TWI527903B - Hybridoma cell line producing monoclonal antibodies against the bont/a, the monoclonal antibodies therefrom, and reagent and elisa kit comprising the same - Google Patents

Description

Botox type A toxin fusion tumor cell line, monoclonal antibody thereof, and immunodetection reagent and kit containing the same

The invention provides a fusion detection cell strain, a monoclonal antibody, and an immunodetection reagent and a kit comprising the monoclonal antibody, wherein the monoclonal resistance system is a specific single against the botulinum toxin type A toxin. Strain antibody.

Botox botulinum is a Gram-positive bacterium that grows in normal temperature, low acid and anoxic environments. Botox is a very anaerobic spore-forming bacterium, and its toxin is the main causative agent. . Botox is widely distributed in anaerobic environments throughout the world, such as soil, vegetables, underwater sludge, animal waste, and spore-contaminated foods.

According to the antigenicity of the botulinum toxin produced, botulinum toxin can be divided into 8 serosubtypes of A, B, C, (Ca, Cb), D, E, F, G, and H. Type A toxin: Common human botulinum toxin poisoning, strong toxicity, can liquefy protein. Type B toxins: common in natural soils, less toxic, partially liquefied proteins. Type C toxin: It is only poisoned to poultry and livestock animals, does not cause disease to humans, and cannot liquefy proteins. Type D toxin: It is only poisoned to poultry and livestock animals. It is rarely pathogenic to humans and cannot liquefy proteins. Type E toxins: mainly exist in fish. Poisoning cases are often associated with fish and seafood, which can cause human poisoning and cannot liquefy proteins. Type F toxin: Founded in Denmark in 1958, the toxin is similar to the A and B types, partially liquefying the protein. G-type toxin: can liquefy protein, but does not cause disease to humans. H-toxin is new in 2013 The serotype was found and the pathogen was unclear. In human cases of botulism, most of the toxins of type A, B and E are common, very few are F and G, and types C and D are common in poultry, livestock or birds.

Among them, type A toxin is the most common toxin causing botulinum poisoning in humans, and because of its strong toxicity and can block the release of the neurotransmitter acetylcholine, the neuron conduction of the poisoned person is blocked, resulting in local or whole body Sexual paralysis and difficulty breathing. It is clinically known as botulism. If there is no good medical care, the mortality rate is quite high. Therefore, botulism cases often require rapid diagnosis and confirmation.

However, generally used methods for detecting botulinum toxin include PCR, ELISA, traditional pathogen isolation culture, and the like. Because botulism cases often require rapid diagnosis and testing, the isolation and toxin identification of traditional botulinum bacilli requires anaerobic culture and animal neutralization tests. The detection of serum toxins takes 5 days, and the bacterial culture of feces takes 14 hours. Days, and anaerobic operation technology requires a special anaerobic operation platform, rather than the general laboratory suction cabinet, and requires a specific anaerobic package for bacterial culture to prevent this air from entering the culture dish, so that the culture dish is maintained. The oxygen environment, so the process of detection is cumbersome and time consuming. However, in the case of botulism, if there is no immediate treatment with the correct antiserum and specific medical methods, it will cause a high mortality rate. In terms of the requirements of quarantine aging, rapid identification and immediate medical care, the traditional types of bacterial culture and identification methods can not meet this demand. The preparation and screening of individual antibodies help to establish rapid Botulinum toxin protein detection technology.

Accordingly, the present invention contemplates providing a fusion bacterium of type A botulinum toxin, a monoclonal antibody, and a test kit/method thereof for rapid detection of botulinum toxin type A poisoning.

In view of the above, the present invention provides a murine fusion tumor cell line which can produce a monoclonal antibody against Botox type A toxin (BoNT/ A ), which can be used in an enzyme immunoassay (ELISA) to detect food samples and Botox type A toxin in biological samples.

