TW201610158A - Hybridoma cell line producing monoclonal antibodies against the ricin, the monoclonal antibodies therefrom, and reagent and ELISA kit comprising the same - Google Patents

Abstract

The present invention provides a hybridoma cell line producing monoclonal antibodies against ricin, the monoclonal antibodies against ricin therefrom, reagent and kit for ELISA, wherein the hybridoma cell line is produced from a parental cell and a myloma cell, and the parental cell is from spleen of the mouse that is immunized by an antigen which is ricin; wherein the hybridoma could produce a monoclonal antibody against A chain of ricin (which is comprising the sequence of SEQ ID NO.1).

Description

Castor bean toxin fusion tumor cell strain, monoclonal antibody thereof, and immunodetection reagent and kit containing the same

The present invention provides an immunodetection reagent and a kit capable of producing a fusion tumor cell strain, a monoclonal antibody, and an antibody comprising the monoclonal antibody, wherein the monoclonal antibody is a monoclonal antibody against ricin.

Ricin is a toxin extracted from a plant called ramie, also known as ricin. The average lethal dose of this toxin to humans is 0.2 mg, but there are some literatures with higher data. This toxin inhibits the process of protein synthesis and damages the organism. In addition, related studies have shown that only 8 toxins from castor seeds can cause fatal toxicity to an adult.

The ricin is a heterodimeric glycoprotein comprising two protein chains with different amino acid sequences, weighing about 32 kDA and 34 kDA, respectively, and the two protein chains are linked by a disulfide bond.

The two protein chains are as follows: A chain (A chain; EC 3.2.2.22) contains 267 amino acids, an N-glycoside hydrolase that cleaves a covalent bond on 28S rRNA. Remove an adenine. Since 28S rRNA is the main substance that constitutes ribosomes, and ribosomes are required for the synthesis of proteins, when rRNA is destroyed, protein synthesis is inhibited; B chain (B chain; EC 3.2.2.22) is an agglutination Prime, its fourth The residue and the A chain are bonded by a disulfide bond. This chain binds to the galactose residue or mannose residue on the cell membrane, assisting the A chain to pass through the cell membrane, enter the cell and act.

Castor bean toxin is one of immunotoxins and is a highly toxic biological toxin that binds to specific molecules on the cell membrane. Human poisoning can be caused by respiratory, oral, wound infection and other routes. It is estimated that the dose of about 1-10 ug/kg body weight can be caused by the path of breathing or injection. The symptoms of poisoning vary depending on the amount of contact and the way of contact. Inhalation of ricin may not cause poisoning symptoms after 8 hours, first of all, respiratory distress, fever, cough, nausea and chest tightness, followed by excessive sweating and pulmonary edema, and may eventually die in hypotension and respiratory failure. Swallowing may cause symptoms within 6 hours, including blood vomiting and diarrhea, and detoxification of the poisoned person, may die of organ failure in a few days, if the poisoned person has not died after 3 to 5 days, usually Will recover. During the First World War, the US military extracted castor bean toxins and used them for military purposes, including the use of ricin toxin as a raw material or covering ricin toxin in the ammunition shell. In addition, the Soviet Union used ricin toxins as a chemical weapon during the Cold War.

Currently, castor bean toxin has been listed as a Class B biological weapon by the US Centers for Disease Control and Prevention, and it takes a while to show symptoms after poisoning, and many symptoms are similar to other diseases, so the poisoned person knows that he is a nettle. Toxin poisoning often exceeds the gold treatment period. Therefore, the preparation and screening of monoclonal antibodies to castor bean toxin can help establish a rapid ricin toxin detection technology to avoid the golden treatment period of sesaminogen poisoning.

Accordingly, the present invention contemplates providing a fusion tumor of ricin toxin, a monoclonal antibody, and a test kit/method thereof, which can rapidly detect whether or not the castor bean is poisoned.

