Monoclonal antibody for specifically recognizing cysts, oocysts and sporangiums of metrosporidium megalanthum and application of monoclonal antibody

Abstract

The invention provides a monoclonal antibody for specifically recognizing cysts and oocysts / sporangiums of sheep sarcocysts and application of the monoclonal antibody, and the monoclonal antibody provided by the invention can generate positive reaction aiming at soluble antigens of adult holoproteins of sarcocysts tenella and soluble antigens of oocysts / sporangiums of the sarcocysts tenella. The two key development stages of adults and oocysts / sporangiums are covered at the same time. A complete parasite life cycle detection system can be established, the clinical sample detection rate is remarkably improved (experiment shows that the clinical sample detection rate is improved by 35-40%), and the detection window period is 7-10 days earlier than that of a traditional method. Meanwhile, the ELISA detection kit and the detection method thereof provided by the invention are high in sensitivity and convenient to operate, can greatly improve the detection efficiency, and have a good industrial application prospect.

Description

Technical Field

[0001] This invention belongs to the field of proteins and their biological detection, specifically relating to a monoclonal antibody that specifically identifies Sarcocystis ovis cysts, oocysts and sporangia and its application. Background Technology

[0002] Sarcocystidia ( Sarcocystis Sarcocystis spp., also known as Sarcocystis, is an important zoonotic parasite belonging to the phylum Apicomplexa, class Sporozoasida, order Eucoccidiorida, family Sarcocystidae, and genus Sarcocystis. Sarcocystis To date, over 200 species of Sarcocystis have been reported, widely parasitizing the muscles of amphibians, reptiles, birds, mammals, and other animals, causing zoonotic sarcocystosis. This disease is widespread, with a high infection rate, and poses a significant threat to both humans and animals. In 1978, the World Health Organization (WHO) convened a World Congress, which for the first time specifically discussed the harm caused by Sarcocystis and its impact on humans and the economy, bringing widespread attention to the disease.

[0003] Currently reported species of Sarcocystis suis in sheep include Sarcocystis tenella (… S. tenella ), giant sarcoplasmosis ( S. gigantica ), White sheep dog living fleshy cysts ( S. arieticanis ) and jellyfish-like sarcoptera ( S. medusiformis Of these, *Sarcocystis tenuis*, *Sarcocystis macrocarpa*, and *Sarcocystis baileyi* have dogs as their definitive host, while *Sarcocystis medusa* has cats as its definitive host. The *Sarcocystis* species that infect goats include *Sarcocystis baileyi* (…). S. capracanis ), domestic goat dogs live on fleshy cysts ( S. hircicanis ) and Sarcocystis molluscum ( S. moule The definitive host for *Salmonella canis* and *Salmonella domestica* is the dog, while the definitive host for *Salmonella molluscum* is the cat. Most sheep infections with *Salmonella* are mixed infections, primarily manifesting as fever, lethargy, diarrhea, weight loss, and difficulty standing. Severe cases can lead to abortion, decreased milk production, and even death.

[0004] Currently, the main methods for detecting Sarcocystis suis include etiological, immunological, and molecular biological methods. Etiological detection primarily relies on microscopy. Cysts in muscle are typically examined directly by pressing smears, while oocysts / sporangia in feces require filtration through four layers of gauze, centrifugation, flotation in saturated saline, and purification before microscopic examination. This method has drawbacks such as low sensitivity, a high risk of missed or false positives, and the need for specialized personnel. Molecular biological detection methods mainly include conventional polymerase chain reaction (PCR), nested PCR, and quantitative real-time PCR. These methods offer advantages such as high sensitivity, good specificity, and the ability to identify the species. However, they are unsuitable for detecting intermediate hosts in live animals and are only applicable to muscle samples from slaughtered animals or oocysts / sporangia in the feces of definitive hosts. Immunological detection methods can be performed on live animals, requiring only blood collection for antibody testing. This method is not only simple to operate but also causes minimal harm to the animal. However, among existing immunological detection methods, there is no suitable antigen that can simultaneously detect Sarcocystis cysts and oocysts / sporangia. There is an urgent need to establish an immunological detection method with high specificity and sensitivity to detect Sarcocystis infection in intermediate and definitive hosts. Summary of the Invention

[0005] To address the aforementioned issues, this invention first provides a monoclonal antibody 5H2 that specifically recognizes Sarcocystis sheep, secreted by the hybridoma cell line 5H2. This monoclonal antibody 5H2 can react not only with soluble antigens of Sarcocystis sheep oocysts / sporangia, but also with soluble antigens of Sarcocystis sheep cysts.

[0006] Furthermore, the present invention provides the application of the above-mentioned monoclonal antibody in the preparation of a reagent for detecting and recognizing Sarcocystis ovis.

[0007] Furthermore, the present invention provides an ELISA kit for detecting Sarcocystis sheep, characterized in that it comprises Sarcocystis sheep oocyst / sporangium whole antigen and the above-mentioned monoclonal antibody 5H2.

[0008] Furthermore, the kit also includes standard negative serum, standard positive serum, carbonate buffer, PBST buffer, PBS buffer containing 1% BSA, HRP-labeled goat anti-mouse IgG, chromogenic solution, and stop solution.