The present invention provides a fusion tumor cell line, and is deposited in the Food Industry Research Institute of the People's Republic of China on June 17, 103, the fusion tumor cell bank accession number is BCRC 960486, and the fusion tumor cell line is composed of a parent. The generation cell is prepared by fusing a myeloma cell line, which is obtained by immunizing a mouse with a botulinum A toxin as an antigen, and extracting one of the spleen cells of the mouse, and The fusion tumor cell strain can produce a single antibody capable of specifically identifying a botulinum toxin type A toxin (BoNT/A), and against Botox type B toxin (BoNT/B) and botulinum toxin E (BoNT) /E) Does not produce a cross reaction. This monoclonal antibody belongs to the IgG1 subtype.

The present invention also provides the aforementioned monoclonal antibody; preferably, the monoclonal antibody described above is capable of specifically recognizing the heavy chain of a botulinum toxin type A toxin having the protein sequence of SEQ ID NO.

The invention further provides an immunoassay kit for detecting an antigen to be tested in an analyte by enzyme-linked immunosorbent assay, wherein the immunoassay kit comprises the monoclonal antibody of the invention, which can be specific The heavy chain of a botulinum toxin type A toxin having the protein sequence of SEQ ID NO. 1 is identified.

Preferably, the immunoassay kit further comprises a detection antibody and a signal generating substance; the detection antibody and the signal generating substance are combined in various ways, and in a preferred embodiment, the conjugate is combined The signal generating substance includes, but is not limited to, a radioactive label, a phosphorescent label, a luminescent label, a fluorescent label, and an enzyme. In a preferred embodiment, the luminescent label is a chemical luminescent label or A biological luminescent label; the aforementioned enzymes include, but are not limited to, Hydrogen Peroxidase, Horseradish Peroxidase (HRP), Alkaline Phosphatase (AP), and Beta -Beta-galactosidase, in a preferred embodiment, is a Horseradish Peroxidase (HRP); the immunoassay kit further comprises a substrate, the receptor is compatible with The enzyme reacts to form a color reaction. The immunoassay kit of the present invention comprises various conventional substances Quality, including, but not limited to, phosphate buffer solution, Tween 20 buffer solution, barrier reagents, such as bovine serum albumin, casein, animal gelatin, microplate, hybrid membrane, test paper, and the like.

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

Preferably, the aforementioned myeloma cell line is derived from an NS-1 cell line.

The botulinum toxin type A toxin monoclonal antibody provided by the present invention, and the fusion tumor cell strain capable of producing the botulinum toxin type A toxin monoclonal antibody, and the immunoassay reagent and the kit for the botulinum toxin type A toxin provide a kind Rapid detection of botulinum toxin type A to avoid poisoning by organism infection.

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

Fig. 1 is a diagram showing the detection of botulinum type A toxin by the monoclonal antibody of the present invention using a lateral chromatograph.

Fig. 2 is a result of detecting a different concentration of botulinum toxin type A by the monoclonal antibody of the present invention using a lateral chromatograph (test paper).

Figure 3 is a side chromatograph (test paper) using the monoclonal antibody of the present invention to detect phosphate buffer solution (as a control), 100 ng/mL ricin, 100 ng/mL botulinum B Results of type toxin (BoNT/B), 100 ng/mL of Botox E-type toxin (BoNT/E), and 100 ng/mL of Botox type A toxin (BoNT/A).

Figure 4 shows the results of using a lateral chromatograph to detect 100 ng/mL Botox type A toxin in milk, ham, tofu, pickles, and juice using the monoclonal antibodies of the present invention.

Fig. 5 is a schematic diagram showing the detection of botulinum toxin type A by the antibody of the present invention by enzyme immunoassay (ELISA).

Figure 6 is a method for detecting serially diluted 1 to 1000 ng/mL of Botox type A toxin (BoNT/A) by using an enzyme immunoassay (ELISA). Results of 1 to 1000 ng/mL of Botox type B toxin (BoNT/B) and 1 to 1000 ng/mL of Botox type E toxin (BoNT/E).