The invention provides a fusion tumor cell line, which is deposited in the Food Industry Research Institute of the Foundation of the Republic of China on June 17, 103, and its registration No. BCRC960487, the fusion tumor cell line is prepared by fusing a parental cell with a myeloma cell line, which is immunized to the mouse by using the ricin toxin as an antigen, and one of the mice The spleen cells are obtained, and the fusion tumor cell strain can produce a monoclonal antibody to specifically recognize the A chain of the ricinus toxin.

The present invention also provides a monoclonal antibody, which is capable of specifically recognizing a ricin toxin A chain having the protein sequence of SEQ ID NO. 1 and having no cross reaction with other similar ricin toxins, for example: chicken mother Bead toxin.

The invention further provides an immunoassay reagent and an immunoassay kit, which are used for detecting an antigen to be tested in an analyte by enzyme-linked immunosorbent assay, characterized in that the immunoassay kit contains the individual strain of the invention. An antibody, which specifically recognizes the A chain of a ricin toxin having the protein sequence of SEQ ID NO. 1, the immunoassay kit further comprising a detection antibody and a signal generating substance; the detection antibody and The signal generating substance is combined by various means. In a preferred embodiment, the signal is conjugated; the signal generating substance includes, but is not limited to, a radioactive label, a phosphorescent label, a luminescent label, and a fluorescent light. In the preferred embodiment, the luminescent label is a chemical luminescent label or a biological luminescent label; the enzyme includes, but is not limited to, Hydrogen Peroxidase, Horseradish Peroxidase (Horseradish Peroxidase, HRP for short), Alkaline Phosphatase (AP), and Beta-galactosidase are preferred. In the embodiment, it is a Horseradish Peroxidase (HRP); the immunoassay kit further comprises a substrate capable of reacting with the enzyme to cause a color reaction. The immunoassay kit of the present invention comprises various substances of the prior art, including, but not limited to, phosphate buffer solution, Tween 20 buffer solution, barrier reagent such as bovine serum albumin, casein, animal gelatin, microplate, hybrid membrane, test paper. Wait.

Preferably, the aforementioned mouse is a BALB/c strain mouse.

Preferably, the aforementioned myeloma cell line is derived from FO myeloma Cell line (CRL-1646 cell line).

1‧‧‧hard body support

10‧‧‧sample pad

20‧‧‧Conjugated pad

30‧‧‧antibody coated membrane

40‧‧‧Absorption pad

50‧‧‧Single antibody A (which recognizes the heavy chain of botulinum toxin type A)

60‧‧‧ Antibody B (which identifies the heavy or light chain of botulinum toxin type A)

70‧‧‧flat plastic case

71‧‧‧Interpretation area window

72‧‧‧Check window

80‧‧‧ Horseradish Peroxidase (HRP)

90‧‧ botulinum toxin type A

100‧‧‧lateral-flow strip

200‧‧‧ solid phase adsorption layer

Figure 1 shows the detection of castor bean toxin by the monoclonal antibody of the present invention using a lateral chromatograph.

Figure 2 is a lateral chromatograph for detecting ricin toxin of the present invention.

Figure 3 is a graph showing the results of detecting the different concentrations of castorago toxin by the monoclonal antibody of the present invention using a lateral chromatograph.

Figure 4 shows the detection of 100 ng/mL of ricin toxin, 100 ng/mL of sylvestrein (Abrin), 100 ng/mL of botulinum A using a single-stranded antibody of the present invention. Results of type toxin, 100 ng/mL of Botox type B toxin.

Fig. 5 is a diagram showing the results of detecting ricin added to milk, coffee, cola, juice, and tea using the monoclonal antibody of the present invention using a lateral chromatograph.

Fig. 6 is a schematic diagram showing the detection of ricin toxin by the monoclonal antibody of the present invention by immunoassay (ELISA).

Figure 7 is a method for detecting 1 to 1000 ng/mL of castoragotoxin, 1 to 1000 ng/mL of arboreal toxin (Abrin), 1 to 1000 ng by immunoassay (ELISA) using the monoclonal antibody of the present invention. Results of /mL of Botox type A toxin and 1 to 1000 ng/mL of Botox type B toxin.