[0009] Furthermore, the present invention provides a method for detecting Sarcocystis ovis using the above-mentioned monoclonal antibody, the method comprising the following steps: (1) Antigen coating: Dilute the whole antigen of Sarcocystis omatis cysts to 6.25 μg / mL with 0.05M carbonate buffer at pH 9.6, add 100 μL of antigen dilution to each well, and coat at 4℃ for 12-16 h.

[0010] (2) Blocking: Wash the microplate with PBST buffer for 5 min each time, 3 times, and shake off the residual liquid in the wells after each wash. Add 100 μL of PBS buffer containing 1% BSA to each well and block at 37℃ for 1 h.

[0011] (3) Serum sample incubation: Repeat the washing steps above, dilute the serum to be tested 1:1 with PBST buffer, and dilute the standard negative serum by the same factor as a control. Add 100 μL to each well and incubate at 37°C for 1 h.

[0012] (4) Monoclonal antibody incubation: Repeat the washing steps above, add 100 μL of monoclonal antibody 5H2 solution diluted 1:500 per well, and incubate at 37°C for 1 h.

[0013] (5) Secondary antibody incubation: Repeat the washing steps above, add 100 μL of HRP-labeled goat anti-mouse IgG diluted 1:4,000 per well, and incubate at 37°C for 1 h.

[0014] (6) Substrate color development: Repeat the washing steps above, add 100 μL of TMB color development solution to each well under dark conditions, and incubate at 37°C for 10 min in the dark.

[0015] (7) Termination and reading: Add 50 μL of stop solution to each well and immediately read the absorbance (OD value) at 450 nm using an ELISA reader.

[0016] (8) Result calculation and judgment: The blocking rate (PI) is calculated as follows: Blocking rate = (OD value of standard negative serum - OD value of test serum) / OD value of standard negative serum × 100%.

[0017] When the blocking rate (PI) is ≤4.95%, it is considered negative; when the PI is ≥7.92% (i.e., the mean plus 3 standard deviations), it is considered positive; when the PI is between 4.95% and 7.92%, it is considered suspicious.

[0018] Beneficial effects The monoclonal antibody provided by this invention can react with both soluble antigens of Sarcocystis ovis oocysts / sporangia and cysts, enabling detection of both the definitive and intermediate parasitic stages of Sarcocystis ovis. This allows for the establishment of a complete parasite life cycle detection system, significantly improving the detection rate of clinical samples (experiments show an improvement of 35-40%), and the detection window period is 7-10 days earlier than traditional methods. Furthermore, the ELISA kit and detection method provided by this invention are highly sensitive, easy to operate, and can greatly improve detection efficiency, showing promising prospects for industrial application. Attached Figure Description

[0019] Figure 1 SDS-PAGE of Sarcocystis ovis cysts and oocysts / sporangia. PAGE electrophoresis image, where lane 1: soluble antigen of Sarcocystis ovis cysts; lane 2: soluble antigen of Sarcocystis ovis oocysts / sporangia; M: protein molecular weight standard.

[0020] Figure 2 SDS-PAGE electrophoresis image of purified monoclonal antibody 5H2. Detailed Implementation

[0021] 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.

[0022] Example 1: Preparation of Sarcocystis ovis cyst complete antigen and oocyst / sporangium complete antigen 1. Preparation of soluble antigen from Sarcocystis ovis cysts Sarcocystis suis cysts were collected from muscle samples of sheep infected with Sarcocystis suis. Purified cysts were obtained through pepsin digestion and density gradient centrifugation, as detailed below: (1) Collect muscle tissue from slaughtered sheep and examine the muscle smears under a microscope.

[0023] (2) For muscle samples that are positive under microscopic examination, remove visible fat, fascia and connective tissue with scissors and tweezers, cut into small pieces, add a small amount of 1% pepsin, and homogenize with a tissue homogenizer.

[0024] (3) Add 1% pepsin at a weight-to-volume ratio of 1:5 and digest the muscle tissue in a 37°C water bath for 4 h. Then, add an appropriate amount of 1 M NaOH solution to adjust the pH of the mixture to neutral (about 7.0-7.4) to terminate the activity of pepsin.

[0025] (4) The neutralized digested mixture is filtered through 100-mesh and 200-mesh sieves in sequence to remove most of the impurities.

[0026] (5) Centrifuge the filtrate at 1,500×g for 15 min at room temperature and discard the supernatant.

[0027] (6) Resuspend the precipitate in PBS, centrifuge again to precipitate, repeat 2-3 times to completely remove the digestion solution.

[0028] (7) Slowly add 5 mL of 60%, 45%, and 30% Percoll solution to a 50 mL centrifuge tube from bottom to top, and slowly add 2 mL of resuspended encapsulation solution to the top layer.

[0029] (8) Centrifuge at 3,000×g for 30 min at room temperature, and carefully aspirate the milky white capsules near the 45%-60% interface with a pipette.

[0030] (9) Add at least 3-5 times the volume of PBS to the collected capsule suspension and mix thoroughly to dilute the Percoll solution.

[0031] (10) Centrifuge at 1,500×g for 15 min and discard the supernatant. Repeat 2-3 times to completely remove the Percoll solution.

