JP2007261959A - Monoclonal antibody of coccidial protozoan and use thereof - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To obtain a monoclonal antibody for inhibiting invasion of a coccidial protozoan to chicken egg cells. <P>SOLUTION: The monoclonal antibody to react with Eimeria tenella of a coccidial protozoan is obtained from a hybridoma prepared by fusing spleen cells obtained from a mouse immunized against a polypide of sporozoite stage of Eimeria tenella of a coccidial protozoan with mouse myeloma cells. The monoclonal antibody inhibits invasion of Eimeria tenella of a coccidial protozoan to chicken egg cells. The hybridoma is (1) 1G4(FERM AP-20811), (2) 3A5(FERM AP-20812), (3) 4C8(FERM AP-20813), (4) 7B12(FERM AP-20814) or (5) 9D6(FERM AP-20815). <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention relates to a monoclonal antibody against Coccidium parasite Eimeria tenella, a hybridoma producing the same, and use thereof.

One of the diseases causing economic losses in the world poultry industry is coccidiosis. The cause of this disease is Eceria spp. Infected with chicken, coccidia parasite that indicates the pathogenic seven are known ^{(Disease of Poultry 9 th Edition,} Iowa State University Press (1991)). Among these, four types of coccidial protozoal infections are a problem. The four types of protozoa in question are Eimeria tenella, Eimeria acervulina, Eimeria maxima and Eimeria necatrix. Among these, Eimeria Tenella and Eimeria Asselbrina have caused the most damage. Coccidiosis occurs mainly from the young to middle chick stages. Infected chicks develop diarrhea, and death in the case of dense infection usually leads to a temporary loss of body weight gain. It is also known that the decrease in gain is prolonged over a long period due to infection with different coccidial protozoa. In chicken life, this period is a period of fattening and rearing, and such a decrease in body weight gain affects feed efficiency and causes serious economic loss. In particular, in the broiler industry where the breeding period is short, coccidium infection is a serious problem. Currently, anti-coccidial agents are used to prevent coccidial infection. Anti-coccidial agents are effective against all coccidiums, but due to the emergence of coccidium-resistant protozoa and problems with residual drugs, the amount used and the period of use are limited, and a drug holiday period is also provided. Moreover, once a poultry house is contaminated with coccidium, it is extremely difficult to clean it completely, and it is necessary to continue using chemicals to prevent contamination. In recent years, live vaccines have been used as effective for preventing coccidiosis. However, if a vaccine strain, which is an active ingredient of a live vaccine, is mutated and rendered toxic, it may contaminate the poultry house. Moreover, even when it does not mutate, coccidium administered as a vaccine is killed by an anti-coccidial agent, and thus cannot be used in combination. As a result, other protozoal infections that have traditionally been suppressed with small amounts of anti-coccidial agents may occur.

In such vaccine research and therapeutic drug research, much information has been obtained from proteins involved in infection, proliferation and development of pathogens.
In the case of viruses, since there are few types of proteins to be expressed, a monoclonal antibody having some function can be obtained relatively easily, and it is easy to obtain a protein or gene having any function using this monoclonal antibody. However, in the case of coccidia protozoa, the types of proteins to be expressed are enormous, and the proteins to be expressed differ depending on the life cycle, and the form and the antigen to be expressed are different at each stage. For this reason, it is extremely difficult to obtain a monoclonal antibody having a target function, and an identification method that does not use a monoclonal antibody, for example, a method for purifying and examining a protozoan protein, a λgt11 library from cDNA, and a coccidium protozoan A method of screening with infected sera, a method of finding an antigen from a coccidial protozoan gene having a high homology with other infection-defense antigen genes known in the genus Eimeria, etc. No. 10-18283, etc.), and at present, proteins are identified by methods involving complicated operations.
However, as described above, it has been difficult to identify a functional protein for coccidium, which has hindered the development of conventional coccidium vaccines and therapeutic agents for coccidium infection.

Japanese Patent No. 3051130 JP-T 9-504604 Japanese Patent Laid-Open No. 10-18283 Disease of Power 9th Edition, Iowa State University Press (1991)

  As a result of intensive studies to obtain a new antigenic protein under such conventional techniques, the present inventors focused on the fact that the invasion time of the coccidial parasite into the intestinal cells is the sporozoite stage or the merozoite stage. As a result of attempting to produce a hybridoma using a worm body, the hybridoma was found to produce a monoclonal antibody having an activity that strongly inhibits the invasion of coccidial protozoa into chicken cells, thereby completing the present invention. It came.

