulosus comprising the monoclonal antibody 4E6 of claim 1.

5. A colloidal gold test strip for detecting E. granulosus comprising the monoclonal antibody 4E6 of claim 1.

6. Use of the monoclonal antibody 4E6 of claim 1, the kit of claim 4, and / or the test strip of claim 5 in detecting E. granulosus components for non-disease treatment or diagnosis purposes, wherein the detection sample of the use is selected from one or more of the following: fecal samples of canids; water samples; food; soil samples; environmental samples.

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