[0032] (11) The obtained Sarcocystis cysts were stored at -80℃ for later use.

[0033] (12) Take the purified Sarcocystis suis cysts, put them into a 1.5 mL centrifuge tube, add 2-3 sterile steel balls to the tube, grind them on a tissue grinder and break them up.

[0034] (13) Take the ground capsules, add 2 mL of PBS buffer, mix well and then use an ultrasonic homogenizer to homogenize until clear (the whole process is carried out on ice).

[0035] (14) Centrifuge at 4℃ and 12,000×g for 10 min, transfer the supernatant to 1.5 mL EP tubes (100 μL per tube), and determine the concentration using a protein quantification kit.

[0036] 2. Preparation of soluble antigens from Sarcocystis ovis oocysts / sporangia Within the definitive host, Sarcocystis is an obligate intracellular parasitic protozoan. Its cysts are digested in the stomach and small intestine, releasing bradyzoites. These bradyzoites invade the small intestinal mucosa, developing into large female gametes and small male gamete mother cells, which further develop into large and small gametes. After fertilization, encapsulated zygotes are produced, forming oocysts. These oocysts undergo sporulation in the lamina propria of the small intestine. A fully sporulated oocyst contains two sporangia, each containing four sporozoites. The sporulated oocysts are easily ruptured, releasing single, free sporangia into the intestinal lumen, which are excreted into the external environment with the definitive host's feces. Therefore, Sarcocystis excreted by the definitive host contains both oocysts and sporangia.

[0037] The collection methods for oocysts / sporangia are as follows: (1) Collect feces from canines in areas where Sarcocystis is endemic, mix with water, and filter through a 100-mesh sieve. Centrifuge the filtrate at 3,000×g for 10 min.

[0038] (2) Discard the supernatant, add saturated salt water to the precipitate and mix well, centrifuge at 1,500×g for 10 min.

[0039] (3) Take the supernatant, add 4 times the amount of water to dilute it, 3,000×g, centrifuge for 10 min to remove salt completely.

[0040] (4) After resuspending the precipitate with a small amount of PBS, take 20 µL of the solution for microscopic examination, and store the remaining oocyst / sporangium fluid at -20℃ for later use.

[0041] The method for preparing soluble antigens from oocysts / sporangia is as follows: (1) Take the above-enriched oocyst / sporangium fluid and add 10 µL of 100× penicillin-streptomycin mixed solution to each 1 mL suspension until the final concentration is 1%.

[0042] (2) Then add 10 µL of nystatin solution with a concentration of 10 mg / mL to a final concentration of 10 mg / L to obtain a suspension of Sarcocystis oocysts / sporangia, and store at 4°C.

[0043] (3) After repeatedly homogenizing the collected oocysts / spore cysts in an ice bath, the mixture was precipitated at 4°C for 48 h and centrifuged at 10,000×g for 1 h.

[0044] (4) Absorb the supernatant, collect and package it to obtain the soluble antigen of Sarcocystis ovodia oocysts / sporangia.

[0045] The protein solutions were concentrated and then identified by SDS-PAGE electrophoresis (see [link to SDS-PAGE analysis]). Figure 1 Protein concentration was determined using a protein quantification kit.

[0046] Example 2: Preparation and Screening of Monoclonal Antibodies To detect intermediate and definitive hosts of Sarcocystis suis, this study first used the whole antigen of Sarcocystis suis cysts as an immunogen to prepare monoclonal antibodies. Then, using soluble antigens of Sarcocystis suis oocysts and / or sporangia as coating antigens, the selected monoclonal antibodies were further screened using ELISA.

[0047] 1. Mouse immunization Six-week-old female Balb / c mice were selected for immunization, and the specific steps are as follows: (1) Primary immunization: Mix the whole antigen of Sarcocystis suis cysts with an equal volume of Freund's complete adjuvant, emulsify it to a state where it does not spread when dropped, and inject it subcutaneously at multiple points on the back of mice, each mouse receiving 200 μL (containing 100 μg of antigen).

[0048] (2) Second immunization: The second immunization was performed two weeks after the primary immunization. The above antigen was emulsified with an equal volume of Freund's incomplete adjuvant to the same state, and injected subcutaneously at multiple points on the back of the mice, with each mouse receiving 200 μL (containing 50 μg of antigen).

[0049] (3) Third immunization: The third immunization is performed two weeks after the second immunization. The immunization method is the same as the second immunization, and each mouse is injected with 200 μL (containing 50 μg of antigen).

[0050] (4) Antibody titer test: After the third immunization, blood was collected from the tail vein of mice, and the serum was separated by centrifugation. The serum antibody titer was detected by ELISA. If the antibody titer reached 1:10,000 or higher, cell fusion could be performed.

[0051] (5) Boosting immunization: If the serum antibody level of mice does not reach the target after the third immunization, a fourth booster immunization should be performed using the same method as the second and third immunizations.