Thus, according to the present invention,
(1) A monoclonal antibody that reacts with Coccidium parasite Eimeria tenella, and can inhibit the entry of Coccidium parasite Eimeria tenella into chicken cells,
(2) The hybridoma producing the monoclonal antibody according to claim 1 or 2 produced by fusing a spleen cell obtained from a mouse immunized with a sporozoite stage worm body of the coccidial protozoan Eimeria tenella and a myeloma cell of the mouse ,
(3) A method for identifying an antigenic protein derived from Coccidia parasite Eimeria tenella using the monoclonal antibody,
(4) a compound derived from the coccidial parasite Eimeria tenella recognized by the monoclonal antibody,
(5) Coccidium protozoa Eimeria tenella infection therapeutic agent comprising the monoclonal antibody according to (1) as an active ingredient,
Is provided.

  Thus, according to the present invention, a novel monoclonal antibody having activity of preventing entry of Eimeria tenella into chicken cells and a hybridoma producing the same can be obtained. The monoclonal antibody of the present invention that recognizes a coccidium protozoa protective antigen can be used to identify a coccidium protozoan antigen. The identified antigen is expected to be used as a vaccine antigen, and the monoclonal antibody itself can be used as a therapeutic agent for coccidiosis.

(1) Monoclonal antibody The monoclonal antibody of the present invention reacts with Eimeria tenella, a coccidial protozoan,
In addition, it can inhibit the entry of Eimeria tenella into chicken cells. In the present invention, “reacts with Eimeria tenella” means that a monoclonal antibody recognizes a protein derived from Eimeria tenella and exhibits an antigen-antibody reaction. The antigen-antibody reaction is confirmed by the indirect enzyme antibody method (ELISA method) using the hybridoma culture supernatant. Specific conditions are as described in Example (2). A monoclonal antibody produced by a hybridoma whose absorbance of the culture supernatant under the conditions of this example is 0.1 or more is preferred.

In the present invention, “to prevent entry of Eimeria tenella into chicken cells” refers to inhibiting entry of protozoa, which are sporozoite stages of Eimeria tenella, into chicken embryo fibroblasts, specifically Confirm that the intrusion is blocked by the following method.
Eimeria sporozoite parasites were mixed with ascitic fluid containing monoclonal antibodies and then added to chicken fetal fibroblasts. The inhibitory effect was measured by the reduction rate of the number of coccidial parasites entering the cells, completely unrelated to the coccidia parasites. When the number of invading cells was 100% when ascites containing a monoclonal antibody that reacts with various antigens and worms were mixed, a significant decrease in the number of worms was observed (that is, ( If the intrusion prevention rate obtained in 4) exceeds 20%), it is determined to be active.

  Examples of such monoclonal antibodies include 1G4 (FERM AP-20811), 3A5 (FERM AP-20812), 4C8 (FERM AP-20813), 7B12 (FERM AP- 20814), 9D6 (FERM AP-20815). The subclass of monoclonal antibodies produced from these hybridomas is IgG1. The monoclonal antibody produced from the hybridoma 3A5 recognizes the epitope of the antigenic protein of 90 to 120 kilodaltons expressed by Eimeria tenella.

The monoclonal antibody of the present invention is produced from a hybridoma prepared by fusing a spleen cell obtained from a mouse immunized with Eimeria tenella at a sporozoite stage and a myeloma cell of the mouse.
Specific examples of coccidial protozoa used for immunization include attacking strains and field isolates such as Eimeria tenella, Eimeria asselbrina, Eimeria maxima, Eimeria necatricks, Eimeria brunetti, Eimeria mitis. The amount of the worm body is usually adjusted to a concentration of 10 ⁴ to 10 ¹⁰ / ml.

  During immunization, the worm body may be killed by freezing and thawing. However, when inoculating mice, there is no risk of infection, so immunization may be carried out by mixing with adjuvant as it is. The adjuvant used to immunize this worm body is not particularly limited, but Freund Complete Adjuvant, Freund Incomplete Adjuvant, Alum, Aracel, Alum, etc. are used. It is done. The mixing ratio of the insect body and the adjuvant is not particularly limited as long as immunity is established, but is usually 1: 1. Examples of animals immunized with coccidiosis include mice, rats, hamsters, and the like, and the strains of these animals are not particularly limited, but preferably Balb / c mice are used. In addition to these animals, human lymph node cells and peripheral lymphocytes can be used.