The related art of immunological principle, cell culture, staining and protein detection according to the present invention can be understood by those skilled in the relevant art, and therefore, the description below will not be completely described.

The monoclonal antibody against botulinum type A toxin provided by the invention, and the fusion tumor cell strain capable of producing the botulinum toxin type A toxin monoclonal antibody, and the immunoassay reagent and the kit for the botulinum toxin type A toxin, The specific implementation is as follows.

An immunoassay kit for detecting an antigen to be tested in a sample to be tested by a sandwich enzyme-linked immunosorbent assay, characterized in that the immunoassay reagent contains the monoclonal antibody of the present invention.

1. Preparation of mouse immunization and anti-botulinum toxin type A antibody:

A fusion cell strain of monoclonal antibodies against botulinum toxin type A, mainly using type A toxin produced by botulinum strain (ATCC7948) as an immunizing antigen, and injecting BALB/c mice to produce an immune response Produce antibodies, then Monoclonal antibodies were obtained via fusion tumor cell lines, which were prepared by the following methods:

A. BALB/C mouse immunization

(a) Three BALB/c mice were taken, and 0.2 mL of Complete Freund's adjuvants and Botox type A toxoid were made into an emulsion, which was administered by intraperitoneal injection, and each mouse was injected. 10 μg of botulinum toxin type A.

(b) After 14 days, 0.2 mL of incomplete Freund’s adjuvants and Botox type A toxoid were made into emulsion A, which were administered by intraperitoneal injection. Into three BALB/c mice. 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, 1 μg of botulinum toxin type A was added, and cell fusion test was performed five days later.

B. Myeloma cell culture:

(a) One week before the mouse was scheduled to be fused, NS1 myeloma cells (Myeloma cells, P3-NS-A-Ag4/1, NS-1) previously stored in liquid nitrogen were taken out and subjected to cell thawing.

(b) The cell preservation bottle was taken out from the liquid nitrogen, quickly placed at 37 ° C, and the cells were nearly completely dissolved. The suspension was suspended in 10 mL of serum-free RPMI 1640 medium, centrifuged at 800 rpm for 5 minutes, and then the supernatant was removed. The osteoblast cells were suspended in 10 mL of RPMI 1640 medium containing 10% fetal bovine serum, cultured in a 25 cm 2 cell culture flask (25T, cell culture flask), and cultured in an incubator containing 5% CO2.

(c) After 16-24 hours, after being overgrown with the cell culture flask, the cells were subcultured to a 75T cell culture flask and continued for one to one passage to maintain the growth activity of the cells.

C. Cell fusion:

(a) After sacrificing the mouse by cervical dislocation, the chest and abdomen were wiped with alcohol and placed on an aseptic table.

(b) Using a sterile instrument to remove the fur and open the muscles to open the abdominal cavity, remove the spleen, place it in a sterile Petri dish containing serum-free RPMI 1640 medium, and aspirate the RPMI 1640 medium with a 10 mL syringe and repeat the RPMI 1640 The culture solution was injected into the spleen of the mouse, and the spleen cells were washed out.

(c) Treated spleen cells were centrifuged at 400 x g for 10 minutes. Discard Clear the solution and repeat the above action once to remove the impurities.

(d) 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 procedure was repeated twice in total.

(e) 5× ^{10 7} myeloma cells and treated mouse spleen cells were mixed, centrifuged at 800 xg for 5 minutes, and the supernatant was discarded. This action was repeated once.

(f) Knock the centrifuged cells, add 1 mL of 50% PEG 1500 in 60 seconds, continue to shake for 60 seconds, then add 1 ml of RPMI 1640 medium containing 10% serum for 60 seconds, for the next 2 minutes. 9 ml of RPMI 1640 medium containing 10% serum was added and centrifuged at 400 xg for 5 minutes.

(g) Discard the supernatant and suspend the fusion tumor cells in 200 mL of 20% serum and 10% OPTI-CLONE (ICN Biomedicals)-HAT-RPMI 1640 medium and add to the 96-well microplate (Microwell-plate). ) Perform fusion cell culture.