[0052] 2. Cell fusion (1) Preparation of feeder cells One healthy Balb / c mouse was selected and euthanized by cervical dislocation. The mouse was then disinfected by immersion in 75% alcohol for 3 minutes. It was then placed in a biosafety cabinet, abdomen up, and fixed on a dissecting board. The abdominal skin was lifted with sterile ophthalmic forceps, and the skin was cut open with scissors, bluntly dissecting to fully expose the peritoneum. The peritoneum was disinfected by wiping with an alcohol swab. RPMI-1640 basal culture medium was drawn into the peritoneal cavity using a 5 mL disposable syringe and injected. The syringe was held in place, and aspiration was repeated several times. The peritoneal fluid was then extracted and transferred to a 15 mL centrifuge tube. The tube was centrifuged at 1,000 × g for 5 minutes. The supernatant was discarded, and the cells were resuspended in an appropriate amount of HAT selection medium and diluted to 1 × 10⁻⁶. 5 Cells / mL. The cell suspension was seeded at 100 μL / well into 96-well cell culture plates and incubated at 37°C in a 5% CO2 incubator for 24 h, during which cell growth was closely observed.

[0053] (2) Culture of myeloma cells (SP2 / 0) Myeloma cells frozen in liquid nitrogen were thawed 20-30 days prior to fusion. They were cultured in complete medium containing 8-nitroguanine (20 μg / mL) to maintain their resistance. The cells were then placed in a 75 cm³ container. 2 Expand and culture in culture flasks. Select cells in the logarithmic growth phase with good morphology and a viability ≥95%. Gently pipette to prepare a cell suspension and transfer to a 50 mL centrifuge tube. Centrifuge at 1,000×g for 5 min and discard the supernatant. Wash twice with RPMI-1640 medium, resuspend in 20 mL RPMI-1640 medium, and count the cells. Finally, collect 2×10⁶ cells. 7One cell is available for use.

[0054] (3) Spleen cell preparation Balb / c mice, immunized with titers and meeting antibody level targets, were euthanized by cervical dislocation and disinfected by immersion in 75% alcohol for 3 min. They were then transferred to a biosafety cabinet, the abdomen was cut open, and the spleen was aseptically removed. Surrounding connective tissue was removed, and the spleen was rinsed thoroughly with RPMI-1640 medium. The spleen was thoroughly ground in a mortar and filtered through a 200-mesh sieve, and the cell suspension was collected. The suspension was centrifuged at 1000×g for 5 min, the supernatant was discarded, and erythrocytes were lysed by adding 0.91% NH4Cl pre-chilled at 4°C and incubating on ice for 5 min. Lysis was then terminated by adding 15 mL of RPMI-1640 medium, followed by centrifugation and discarding of the supernatant. The cells were washed twice more with RPMI-1640 medium and finally resuspended in 20 mL of RPMI-1640 medium for cell counting. 1×10⁶ cells were collected. 8 One spleen cell is prepared for use.

[0055] (4) Fusion of myeloma cells with spleen cells Myeloma cells and spleen cells were mixed at a ratio of 1:5 and washed with RPMI-1640 medium. The mixture was then centrifuged at 1200×g for 8 min. The supernatant was discarded, and residual liquid in the tube was blotted with filter paper. The tube bottom was gently tapped to loosen the cell clumps. The tube was placed in a 37°C water bath, and 1 mL of preheated 50% PEG solution was slowly added over 90 s. After gentle shaking to mix, the mixture was allowed to stand for 1 min. Subsequently, every 2 min, 1 mL, 2 mL, 3 mL, 4 mL, 5 mL, and 10 mL of preheated RPMI-1640 medium were added sequentially to terminate the confluence, gently shaking to mix after each addition. The mixture was centrifuged at 1000×g for 10 min, and the supernatant was discarded.

[0056] 3. Culture of hybridoma cell lines The fused cells were gently suspended in an appropriate amount of HAT medium and transferred to 96-well plates containing feeder cells (100 μL / well) and cultured at 37°C in a 5% CO2 incubator. Starting from day 3 post-fusion, cells were observed and recorded daily under an inverted microscope, including cell growth status, medium contamination, and feeder cell condition. From day 5 onwards, the HAT medium was replaced with a half-replacement every 2-3 days, observing for hybridoma cell cloning. After approximately 10 days, once the hybridoma cells had stabilized, HT medium was used. After three cloning cycles, the HT medium was replaced with standard RPMI-1640 complete medium.

[0057] 4. Screening of antibody-secreting hybridoma cells (ELISA method) On day 8 after cell culture, based on the observation results of cell clones, all wells with cell clone growth were selected for detection.

[0058] The ELISA testing steps are as follows: (1) Antigen coating: Dilute the whole antigen of Sarcocystis omatis cysts to 0.2 μg / mL with coating solution, add 100 μL to each well of a 96-well ELISA plate, and coat overnight at 4°C. (2) Blocking: Discard the coating solution, wash the plate 3 times with PBST for 3 min each time, and pat dry. Add 200 μL of 5% skim milk (prepared with 1×PBS) blocking solution to each well and incubate at 37°C for 1 h. (3) Add test samples: Repeat the above method to wash the plate. Under aseptic conditions, aspirate the supernatant of the hybridoma cells that have grown in clusters on the cell culture plate and add it to the ELISA plate, 100 μL per well. At the same time, use myeloma cell supernatant as a negative control. Incubate at 37℃ for 1 h.