The present invention discloses a fusion tumor cell strain, a monoclonal antibody thereof, and an immunological reagent kit prepared by using the same monoclonal antibody, wherein The related immunological principles, cell culture, staining, and protein detection techniques have been known to those of ordinary skill in the art, and therefore will not be fully described in the following description.

The monoclonal antibody against castor bean toxin produced by the present invention, the monoclonal antibody against ricinus toxin, the fusion tumor cell strain capable of producing the monoclonal antibody against ricinoleic acid, and the immunodetection reagent and the kit of castor bean toxin are embodied. For example, below.

1. Preparation of castor bean toxin: The present invention uses castor bean toxin as an antigen, and the preparation method is as follows:

(a) 200 g of the shelled castor bean was immersed in 5% acetic acid overnight, and then broken up by a juice machine, and the residue was removed by centrifugation to obtain a supernatant.

(b) The supernatant of the step (a) is added to a sulfated amine to a concentration of 60% to obtain a precipitated protein, which is then centrifuged to obtain a precipitate.

(c) The phosphate buffer solution was added to the precipitate in step (b) to completely dissolve it, and the dissolved protein was dialyzed against a phosphate buffer solution.

(d) The sepharose 6B (5 x 24 cm) chromatography column was first equilibrated with a phosphate buffer solution. The protein after dialysis in step (c) is then injected into a sepharose 6B chromatography column, followed by washing the column with 1-1.5 liters of phosphate buffer.

(e) The protein adsorbed on the sepharose 6B chromatography column was washed with 200 mL of a phosphate buffer solution containing 0.1 M galactose.

(f) The protein obtained in the above step (e) was concentrated to 5 mL by a centrifugal concentration tube.

(g) Sephacryl S-200 (5x60cm) molecular sieve chromatography column was first balanced with phosphate buffer solution. The protein concentrated in step (f) was injected into a pre-equilibrated sephacryl S-200 chromatography column.

(h) The column was washed with a phosphate buffer solution, and proteins of different absorption peaks were collected.

(i) Analyze the protein purity of different absorption peaks in step (h) by 8% SDS-PAGE electrophoresis. The ricin toxin containing 66KDA was taken and 100 ug was dialyzed against 1% formalin-phosphate solution for 10 days. A ricin toxin is used as an antigen for the present invention.

2. Preparation of a fusion cell strain of mouse immunization and anti- ricin toxin monoclonal antibody:

(a) Three BALB/c mice were taken, 0.2 mL of Complete Freund's adjuvants and the castor bean toxoid prepared in Example 1 were made into an emulsion, and each was administered by intraperitoneal injection. Mice were injected with 10 μg of castor bean toxoid.

(b) After 14 days, 0.2 mL of incomplete Freund's adjuvants and the castor bean toxoid prepared in Example 1 were made into emulsion A, and 3 BALB/ were separately administered by intraperitoneal injection. c mice in vivo. On the 14th day and the 28th day, the emulsion A was additionally applied and the blood of the mice was collected by eye socket blood collection. The blood of the mice was allowed to stand and centrifuged to obtain serum, and then the antibody price was changed by ELISA. 1:32000, 10 μg of castor bean toxin prepared in Example 1 was additionally applied, and cell fusion test was performed five days later.

(c) After the spleen cells of the mouse and the FO myeloma cell line (CRL-1646 cell line) were subjected to cell fusion, the fusion tumor cell line was selected by immunosorbent assay ELISA and Western blotting method to obtain the fusion tumor of the present invention. Cell line.