D. Culture of fusion tumor cells:

The cells after fusion were cultured in a 96-well microplate, and a total of ten plates were used. After 5-7 days, the cell growth was changed. 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.

E. Screening of fusion tumors:

(a) Further analysis of whether the fusion tumor cells have antibody production against botulinum toxin type A is fusion tumor screening. The method used was as follows: Enzyme-Linked Immunosorbant Assay (ELISA), Western blotting (Western blot).

(b) Using an enzyme-linked immunosorbent assay (ELISA) to detect the ability to secrete antibodies Fusion of tumor cells.

(c) First, the botulinum toxin type A 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). ), placed at 4 ° C overnight, so that botulinum toxin type A can be bound 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). After the immunoassay plate was washed three times with PBST, the fusion tumor cell culture solution (50 μL/well as 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, added 100μL / well coloring agent 3,3,5,5-tetramethylbenzidine (3,3 ', 5,5' -tetra methylbenzidine , 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.

(d) In order to further analyze the specificity of the secreted antibody, and determine that it can react with the light chain or heavy chain of botulinum toxin type A, it will be reacted by enzyme immunoassay. Tumor cell lines were analyzed by Western blot analysis. First, botulinum toxin type A, electrophoresis by sodium dodecyl sulfate-polypropylene guanamine colloid (SDS-PAGE) After the protein was removed, it was transferred to nitrocellulose paper (NC paper) for analysis by Western blotting.

(e) Washing the nitrocellulose paper three times with PBST (containing 0.1% Tween 20 in PBS), adding 1% BSA (dissolved in PBST), and blocking to cover the vacancies not bound by antigen (37 ° C, 1 hour). After the nitrocellulose paper was washed three times with PBST, the fusion tumor cell culture solution (1 mL/well as a grade 1 antibody) was added and reacted at 37 ° C for 1 hour. The nitrocellulose paper 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 (10 mL/well). ), reacted at 37 ° C for 1 hour. Finally, after washing the nitrocellulose paper five times with PBST, 10 mL/well of the color former was added, and the reaction was carried out at 25 ° C for 10 minutes.

Thereafter, deionized water was added to terminate the reaction, and the molecular weight of the antibody-bound toxin site was observed.

F. Fusion tumor cell monoculture:

(a) A fusion tumor cell line having an antibody against botulinum toxin type A will be subjected to a limiting dilution method.

(b) Take 30 HT RPMI 1640 broths, which will have fusion cells with botulinum toxin type A antibody, plus 200 ml of 20% serum and fusion tumor reaction reagent (Roche), each added to a small 100 μl Mouse spleen cells or macrophages were used as feeder cells, and cultured in a 96-well microplate, and monogenic formation was observed for 10-14 days.

(c) The monogenic cell population obtained after 10-14 days is the fusion tumor cell of the present invention (Accession No. BCRC 960486).

G. Characterization of individual antibody types:

(a) Monoclonal resistance produced by spleen cell fusion tumors of pathogenically immunized mice The species or subspecies of the body will be determined using the mouse fusion tumor subspecies component Iso-strip (Roche).

(b) Iso-strip is used to distinguish the individual antibodies from IgA, IgG1, IgG2a, IgG2b, IgG3 or IgM in the mouse antibody class, and the detailed steps will be based on the manufacturer's manual.

H. Production of ascites:

0.5 mL of pristane was injected into the peritoneal cavity of the mouse, and after 7 days, 5× ^{10 6} to 1×10 ^{7 of the} aforementioned fusion tumor cells of the present invention (Accession No. BCRC 960486) were injected into the peritoneal cavity of the mouse, about 7 to 14 days later. 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. The ascites was centrifuged at 4500 rpm for 10 minutes, and the supernatant was taken and dispensed into a 1.5 mL vessel, and stored in an environment of -20 ° C.