[0059] (4) Add secondary antibody: Repeat the above method to wash the plate, dilute the horseradish peroxidase (HRP)-labeled goat anti-mouse IgG antibody to 10,000 times, and add 100 μL to each well. Incubate at 37°C for 1 h.

[0060] (5) Color development: Repeat the above method to wash the plate, add 100 μL of TMB substrate color development solution to each well, and develop the color at 37℃ in the dark for 10-15 min.

[0061] (6) Termination: Add 50 μL of stop solution (2M H2SO4) to each well to terminate the reaction.

[0062] (7) Reading: Use an ELISA reader to read the absorbance (OD value) of each well at a wavelength of 450 nm.

[0063] (8) Result determination: The OD value of the test well is compared with the OD value of the negative control well (P / N). When the ratio is ≥2.1, it is determined to be positive. Select the positive well with the highest P / N value, mark it and clone the corresponding hybridoma cells.

[0064] 5. Cloning of target hybridoma cells (limiting dilution method) Twenty-four hours prior to the experiment, feeder cells were prepared using standard methods and seeded into new 96-well plates. Strongly positive monoclonal wells were selected, old culture medium was discarded, and fresh RPMI-1640 medium was added. The cells were gently pipetted to mix and prepare a single-cell suspension. The suspension was centrifuged at 1,000×g for 5 min, the supernatant was discarded, and the cells were resuspended for cell counting. The cell suspension was diluted with HT medium at four concentration gradients: 1,000 cells / mL, 100 cells / mL, 10 cells / mL, and 5 cells / mL. Cells at different concentrations were seeded into 96-well plates at 100 μL per well. Column A was seeded at 1,000 cells / mL (100 cells / well), column B at 100 cells / mL (10 cells / well), column CG at 10 cells / mL (1 cell / well), and column HL at 5 cells / mL (0.5 cells / well). Cells were cultured at 37℃ and 5% CO2 for 4-5 days, and cell growth and colony numbers were recorded under an inverted microscope. 150 μL of fresh HT medium was added, and cultured for another 10-14 days until approximately 50% of the colonies covered the bottom of the wells. For wells with well-grown single clones, the antibody titer in the cell supernatant was detected using the aforementioned ELISA method. High-titer positive single clones were selected, and the limiting dilution method was repeated for subcloning (generally requiring 3-4 rounds) until the positive rate of all subcloning wells reached 100%. Finally, three hybridoma cell lines that stably secreted specific antibodies were obtained: 2D5, 5H2, and 6G7.

[0065] 6. Antigen-specific screening of positive clone cell lines To further screen hybridoma cell lines with specific recognition ability for the soluble antigens of Sarcocystis mutata oocysts / sporangia, this study continued to use ELISA screening methods to coat enzyme-linked immunosorbent assay (ELISA) plates with the soluble antigens of Sarcocystis mutata oocysts / sporangia, identifying the recognition ability of the three positive hybridoma cell lines (2D5, 5H2, and 6G7) for the oocyst / sporangia antigens. Ultimately, it was found that both 5H2 and 6G7 could react with both Sarcocystis mutata cyst and oocyst / sporangia whole antigens, suggesting that a highly expressed homoantigen may exist in the mixed antigen of cysts and oocysts / sporangia. Among them, 5H2 had the highest titer, with a detection limit of 1:102,400.

[0066] 7. Amplification and cryopreservation of positive cell lines The selected 5H2 positive cells were transferred from 96-well plates to 24-well plates for expansion culture. RPMI-1640 complete medium was used as the culture medium, and cell growth was observed regularly. When the cells covered approximately 80-90% of the well bottom, were in logarithmic growth phase, and exhibited good growth and regular morphology, they were gently pipetted with complete medium to detach from the well bottom. The cell suspensions from 2-3 wells were combined into centrifuge tubes, centrifuged at 3000×g for 5 min, and the supernatant was discarded. The cells were resuspended in pre-chilled cell cryopreservation medium and aliquoted into labeled cryovials, clearly labeled with the cell line name and cryopreservation date. The cryovials were placed in a programmed cooling box and stored at -80℃ for 6 h, then transferred to liquid nitrogen for long-term storage, establishing a master cell bank (MCB).

[0067] 8. Stability assessment and resuscitation verification of cell lines The established cell bank underwent regular resuscitation and passage, and the antibody secretion titers of the cell culture supernatants from different passages were measured, recording their stability. After one month of cryopreservation, one vial of 5H2 cryopreserved cells was removed from liquid nitrogen and rapidly thawed at 37°C. The cells were aseptically transferred to centrifuge tubes containing basal culture medium and centrifuged at 1000×g for 5 min. The supernatant was discarded, and the cells were resuspended in 5 mL of complete culture medium and seeded into cell culture flasks. The cells were then revived and cultured at 37°C with 5% CO2. After the cells stabilized (approximately 3-5 days), the supernatant was collected. The antibody secretion capacity of the cell supernatants before and after thawing was simultaneously detected using the same ELISA method to evaluate the cryopreservation effect. The results confirmed that the antibody titers secreted by the 5H2 cells after thawing were not significantly different from those before cryopreservation, demonstrating that this hybridoma cell line has good antibody secretion stability and cryopreservation tolerance.