(d) Myeloma cell culture: One week before the mouse was scheduled to be fused, FO myeloma cells (Myeloma cells, CRL-1646 cell line) previously stored in liquid nitrogen were taken out, and the cells were thawed. The cell preservation bottle was taken out from the liquid nitrogen, quickly placed at 37 ° C to warm, until the cells were nearly completely dissolved, suspended in 10 ml of serum-free RPMI 1640 medium, centrifuged at 800 rpm for 5 minutes, and then the supernatant was removed to 10 ml. The osteosarcoma cells were suspended in RPMI 1640 culture medium containing 10% fetal calf serum, and cultured in a 25 cm ² cell culture flask (25T, cell culture flask), and cultured in an incubator containing 5% CO2. After 16-24 hours, after it was overgrown with the cell culture flask, it was subcultured to a 75T cell culture flask and continued for one to one passage to maintain the growth activity of the cells.

(e) Cell fusion: After sacrificing the mouse by cervical dislocation, the chest and abdomen were wiped with alcohol and placed on an aseptic table. Use a sterile instrument to remove the fur and open the muscles to open the abdominal cavity, remove the spleen, place it in a sterile culture dish containing serum-free RPMI 1640 medium, and aspirate the RPMI 1640 medium with a 10 mL syringe and repeat the injection of RPMI 1640 medium. The spleen of the mouse rushes out of the spleen cells. The treated spleen cells were centrifuged at 400 x g for 10 minutes. Discard the supernatant and repeat the above action once to remove the impurities. The well-activated myeloma cells were photographed from the culture flask, centrifuged at 400 x g for 10 minutes, and the supernatant was removed, and the serum-free RPMI 1640 culture solution was suspended. This operation was repeated twice in total. 5×107 myeloma cells and treated mouse spleen cells were mixed, centrifuged at 800 x g for 5 minutes, and the supernatant was discarded. This action was repeated once. The centrifuged cells were knocked loose, 1 ml of 50% PEG 1500 was added in 60 seconds, shaking was continued for 60 seconds, then 1 ml of RPMI 1640 medium containing 10% serum was added in 60 seconds, and 9 ml was added in the next 2 minutes. 10% serum of RPMI 1640 medium was centrifuged at 400 xg for 5 minutes. The supernatant was discarded, and the fusion tumor cells were suspended in 200 ml of 20% serum and 10% OPTI-CLONE (ICN Biomedicals)-HAT-RPMI 1640 medium, and added to a 96-well microplate (Microwell-plate) for fusion. Tumor cell culture.

(f) Culture of fusion tumor cells: The cells obtained by fusion were cultured in a 96-well microplate, and a total of ten dishes were taken, and the cells were changed as needed after 5-7 days. When changing the liquid, 80-100 μL is taken out, and a new RPMI 1640 medium containing HAT and serum is added as the principle of not exceeding one-half of the original volume.

(g) Screening of fusion tumors: further analysis of whether the fusion tumor cells have antibody production against ricin toxin is fusion tumor screening. The method used was as follows: Enzyme-Linked Immunosorbant Assay (ELISA), Western blotting (Western blot).

(h) Enzyme-linked immunosorbent assay (ELISA) is used to detect fusion tumor cells having the ability to secrete antibodies. First, the ricin obtained in Example 1 was diluted to 1 μg/ml with a coating buffer (15 mM Na2CO3, 35 mM NaHCO3, 0.02% NaN3, pH 9.6), and then added to a 96-well ELISA immunoassay plate (100 μl/well). Place at 4 ° C overnight to allow ricin to bind to the ELISA immunoassay plate. The next day, after washing the ELISA immunoassay plate three times with PBST (containing 0.1% Tween 20 in PBS), 1% BSA (dissolved in PBST) (200 μl/well) was added for blocking to cover the antigen-free binding. Vacancies (37 ° C, 1 hour).

(i) After the immunoassay plate was washed three times with PBST, the fusion tumor cell culture solution (50 μl/well was a grade 1 antibody) was added, and the reaction was carried out at 37 ° C for 1 hour. The immunoassay plate was washed five times with PBST, and a horseradish peroxidase (HRP) marker level 2 antibody (goat anti-mouse diluted 1:5000 in 1% skim milk) was added (50 μl/well). ), reacted at 37 ° C for 1 hour. After the final wash immunoassay plate with PBST five times, was added 100μl / well coloring agent 3,3,5,5-tetramethylbenzidine (3,3 ', 5,5' -tetramethylbenzid ine , of TMB for short), in The reaction was carried out at 25 ° C for 15 minutes. Thereafter, the reaction solution was terminated by adding a 0.16 M sulfuric acid solution to terminate the solution, and the absorbance at 450 nm (OD450) was measured by an enzyme immunoassay analyzer.