  Mouse myeloma cells used in the present invention are P3 / X63-Ag8. U1 (P3U1), Sp2 / 0-Ag14 (SP2), P3 / NSI-1-Ag8.653, P3 / X63-Ag8 (X63), and the like, preferably P3 / X63-Ag8. U1 (P3U1). The spleen cells to be fused may be of any animal species, but in view of the growth state of the cells after fusion, cells of the same type as myeloma are preferred.

  The polyethylene glycol used for cell fusion has a polymerization average molecular weight of 1,000 to 4,000, preferably 1,000 to 2,000. At this time, it is more preferable to add 30% (w / v) of dimethyl sulfoxide (DMSO).

The method for confirming the antibody productivity of a fused cell (hybridoma) is usually a hybridoma whose antibody productivity has been confirmed using a gel precipitation reaction between a cell culture supernatant and an anti-mouse immunoglobulin or an indirect enzyme antibody method (ELISA method). As for the antibody produced by the hybridoma by the ELISA method in which the coccidial parasite is immobilized or the chicken fetal fibroblast in which the coccidial parasite has entered is confirmed to react with the coccidiotic parasite To do. In the obtained hybridoma, 10 ⁵ to 10 ⁹ cells in the logarithmic growth phase were suspended in 1-2 ml Dulbecco's MEM (DMEM) containing 10% DMSO, 10% fetal calf serum (FCS), and −80 It can be stored by a conventional method for storing cultured cells, such as freezing at 2 ° C. for 2 hours or longer and storing in liquid nitrogen.

  Further, the monoclonal antibody of the present invention can be obtained by Western blotting in which a protein extracted from coccidiosis is developed by SDS-polyacrylamide gel electrophoresis, blotted on a filter membrane and then reacted with the monoclonal antibody, or genomic DNA fragments of coccidiosis are obtained. An antigen protein derived from coccidia parasite can be identified by using, for example, a method for selecting a phage that expresses an antigen to which a monoclonal antibody reacts from the inserted λgt-11 library.

  All of the monoclonal antibodies obtained by the above-described methods recognize compounds derived from coccidia protozoa. However, a conventional method for obtaining a compound having a recognition site such as an epitope, for example, the monoclonal antibody obtained by the present invention. Adsorbs compounds derived from protozoan parasites using affinity column chromatography that adsorbs coccidium protozoa crushed extract to the antibody-immobilized column or a complex of monoclonal antibody and immunoglobulin-adsorbed protein such as protein A. And immunoprecipitation method.

  Furthermore, the monoclonal antibody of the present invention can be used as a therapeutic agent for coccidiosis. This monoclonal antibody can be used by mixing with a pharmaceutically acceptable carrier (physiological saline, phosphate buffered saline, etc.) and can be administered orally or parenterally. The dose varies depending on the age of the chicken, the dosage form, etc., but is usually about 1 to 500 μg per bird.

  The monoclonal antibody of the present invention will be specifically described below with reference to examples, but the present invention is not limited to these examples.

(1) coccidia oocysts Eimeria tenella field isolate Rt strains immune 10 ⁸ to mice protozoa recovered from fecal test infection chickens After purification of oocysts, and PBS was added 10 ml, oocysts suspension A liquid was prepared. About 3 times the amount of 30% sodium hypochlorite solution was added to this oocyst suspension, transferred to a sterilized centrifuge tube, and allowed to stand at room temperature for 10 minutes. p. m. And centrifuged for 10 minutes. After removing the supernatant, the precipitate was suspended in 50 ml of PBS, and 1500 r. p. m. And centrifuged for 10 minutes to remove the supernatant. After repeating this washing operation three times to remove hypochlorous acid, oocysts were suspended in about 3 ml of sterile PBS, and then placed in a Teflon (registered trademark) homogenizer (5 ml). p. m. Was processed at a speed of 10 to 15 minutes to break the outer wall of the oocyst into a sporocyst. Next, 25 ml of trypsin solution (0.3% trypsin-PBS) was added to the released sporocysts, and further 3 ml of chick bile was added and incubated at 40 ° C. for 1.5 hours. Heating was stopped when the sporozoite shelling rate reached 80 to 90% or more, and 1000 r. p. m. And centrifuged for 5 minutes. The precipitate was washed with PBS and then suspended in PBS to obtain a sporozoite antigen of coccidia protozoa for immunization. Sporozoites prepared in PBS at 10 ⁶ cells / ml are frozen in an ultra-low temperature freezer at -80 ° C. and then thawed, mixed with an equal amount of Freund Complete Adjuband (FCA), and ¼ volume is used for one 7 Immunity was immunized subcutaneously on the back of week-old Balb / C mice. Four weeks later, the sporozoite antigen prepared in the same manner was mixed with Freund's complete adjuvant (FIA), and a 1/4 amount was boosted subcutaneously in the back.