[0068] The above-mentioned positive cells were expanded and cultured in 5H2 and then sent to a gene company for sequencing. The results showed that: The heavy chain variable region sequence of 5H2 is as follows: VQQLQEAGLVQGSQLSLTCSCAAVTGYFSSDAYAMSWVIRPEKTLEWVASISGGSATSYHDSVKGSITDNAKANLYLLNSVTSEDTAMYYYCARQAGNYDGSFGFDQGTLVVSA (SEQ ID NO.1); HCDR1 is: SDAYAMS (SEQ ID NO.3); HCDR2 is: SISGGSATSYHDSVKG (SEQ ID NO.4); HCDR3 is: RQAGNYDGSFG (SEQ ID NO.5). The light chain variable region sequence is as follows: AVTQSPALTTEKVATVTFTCRSSISAVSTTYANWVQRPGDHLFTPWLIGGTAHLAISGVPARFGSLIGDKALTITISMAEAEAYFCALWASDPWVFGGAGTKLTVL (SEQ ID NO. 2); LCDR1 is: RSSISAVSTTYAN (SEQ ID NO.6); LCDR2 is: GTAHLAIS (SEQ ID NO.7); LCDR3 is: ALWASDPWV (SEQ ID NO.8).

[0069] Example 3: Establishment of an ELISA detection kit and method for detecting Sarcocystis ovis. The ELISA kit includes soluble antigen of Sarcocystis suis cysts, standard negative serum, standard positive serum, monoclonal antibody 5H2, 0.05M carbonate buffer at pH 9.6, PBST buffer, PBS buffer containing 1% BSA, HRP-labeled goat anti-mouse IgG, chromogenic solution, and stop solution.

[0070] The concentration of soluble antigen from *Sargassum fusiforme* cysts was 1 mg / mL, and the solvent was 0.01 mol / L PBS buffer (pH 7.2). Standard positive and negative sera were prepared according to standard methods in the art. Monoclonal antibodies were prepared using PBS buffer to a final concentration of 2 mg / mL. The stop solution was a 2 mol / L aqueous sulfuric acid solution. HRP-labeled goat anti-mouse IgG and TMB chromogenic solution were commercially available reagents.

[0071] 1. Screening for optimal antigen coating concentration and optimal dilution of serum to be tested The optimal antigen coating concentration and optimal serum dilution were screened using the checkerboard method. The specific steps are as follows: (1) Coating: The whole antigen of Sarcocystis omatis cysts was diluted to 50, 25, 12.5, 6.25 and 3.125 μg / mL with carbonate buffer at pH 9.6 and 0.05 M, respectively. Each concentration was used in duplicate wells with 100 μL per well. The mixture was coated overnight at 4°C.

[0072] (2) Blocking: Wash the microplate with PBST buffer for 5 min each time, for a total of 3 washes. After each wash, shake off any remaining liquid in the plate. Add 100 μL of PBS buffer containing 1% BSA to each well and block at 37°C for 1 h.

[0073] (3) Competitive incubation: Repeat the above method to wash the plate, and dilute the standard positive serum and standard negative serum at 1:1, 1:2 and 1:4, and add 100 μL to each well of the ELISA plate. Incubate at 37℃ for 1 h.

[0074] (4) Monoclonal antibody incubation: Repeat the above method to wash the plate, dilute the monoclonal antibody 5H2 at 1:500, add 100 μL to each well, and incubate at 37℃ for 1 h.

[0075] (5) Secondary antibody incubation: Repeat the above method to wash the plate, dilute HRP-labeled goat anti-mouse IgG at a ratio of 1:5,000, add 100 μL to each well, and incubate at 37°C for 1 h.

[0076] (6) Color development: Repeat the above method to wash the plate. Under light-protected conditions, add 100 μL of TMB solution to each well and incubate at 37°C for 10 min in the dark.

[0077] (7) Termination and reading: Add 50 μL of stop solution to each well and immediately read the absorbance (OD value) at 450 nm using an ELISA reader.

[0078] (8) Result calculation and judgment: The blocking rate is calculated as follows: Blocking rate = (OD value of standard negative serum - OD value of test serum) / OD value of standard negative serum × 100%.

[0079] The results showed that the PI value was highest when the whole antigen of Sarcocystis ovis cysts was 6.25 μg / mL and the serum to be tested was diluted 1:1 (see Table 1).

[0080] Table 1 Screening of optimal antigen coating concentration and optimal dilution of serum to be tested

[0081] Where P is the OD value of the serum to be tested, N is the OD value of the standard negative serum, and PI (%) is the blocking rate.

[0082] 2. Optimization of serum incubation time The ELISA experiment was repeated to determine the optimal incubation time for serum (0.5 h, 1 h, 1.5 h). The coating concentration of *Sargassum fusiforme* cyst antigen was 6.25 μg / mL, and the serum dilution was 1:1. The serum incubation time with the largest difference in OD values ​​and the highest PI value (ratio) between the standard positive and negative sera was selected as the optimal reaction time. The results showed that the optimal serum incubation time was 1 h.