(j) In order to further analyze the specificity of the secreted antibody in the fusion tumor cells, and determine that it can react with ricin, it will be immunoassay by enzyme (ELISA), a fusion cell strain with reaction was analyzed by Western blotting. First, ricin was separated by protein electrophoresis after sodium dodecyl sulfate-polypropylene guanamine colloid (SDS-PAGE), and then transferred to nitrocellulose paper (NC paper) for analysis by Western blotting method. .

(k) Single cell formation of fusion tumor cells (Accession No. 8C12.1): A fusion tumor cell line having an antibody against ricin toxin was used by a limiting dilution method. A fusion tumor cell line having anti-ricin antibody will be taken from 30 cells and 200 ml of HT RPMI 1640 medium containing 20% serum and fusion tumor reaction reagent (Roche), and cultured in a 96-well microplate for 10-14 days. Observe whether there is a single plant formation.

3. Preparation of monoclonal antibodies against castor bean toxin:

(a) 0.5 mL of pristane was injected into the peritoneal cavity of the mouse, and after 7 days, 5 x 106 to 1 x 107 of the aforementioned fusion tumor cells of the present invention (Accession No. 8C12.1) were injected into the peritoneal cavity of the mouse, about 7 to 14 After the day, the abdomen of the mouse will gradually enlarge, and then ascites will be collected every 2 to 3 days, about 2 mL each time, and about 5-10 mL of ascites can be collected from each mouse.

(b) After collecting the collected ascites at 1500 rpm for 10 minutes, the supernatant was collected and filtered through a 0.45 μm filter to obtain a solution B.

(c) Protein G-sepharose was equilibrated with 50 mM Tris buffer pH 7.0, then solution B was passed through a column of protein G-sepharose, and purified by 5 times purification volume of 50 mM Tris buffer at pH 7.0. The solution was washed with water and washed with 0.1 M, pH 3.0 citrate buffer, and the single antibody was washed out and neutralized with 1 M, pH 9.0 Tris-HCl buffer, and stored, and the result was obtained. The ricinus toxin monoclonal antibody was invented.

4. Detection of ricin toxin by the monoclonal antibody of the present invention: using the monoclonal antibody of the present invention to detect ricin toxin, which can be Figure 2 shows.

(a) The monoclonal antibody 8C12.1 and Horseradish Peroxidase (HRP), which have the best capture ability, are formed into a solution C.

(b) Take 100 uL (100 ng/mL) of ricin toxin and solution C, and add it to the monoclonal antibody containing the present invention treated with 1% BSA phosphate buffer solution, and coat it in a 96-well dish. The reaction was carried out for 120 minutes at 37 ° C.

(c) The 96-well disc culture dish (i.e., the solid phase carrier) of the above step (2) was washed 5 times with a phosphate buffer solution containing 0.05% Tween-20 at pH 7.4, and then 100 uL of the color former 3, 3 was added. 5,5-Tetramethylbenzidine (3,3',5,5'-tetramethylbenzidine, or TMB for short) was reacted at 25 ° C for 15 minutes. Thereafter, 100 uL of a stop solution of 0.16 M sulfuric acid solution was added to terminate the reaction, and the color reaction supernatant was aspirated and added to the above 96-well plate, and the absorbance at a wavelength of 450 nm was read by an ELISA reader.

5. Immunological detection reagent containing monoclonal antibody against ricinus toxin: The present invention has the ability to detect ricin with an anti-rice toxin monoclonal antibody, which can be verified by lateral chromatographic detection. As shown in the first figure, in the lateral chromatography tester 100, a single antibody A (which can recognize the A chain of ricin) is coated on an antibody coated membrane 30, and the colloidal gold is added. The colloid gold particles are combined with the anti- ricin toxin single/multiple antibodies and added to the conjugated pad region.