I. Purification of monoclonal antibodies

Protein G-sepharose was equilibrated with proteinG IgG binding buffer (Thermo), then solution B was passed into the column of protein G-sepharose, washed with 5 times purified column volume of proteinG IgG binding buffer, and then with IgG Elution buffer. (Thermo) washed out the antibody, measured the protein concentration with Bio-Rad protein assay reagent, neutralized its pH to pH 7 with 1 M, pH 9.0 Tris-HCl buffer, and stored it in a divided form to obtain the type A meat of the present invention. Toxin-producing monoclonal antibody.

J. Characterization of cross-reactivity of individual antibodies to different antigens:

After the monoclonal antibody which has been determined to be antigenic is purified by mass production, it is further determined by immunoblotting method whether or not the other antigens will cross-react. The main points are as follows: different pathogens are dissolved in the dissolution buffer, and the proteins are separated by SDS-PAGE and transferred to Nitrocellulose membrane (Hybond-C Super; Amershan), containing 5%. After the non-specific binding of the skim milk powder PBS was blocked, the goat monoclonal antibody and the HRP enzyme-conjugated goat anti-mouse immunoglobulin were reacted, and the 4-chloro-1-naphthol color was changed.

2. Detection of Botox type A toxin by the monoclonal antibody of the present invention:

(a) Single antibody 7-3.2 was plated in a 96-well disc culture dish, which was produced by fusion tumor cells (registered label BCRC 960486).

(b) 100 uL (100 ng/mL) of Botox type A was added to a 96-well plate culture dish containing the monoclonal antibody of the present invention treated with 1% BSA phosphate buffer solution, and placed in a 37 ° C environment for 60 minutes. .

(c) Wash 5 times with a phosphate buffer solution containing 0.05% Tween-20 at pH 7.4.

(d) adding other different strains of botulinum toxin type A antibody and horseradish peroxidase (HRP) to form solution C, and adding solution C to the 96-well plate of the above reaction, and placing it at 37 ° C Reaction for 60 minutes.

(e) The 96-well disc culture dish (i.e., the solid phase carrier) of the above step (d) 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.

3. Immunoassay reagent containing monoclonal antibody against botulinum A-type toxin:

The invention contains an anti-botulinum toxin type A toxin monoclonal antibody 7-3.2 The ability to detect botulinum type A toxins can be verified by lateral chromatographic detection.

Five of the above-mentioned processed lateral chromatographic testers 100 were placed on a sample pad 30 with a phosphate buffer solution (without botulinum A-type toxin as a control group), 10 ng/ mL, 50ng/mL, 100ng/mL, 500ng/mL of botulinum toxin type A toxin 100uL, the test liquid will move to the absorption pad 40 (absorption pad), wait for 10 minutes and observe the detection result, the result is the second The figure shows. The C segment is a control stripe, and the T segment is a streak displayed by the botulinum toxin type A toxin in the sample combined with the monoclonal antibody of the present invention. The higher the concentration of the botulinum toxin type A toxin, the deeper the streak of the T segment, indicating that the monoclonal antibody of the present invention can specifically recognize the botulinum toxin type A 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, 100 ng/s were respectively added to the sample pad 30. mL of Botox type B toxin (BoNT/B), 100 ng/mL of Botox E (BoNT/E), 100 ng/mL of Botox type A (BoNT/A), the result is shown in Figure 3. As shown, the results showed that only the botulinum toxin type A toxin appeared as a sample in the T segment, indicating that the monoclonal antibody of the present invention can specifically recognize the botulinum toxin type A toxin.

Next, in the same manner, five of the processed lateral chromatographic testers 100 were taken, and 100 ng/mL of type A toxin milk, ham extract, and tofu extract were respectively added to the sample pad 30. 100 ng/mL kimchi extract and juice, the results are shown in Figure 4, the results show that milk, ham, tofu, kimchi, juice can be detected to contain Botox type A toxins, and The reaction line of tofu extract is the most obvious.