[0083] 3. Optimization of the optimal working concentration of monoclonal antibodies The ELISA experiment was repeated to determine the optimal working concentrations of the monoclonal antibody (1:500, 1:1,000, 1:2,000, 1:4,000). The coating concentration of the Sarcocystis suis cyst antigen was 6.25 μg / mL, the serum dilution was 1:1, and the serum incubation time was 1 h. The working concentration with the largest difference in OD values ​​between positive and negative sera and the highest PI value was selected as the optimal working concentration of the monoclonal antibody. The results showed that the optimal dilution for 5H2 was 1:500 (see Table 2).

[0084] Table 2 Screening for optimal monoclonal antibody dilutions

[0085] 4. Optimization of the optimal incubation time for monoclonal antibodies The ELISA experiment was repeated to determine the optimal incubation time for the monoclonal antibody (0.5 h, 1 h, 1.5 h). The coating concentration of the Sarcocystis suis cyst antigen was 6.25 μg / mL, the serum dilution was 1:1, the serum incubation time was 1 h, and the monoclonal antibody dilution was 1:500. The incubation time of the monoclonal antibody with the largest difference in OD values ​​between positive and negative serum samples and the highest PI value was selected as the optimal incubation time. The results showed that the optimal incubation time for the monoclonal antibody 5H2 was 1 h.

[0086] 5. Optimization of the optimal working concentration of enzyme-labeled secondary antibody The ELISA experiment was repeated to determine the optimal working concentrations (1:4,000, 1:5,000, 1:6,000) of the enzyme-labeled secondary antibody (HRP-IgG). Specifically, the coating concentration of the Sarcocystis suis cyst antigen was 6.25 μg / mL, the serum dilution was 1:1, the serum incubation time was 1 h, the primary antibody dilution was 1:500, and the monoclonal antibody incubation time was 1 h. The reaction conditions with the largest difference in OD values ​​between positive and negative sera and the highest PI value were selected as the optimal reaction conditions. As shown in Table 3, the optimal dilution of the enzyme-labeled secondary antibody was 1:4,000.

[0087] Table 3 Screening of optimal dilutions for enzyme-labeled secondary antibodies

[0088] 6. Optimization of the optimal incubation time for enzyme-labeled secondary antibodies The ELISA experiment was repeated to determine the optimal incubation times (0.5 h, 1 h, 1.5 h) for the enzyme-labeled secondary antibody. The coating concentration of the Sarcocystis suis cyst antigen was 6.25 μg / mL, the serum dilution was 1:1, and the serum incubation time was 1 h. The monoclonal antibody dilution was 1:500, and the incubation time was 1 h. The enzyme-labeled secondary antibody dilution was 1:4,000. The reaction conditions with the largest difference in OD values ​​between positive and negative sera and the highest PI value were selected as the optimal reaction conditions. The results showed that the optimal incubation time for the enzyme-labeled secondary antibody was 1 h.

[0089] 7. Optimization of optimal substrate reaction time The ELISA experiment was repeated to determine the optimal reaction time (5 min, 10 min, 15 min) for the substrate (TMB solution). The coating concentration of the Sarcocystis suis cyst antigen was 6.25 μg / mL, the serum dilution was 1:1, and the serum incubation time was 1 h. The monoclonal antibody dilution was 1:500, and the incubation time was 1 h. The enzyme-labeled secondary antibody dilution was 1:4,000, and the incubation time was 1 h. The reaction conditions with the largest difference in OD values ​​between positive and negative sera and the highest PI value were selected as the optimal reaction conditions. The results showed that the optimal reaction time for the substrate was 10 min.

[0090] 8. Finalization of the ELISA method After optimizing multiple conditions of the ELISA method, the final steps are as follows: (1) Antigen coating: Dilute the whole antigen of Sarcocystis omatis cysts to 6.25 μg / mL with 0.05M carbonate buffer at pH 9.6, add 100 μL of antigen dilution to each well, and coat at 4℃ for 12-16 h.

[0091] (2) Blocking: Wash the microplate with PBST buffer for 5 min each time, 3 times, and shake off the residual liquid in the wells after each wash. Add 100 μL of PBS buffer containing 1% BSA to each well and block at 37℃ for 1 h.

[0092] (3) Serum sample incubation: Repeat the washing steps above, dilute the serum to be tested 1:1 with PBST buffer, and dilute the standard negative serum by the same factor as a control. Add 100 μL to each well and incubate at 37°C for 1 h.

[0093] (4) Monoclonal antibody incubation: Repeat the washing steps above, add 100 μL of monoclonal antibody 5H2 solution diluted 1:500 per well, and incubate at 37°C for 1 h.

[0094] (5) Secondary antibody incubation: Repeat the washing steps above, add 100 μL of HRP-labeled goat anti-mouse IgG diluted 1:4,000 per well, and incubate at 37°C for 1 h.

[0095] (6) Substrate color development: Repeat the washing steps above, add 100 μL of TMB color development solution to each well under dark conditions, and incubate at 37°C for 10 min in the dark.

[0096] (7) Termination and reading: Add 50 μL of stop solution to each well and immediately read the absorbance (OD value) at 450 nm using an ELISA reader.

[0097] (8) Result calculation and judgment: The blocking rate is calculated as follows: Blocking rate = (OD value of standard negative serum - OD value of test serum) / OD value of standard negative serum × 100%.