(2) Preparation of hybridoma The spleen was removed from the mouse 3 days after the additional immunization, loosened with tweezers and scissors in DMEM without FBS, and then isolated spleen cells were passed through a sterilized stainless steel mesh with a diameter of 100 μm. Obtained. The cells were further washed 3 times with DMEM without FBS.
On the other hand, mouse myeloma cells P3U1 are grown in DMEM supplemented with 10% FBS containing 10-8 M 8-azaguanine (2-amino-6-oxy-8-azapurine) and cultured until 10 ⁷ or more can be recovered. Then, it was further washed 3 times with DMEM not containing 8-azaguanine and FBS and recovered.
Next, add 10 ⁷ P3U1 and 10 ⁸ spleen cells to a sterile Spitz tube, mix, centrifuge at 1,600 rpm for 5 minutes to completely remove the supernatant, vortex, A polyethylene glycol solution for fusion {polyethylene glycol 1 g (average molecular weight 1,000), DMSO 0.3 ml, DMEM 0.9 ml} was added and pipetted 2 to 3 times. Then, while shaking lightly with the Spitz tube inclined, DMEM without FBS was added in 9 ml portions at 30 second intervals. The cell suspension was centrifuged at 1,000 rpm for 10 minutes, the supernatant was discarded, and DMEM containing 10% FBS was added so that the number of P3U1 cells was 10 ⁵ to 10 ⁶ cells / ml. This cell suspension was dispensed into a flat-bottom 96-well plate at 0.1 ml / well and cultured in a CO ₂ incubator at 37 ° C., 5% CO ₂ , 100% humidity. On the next day, DMEM • HAT (DMEM containing 100 μM hypoxanthine, 0.4 μM aminopterin, and 16 μM thymidine) supplemented with 10% FBS at 0.1 ml / well was added. The medium was aspirated 1 ml at a time. Instead, the medium was changed by adding DMEM / HAT at 0.1 ml / well, and thereafter, the medium was changed every 2 to 3 days. Unfused myeloma cells and spleen cells died within one week, and only the fused cells grew in each well to form colonies. From the 10th day after the fusion, the cells were cultured in a medium (DMEM / HT) from which only aminopterin was removed from DMEM / HAT to obtain fused cells (hybridomas) in which spleen cells and myeloma cells were fused.

(3) Selection of monoclonal antibody-producing hybridoma that reacts with coccidial protozoa When the thus obtained hybridoma occupies an area of 40% or more of the well, 100 μl of the culture supernatant is taken, and hybridoma antibody production is performed as a primary screening. Sex was examined by the indirect enzyme antibody method. First, anti-mouse immunoglobulin was diluted to 20 ng / ml in a bicarbonate buffer (15 mM Na ₂ CO ₃ , 35 mM NaHCO ₃ , pH 9.8), dispensed in 50 μl aliquots into a 96-well plate, and 1 Incubated for hours. After incubation, each well was washed 3 times with PBS containing 0.05% Tween 20 (pH 7.3 or less, referred to as PBS-TW), and then filled with Block Ace (Dainippon Pharmaceutical) diluted 4-fold with PBS. It incubated at room temperature for 1 hour, and blocked the protein non-adsorbed part of the well. After washing 3 times with PBS-TW, 50 μl each of the hybridoma culture supernatant was added and incubated at room temperature for 1 hour. After washing 3 times with PBS-TW, 50 μl of peroxidase-labeled anti-mouse immunoglobulin diluted 1,000 times with PBS was dispensed and incubated at room temperature for 1 hour. After washing 3 times with PBS-TW, ABTS {2,2′-azino-bis (3-ethylbenzthiazoline-6-sulfonic acid)} which is a substrate of peroxidase was added and incubated at room temperature for 10 minutes.