4. Immunoassay reagent containing monoclonal antibody against botulinum A type toxin:

The invention contains the anti-botulinum A toxin monoclonal antibody The plaque detection reagent can detect the botulinum toxin type A toxin in the sample to be tested. In the preferred embodiment, the botulinum toxin type A toxin is detected by sandwich immunoassay (Sandwich ELISA), and the schematic diagram is shown in FIG. The immunoassay reagent comprises a monoclonal antibody A 50 which recognizes the heavy chain of the botulinum type A toxin (SEQ ID NO. 1) and is coated on the solid phase carrier 200 as a botulinum toxin type A toxin 90 substances.

Detection of serially diluted 1 to 1000 ng/mL of Botox type A toxin (BoNT/A) and 1 to 1000 ng/mL of Botox type B toxin (BoNT) by the above-mentioned ELISA /B), 1~1000ng/mL of Botox E-toxin (BoNT/E), and read the value at OD450nm wavelength. The results are shown in Figure 6. The results show that only Botox A The toxin can be detected by the immunodetection reagent of the present invention.

In addition, the ELISA detection reagent of the present invention is used to detect the content of botulinum A in the market, and the results are shown in Table 1. The results show that pudding, yogurt, ham, hot dog, canned squid, dried bean curd can be used. The botulinum toxin type A toxin was measured at 1,000 pg/mL.

【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: BCRC960486

<110> National Defense Medical College

<120> Botulinum A-type toxin fusion tumor cell strain, monoclonal antibody thereof, and immunodetection reagent and kit comprising the monoclonal antibody

<160> 1

<170> PatentIn version 3.3

<210> 1

<211> 448

<212> PRT

<213> Clostridium botulinum

<400> 1

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

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

80‧‧‧ Horseradish Peroxidase (HRP)

90‧‧ botulinum toxin type A

200‧‧‧ solid phase adsorption layer

Claims (17)

A fusion tumor cell line having the accession number BCRC 960486, the fusion tumor cell line being produced by fusing a parental cell with a myeloma cell line, the parental cell line comprising a botulinum toxin type A ( BoNT/A) is immunized to a mouse as an antigen, and one of the spleen cells of the mouse is taken out, and the fusion tumor cell strain can produce a monoclonal antibody capable of specifically identifying a botulinum toxin type A ( The amine end of the heavy chain of BoNT/A). The monoclonal antibody produced by the fusion tumor cell line can specifically recognize the heavy chain of a botulinum toxin type A toxin (BoNT/A) having the SEQ ID of SEQ ID: Protein sequence of NO.1. The fusion tumor cell line of claim 1 is a BALB/c strain mouse. The fusion tumor cell strain according to claim 1, wherein the myeloma cell line is derived from a BALB/c mouse or an NS-1 cell. A botulinum toxin type A toxin (BoNT/A) monoclonal antibody, which is characterized in that the monoclonal antibody resistance system is produced by the fusion tumor cell strain according to any one of claims 1 to 4, and is capable of specificity. Identification of a botulinum toxin type A toxin (BoNT/A) and no non-specific reaction to botulinum toxin type A toxins. The monoclonal antibody of claim 5, which is capable of specifically recognizing the heavy chain of a botulinum toxin type A toxin (BoNT/A) having 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 comprises a monoclonal antibody of claim 5 of the patent application. An immunoassay kit for detecting an antigen to be tested in an analyte by enzyme-linked immunosorbent assay (ELISA), characterized in that the immunoassay kit contains a single plant of 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 a radioactive label, a phosphorescent label, a luminescent label, a fluorescent label, and an 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 11, 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 associated with the sample The antigen is subjected to an immune reaction to form an immune complex on the solid phase carrier; a detection reagent is provided, the detection reagent is operable to immunologically react with the immune complex, and generate a signal; and detecting the Signal. For example, in the immunoassay method of claim 15, the sample antigen is derived from Botox type A toxin (BoNT/A). For example, in the immunoassay method of claim 15, the detection reagent comprises a detection antibody and a signal generating substance.