[0098] 9. Determining the critical value By testing 85 negative serum samples and following established ELISA testing procedures, the cutoff values ​​for the kits in this study were determined. The specific criteria were as follows: a blocking rate (PI) ≤ 4.95% was considered negative; a PI ≥ 7.92% (mean plus three standard deviations) was considered positive; and a PI between 4.95% and 7.92% was considered suspicious.

[0099] 10. Repeatability analysis of ELISA kits Soluble antigen from the same batch of *Salmonella ovis* cysts and monoclonal antibody 5H2 were used to detect positive and negative sera of *Salmonella ovis* according to the ELISA method established in this invention. Each serum sample was tested in triplicate, and the intra-batch coefficient of variation was calculated. Three batches of purified *Salmonella ovis* cyst-soluble antigen and monoclonal antibody 5H2 were used to detect positive and negative sera of *Salmonella ovis* according to the ELISA method established in this invention. Each serum sample was tested in triplicate, and the inter-batch coefficient of variation was calculated. The results showed that the coefficients of variation for both intra-batch and inter-batch replicates were less than 10%, indicating that this kit has good reproducibility.

[0100] 11. Sensitivity analysis of ELISA kits Serum samples positive for *Nasporula mutatis murineis* were randomly selected and serially diluted with PBST buffer (1:1, 1:2, 1:4, 1:8, 1:16, 1:32, 1:64). The ELISA method established in this invention was compared with an indirect ELISA method previously established based on the *Nasporula mutatis murineis* 14-3-3 protein. The results showed that the method of this invention could still detect positive results when the serum was diluted to 1:32, while the upper limit of detection for the indirect ELISA method was 1:16, indicating that the ELISA method established in this invention has higher sensitivity.

[0101] 12. Specificity analysis of ELISA kits Positive serum samples from sheep infected with common parasites, including Toxoplasma gondii, Echinococcus granulosus, Babesia, and Fasciola hepatica, were collected and tested using the ELISA method established in this invention to verify the specificity of the kit. The results showed that only serum samples positive for Sarcocystis suis reacted, while the others were negative, indicating that this kit has good specificity.

[0102] 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 5H2 that specifically recognizes Sarcocystis ovis, characterized in that... The monoclonal antibody 5H2 is secreted by the hybridoma cell line 5H2. This monoclonal antibody 5H2 can react not only with the soluble egg antigens (SEA) excreted by the definitive host (dog), but also with the cyst soluble antigens in the muscle of the intermediate host (sheep).

2. The use of the monoclonal antibody 5H2 as described in claim 1 in the detection of Sarcocystis ovis oocysts / sporangia and cysts.

3. An ELISA kit for detecting Sarcocystis sheep, characterized in that... It includes the whole antigens of Sarcocystis ovogina oocysts / sporangia and cysts and the monoclonal antibody 5H2 as described in claim 1.

4. The ELISA kit as described in claim 3, further comprising standard negative serum, standard positive serum, carbonate buffer, PBST buffer, PBS buffer containing 1% BSA, HRP-labeled goat anti-mouse IgG (HRP-IgG), chromogenic solution and stop solution.

5. A method for detecting Sarcocystis omatis using the monoclonal antibody 5H2 as described in claim 1 or the ELISA kit as described in claim 3, the method comprising the following steps: (1) Antigen coating: Dilute the whole antigen of Sarcocystis omatis cysts to 6.25 μg / mL with 0.05M carbonate buffer at pH 9.6, add 100 μL of antigen dilution to each well, and coat at 4℃ for 12-16 h; (2) Blocking: Wash the microplate with PBST buffer for 5 min each time, wash 3 times, and shake off the residual liquid in the well after each wash. Add 100 μL of PBS buffer containing 1% BSA to each well and block at 37℃ for 1 h. (3) Serum sample incubation: Repeat the above washing steps, dilute the serum to be tested with PBST buffer 1:1, and dilute the standard negative serum by the same factor as a control. Add 100 μL to each well and incubate at 37°C for 1 h. (4) Monoclonal antibody incubation: Repeat the washing steps above, add 100 μL of monoclonal antibody 5H2 solution diluted 1:500 per well, and incubate at 37°C for 1 h; (5) Secondary antibody incubation: Repeat the washing steps above, add 100 μL of HRP-labeled goat anti-mouse IgG diluted 1:4,000 per well, and incubate at 37°C for 1 h; (6) Substrate color development: Repeat the above washing steps, add 100 μL of TMB color development solution to each well under dark conditions, and incubate at 37°C for 10 min in the dark; (7) Termination and reading: Add 50 μL of stop solution to each well and immediately read the absorbance (OD value) at 450 nm using a microplate reader. (8) Result calculation and judgment: The blocking rate (PI) is calculated as follows: Blocking rate = (OD value of standard negative serum - OD value of test serum) / OD value of standard negative serum × 100%; When the blocking rate (PI) is ≤4.95%, it is considered negative; when the PI is ≥7.92% (i.e., the mean plus 3 standard deviations), it is considered positive; when the PI is between 4.95% and 7.92%, it is considered suspicious. The detection method described herein is not for disease diagnosis purposes.

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