Using an immunoreader, the absorbance at a wavelength of 405 nm was measured, and the hybridoma culture supernatant corresponding to the well exhibiting an absorbance of 0.1 or higher was subjected to the following reactivity as a secondary screening for coccidium protozoa. I investigated. Thawed sporozoites worms 10 ⁶ Eimeria tenella, after confirming that the survival worms must not have, washed 3 times with PBS, 96-well plates were diluted to 1 [mu] g / ml in bicarbonate buffer 50 μl each was dispensed and incubated at room temperature for 1 hour. After washing 3 times with PBS-TW, the well was filled with Block Ace diluted 4-fold with PBS and incubated at room temperature for 1 hour to block the protein unadsorbed portion of the well. After washing 3 times with PBS-TW, 50 μl each of the hybridoma culture supernatant was added and incubated at room temperature for 1 hour. After washing 3 times with PBS-TW, 50 μl of biotinylated anti-mouse immunoglobulin diluted 1,000 times with PBS was dispensed and incubated at room temperature for 1 hour. After washing 3 times with PBS-TW, avidin-conjugated peroxidase was diluted 1,000 times with PBS, 50 μl each was added, and incubated at room temperature for 1 hour. After washing 3 times with PBS-TW, ABTS {2,2′-azino-bis (3-ethylbenzthiazoline-6-sulfonic acid)} was added and incubated at room temperature for 10 minutes. Using an immunoreader, the absorbance at a wavelength of 405 nm was measured, and a hybridoma corresponding to a well exhibiting an absorbance of 0.1 or more was cloned by a limiting dilution method.

(3) Ascites of hybridoma Next, 0.5 ml of pristane (2,6,10,14-tetramethylpentadecane) was intraperitoneally administered 4 weeks ago for the purpose of obtaining a large amount of monoclonal antibody produced by the cloned hybridoma. Approximately 10 ⁸ hybridomas were transplanted into the prepared mice. Ascites collected in the abdominal cavity after 1-3 weeks was collected with a syringe. The collected ascites was centrifuged at 3,500 rpm for 20 minutes to collect the supernatant, and treated at 56 ° C. for 30 minutes to be inactivated. These were stored at −80 ° C. until use.

(4) Inhibition activity of monoclonal antibody coccidium into embryonic fetal fibroblasts The Eimeria tenella sporozoite parasite prepared in the above (1) was prepared to 1000 sporozoites / ml in Eagle's MEM medium. The ascites obtained and stored in (3) above was diluted 10 times, 50 times, and 250 times. 1 ml of the prepared Teneras polozoite and 1 ml of ascites were mixed well and incubated at 41 ° C. for 1 hour. As a negative control, ascites of 35A6 hybridoma (Japanese Patent Laid-Open No. 9-103294) that reacts with Mycoplasma gallisepticum antigen, which is an antigen unrelated to coccidium, was mixed with Teneras polozoite at the same mixing ratio, and Eagle MEM. 2 ml of a sporozoite suspension prepared to 500 sporozoite / ml was used. After incubation, it was added to cultured chicken fetal fibroblasts confluent in a 9 cm diameter cell culture dish. After incubating at 41 ° C. for 1 hour in a carbon dioxide incubator for cells, it was washed three times with Eagle's MEM medium not containing fetal calf serum to wash away sporozoites that were not adsorbed on the cells. After washing, 10 ml of -20 ° C methanol was added to the cells and incubated for 10 minutes. After discarding methanol, the air-dried plate was filled with dish with Block Ace diluted 4 times with PBS, and incubated at room temperature for 1 hour to block the protein unadsorbed portion of the well. After washing 3 times with PBS-TW, the dish was filled with 10 ml of 6D12 anti-coccidial monoclonal antibody (chicken type) (J. Pasitol., 84, 654-656 (1998)), and incubated at 37 ° C. for 1 hour. After washing 4 times with PBS, a FITC-labeled anti-chicken IgG (Sigma) antibody diluted 100-fold with PBS was added and incubated at 37 ° C. for 30 minutes. The labeled antibody was washed thoroughly 4 times with PBS, and the number of worms emitting fluorescence was counted using an inverted phase contrast fluorescence microscope. Counts the number of sporozoite parasites that have invaded chicken embryo fibroblasts in the whole dish, and blocks the percentage that was subtracted from 100% when the monoclonal antibody was treated when the number of invading sporozoites in the negative control was 100 Rate. The results are shown in Table 1.