Five of the above-mentioned processed lateral chromatographic testers 100 were placed, and a phosphate buffer solution (without ricin toxin as a control group), 10 ng/mL, 50 ng was added to the sample pad 10, respectively. /mL, 100ng/mL, 500ng/mL, the liquid will move to the absorption pad 40, and the detection result will be observed. The result is shown in Fig. 2. The C region is a control stripe, and the T region is a streak displayed by the combination of the castor bean toxin in the sample and the monoclonal antibody of the present invention. As the concentration of the castor bean toxin is higher, the fringes of the T region are deeper, indicating that the monoclonal antibody of the present invention can specifically recognize the castor bean toxin.

Similarly, five of the above-mentioned processed lateral chromatographic testers 100 were placed, and a phosphate buffer solution (as a control group), 100 ng/mL of ricin, and 100 ng/s were respectively added to the sample pad 10. mL of chicken arboreal protein (Abrin), 100 ng/mL of botulinum toxin type A toxin, and 100 ng/mL of botulinum toxin type B toxin. The results are shown in Fig. 3. The results show that only ricinus toxin is used as The sample showed streaks in the T region, indicating that the monoclonal antibodies of the present invention can specifically recognize the ricin toxin.

Next, in the same manner, five of the processed lateral chromatographic testers 100 were taken, and 100 ng/mL of milk mixed with castor bean toxin, 100 ng/mL of coffee, and 100 ng were respectively added to the sample pad 10. /mL of cola, 100ng/mL of juice, 100ng/mL of tea, the results shown in Figure 4, the results show that milk, coffee, cola, juice, tea can be detected to add added castor bean toxin, Among them, coffee color reaction is the strongest.

6. An immunoassay reagent containing an antibody against ricinus toxin monoclonal antibody: the present invention comprises an immunoassay reagent for anti-ricin drug monoclonal antibody, which can detect ricin toxin in a sample to be tested, in a preferred embodiment, The ricin is detected by a Sandwich ELISA. As shown in Figure 5, the immunoassay reagent contains a monoclonal antibody 50, which recognizes the A chain of ricin (TO SEQ ID NO. 1). And coated on the solid phase carrier 200 as a substance that captures ricin 90, and the identifiable monoclonal antibody 60 is linked to horseradish peroxidase (HRP) 80, which can be grasped by identifying castor bean toxin. Live ricin.

1 to 1000 ng/mL of ricin toxin, 1 to 1000 ng/mL of mutin (Abrin), 1 to 1000 ng/mL of botulinum toxin A, and 1 to 1000 ng/mL were detected by the above ELISA. The botulinum type B toxin was read at a wavelength of OD450 nm, and the results are shown in Fig. 6. As a result, only ricin toxin was detected by the immunodetection reagent of the present invention.

Further, the detection reagent of the present invention is used to detect the ricin toxin added to the commercially available beverage, and the results are shown in Table 1. The results show that whole milk, 5% skim milk, yogurt, 10% honey, 10% Coca-Cola, 10% oatmeal, 10% tomato sauce, 10% vegetable juice, 10% grape juice, and 10% orange juice can detect at least 1 ng/mL of additional added castor bean toxin.

Symbols :+++, OD.>1.0;++,OD.0.5~1.0;+,OD.0.10~0.49;-,OD.<0.1.

The detection reagent of the present invention is applied to a simulated clinical specimen to detect ricin toxin in a normal human body and a balb/c strain mouse, and the results are shown in Table 1, indicating that the volunteer who takes the normal body is from the forearm. Serum, normal urine and feces of venous blood collection: or serum obtained from the eye socket of mice, normal urine and feces, each diluted with phosphate buffer at a volume ratio of 1:10, added with fixed concentration of castor Toxins, detected by this test reagent, showed that the ten-fold dilution of serum, urine and feces in mice or humans did not inhibit the sensitivity of this assay (as shown in Table 2). Clinically simulated specimens do not produce false positive results in this assay.

Symbols :+++, OD.>1.0;++,OD.0.5~1.0;+,OD.0.10~0.49;-,OD.<0.1.

【Biomaterial Storage】 Domestic registration information [please note according to the registration authority, date, number order]

Depository institution: Food Industry Development Research Institute

Hosting date: June 17, 103

Registration number: BCRC960487

<110> National Defense Medical College

<120> A ricinus toxin fusion tumor cell strain, a monoclonal antibody thereof, and an immunodetection reagent and a kit comprising the monoclonal antibody

<160> 1

<170> PatentIn version 3.3

<210> 1

<211> 310

<212> PRT

<213> Ricinus communis

<400> 1

Claims (17)

A fusion tumor cell line, the accession number is BCRC960487, and the fusion tumor cell line is prepared by fusing a parental cell with a myeloma cell line, and the parental cell line is immunized by using a ricin toxin as an antigen. a mouse, and one of the spleen cells of the mouse is taken out, and the fusion tumor cell strain can produce a single antibody capable of specifically identifying a ricin (Ricin) A chain, and is similar to ricin toxin No non-specific reactions are produced. The fusion tumor cell line according to claim 1, wherein the A chain of the ricin (Ricin) has the protein sequence of SEQ ID NO. The fusion tumor cell line of claim 1 is a BALB/c strain mouse. The fusion tumor cell line according to claim 1, wherein the myeloma cell line is derived from a FO mouse myeloma cell line CRL-1646. A ricin toxin monoclonal antibody, which is produced by the fusion tumor cell strain according to any one of claims 1 to 4, and is capable of specifically identifying a ricin (Ricin) The A chain does not produce a non-specific reaction to ricin toxin. A monoclonal antibody according to claim 5, wherein the A chain of the ricin (Ricin) has the protein sequence of SEQ ID NO. An immunoassay reagent for detecting an antigen to be tested by an enzyme-linked immunosorbent assay (ELISA), characterized in that the immunoassay reagent contains a monoclonal antibody as in claim 5 of the patent application. An immunoassay kit for detecting an antigen to be tested in an analyte by an enzyme-linked immunosorbent assay (ELISA), characterized in that the immunoassay kit contains a single plant as in claim 5 antibody. The immunoassay kit of claim 8 further comprises a detection antibody and a signal generating substance. For example, in the immunoassay kit of claim 9, the detection antibody and the signal generating substance are conjugated through a conjugate. For example, in the immunoassay kit of claim 9, the signal generating substance is selected from the group consisting of radioactive labeling, phosphorescent labeling, luminescent labeling, fluorescent labeling, and enzyme. The immunodetection kit of claim 11, wherein the luminescent marker is a chemical luminescent marker or a bioluminescent marker. The immunoassay kit according to claim 11, wherein the enzyme is selected from the group consisting of: Hydrogen Peroxidase, Horseradish Peroxidase (HRP) , alkaline phosphatase (Alkaline Phosphatase, abbreviated as AP) and Beta-galactosidase (Beta-galactosidase). The immunoassay kit of claim 17, further comprising a substrate capable of reacting with the enzyme to cause a color reaction. An immunoassay method for detecting by an enzyme-linked immunosorbent assay (ELISA), comprising the steps of: providing a solid phase carrier; providing a monoclonal antibody having a patent application scope a feature according to item 5; applying the monoclonal antibody to the solid phase carrier; providing the same antigen, the sample antigen system is specifically bound to the monoclonal antibody; and providing a sample to be tested, The sample to be tested is operatively immunoreactive with the sample antigen to form an immune complex on the solid phase carrier; and a detection reagent is provided, the detection reagent is operable to immunize the immune complex Reacting and generating a signal; and detecting the signal. For example, in the immunoassay method of claim 15, the sample antigen is derived from the toxin protein extracted from castor bean. For example, in the immunoassay method of claim 15, the detection reagent comprises a detection antibody and a signal generating substance.