  Ascites fluid containing monoclonal antibodies produced by 1G4, 3A5, 4C8, 7B12, and 9D6 extremely prevented the entry of Eimeria tenella sporozoites into chicken embryo fibroblasts. From these results, hybridoma 1G4 (FERM AP-20811), hybridoma 3A5 (FERM AP-20812), hybridoma 4C8 (FERM AP-20813), hybridoma 7B12 (FERM AP-20814), and hybridoma 9D6 (FERM AP-20815) were obtained. It was found that the monoclonal antibody produced was an infection protective antibody, and the recognized antigen was an infection protective antigen.

(5) Determination of isotype of antibody produced by each hybridoma Next, the class or subclass of the antibody produced by these hybridomas was determined by ISOTYPE Ab-STATTM (SangStat Medical Corporation). As a result, all hybridomas were confirmed to produce IgG1 antibodies.

(6) Measurement of Molecular Weight of Antigen Recognized by Monoclonal Antibody In order to measure the molecular weight of Eimeria tenella sporozoite antigen recognized by the monoclonal antibody, Western blotting was performed as shown below. First, 100 μg of Eimeria tenella sporozoite antigen obtained by the method described in (1) above was subjected to SDS-polyacrylamide gel electrophoresis according to Laemmli's method (Laemmli UK, Nature 227, 680-685, 1970). Cellular proteins were developed according to molecular weight. Next, the protein developed on the polyacrylamide gel is electrically applied to a hydrophobic filter membrane (pore diameter 0.22 μm) wetted with a transfer buffer (10% methanol, 0.025 M Tris, 0.192 M glycine). Blotted. This filter membrane was washed 3 times with PBS-TW and then immersed in Block Ace at room temperature for 1 hour to block the protein non-adsorbed portion of the membrane. After washing three times with PBS-TW, the membrane was immersed in ascites containing each monoclonal antibody diluted 1,000 times with PBS, and incubated at room temperature for 1 hour. After washing 3 times with PBS-TW, the membrane was immersed in biotinylated anti-mouse immunoglobulin diluted 1,000 times with PBS and incubated at room temperature for 1 hour. After washing 3 times with PBS-TW, the membrane was immersed in avidin-bound alkaline phosphatase diluted 1,000 times with PBS and incubated at room temperature for 1 hour. After washing 3 times with PBS-TW, the membrane was immersed in 5-bromo-4-chloro-3-indolyl phosphate (BCIP) and nitroblue tetrazolium chloride (NBT), which are chromogenic substrates for alkaline phosphatase, and at room temperature. Incubated for 10 minutes. As a result, the monoclonal antibody produced by 4C8 reacted with a protein having a molecular weight of about 50 kilodaltons. From this result, it was estimated that this protein having a molecular weight of about 50 kilodaltons is a protective antigen for Eimeria tenella.

Claims (9)

A monoclonal antibody that reacts with Coccidium parasite Eimeria tenella and can inhibit the entry of Coccidium parasite Eimeria tenella into chicken cells. The monoclonal antibody according to claim 1, wherein the subclass is IgG1. The hybridoma producing the monoclonal antibody according to claim 1 or 2, produced by fusing a spleen cell obtained from a mouse immunized with a sporozoite stage worm of the coccidial parasite Eimeria tenella and a myeloma cell of the mouse. The following hybridomas (1) to (5).
(1) 1G4 (FERM AP-20811)
(2) 3A5 (FERM AP-20812)
(3) 4C8 (FERM AP-20813)
(4) 7B12 (FERM AP-20814)
(5) 9D6 (FERM AP-20815) A method for identifying an antigenic protein derived from the coccidial parasite Eimeria tenella using the monoclonal antibody according to claim 1. A compound derived from the coccidial parasite Eimeria tenella recognized by the monoclonal antibody according to claim 1. 7. The compound according to claim 6, wherein the compound derived from Coccidia parasite Eimeria tenella is a protein. The compound according to claim 6 or 7, which is obtained by the method according to claim 5. A coccidial protozoan Eimeria tenella infection therapeutic agent comprising the monoclonal antibody according to claim 1 or 2 as an active ingredient.