KR102124258B1 - Staphylococcal enterotoxin B diagnostic detection kit using rapid immunochromatography, its specific antibody and antibody-producing cell lines - Google Patents

Abstract

The present invention is a Staphylococcal Enterotoxin B (Staphylococcal Enterotoxin B, SEB) specific monoclonal antibody, a novel cell line (#C1, 2d 25-2) producing the antibody and an immunochromatography kit for detecting SEB using the same It is about. The kit exhibits good sensitivity to SEB and no cross-reactivity to other pathogens and interferers.

Description

Staphylococcal enterotoxin B diagnostic detection kit using rapid immunochromatography, its specific antibody and antibody-producing cell lines} Staphylococcal enterotoxin B diagnostic detection kit using rapid immunochromatography

The present invention is a Staphylococcus aureus B antibody, a novel cell line for producing the antibody (#C1, 2d 25-2), and an antibody for detecting Staphylococcus aureus toxin B containing improved antibody and improved sensitivity and specificity Chromatography kits.

Staphylococcus aureus produces many exotoxins, one of which is Staphylococcal Enterotoxin B (SEB). The toxin is referred to as an exotoxin because it is a substance secreted from cells, and is also referred to as an enterotoxin because it mainly affects the intestine after ingestion. SEB is a fever toxin that is a common cause of food poisoning, and is produced by ingestion of poorly treated food and manifests itself by eating it. The toxin has various biological properties, and the symptoms that appear when exposed through the respiratory tract exhibit very different symptoms than those absorbed through the digestive tract. However, exposure to either route causes very serious symptoms in the human body.

SEB is a very common cause of food poisoning. When the respiratory system is exposed to this toxin, fewer people die from this toxin, but more than 80% of those exposed have clinical symptoms and the toxin was terminated in 1969 because it is difficult to move for a week or two. It was one of seven weapons developed by the American Biochemical Weapon Program.

In addition, SEB is a strong immune-stimulating super antigen. Superantigen directly stimulates a large number of T-lymphocytes by binding to the monocyte's MHC type II receptor, which rapidly produces inflammatory cytokines (tumor necrosis factor, interferon gamma, interleukin-1, interleukin-2). . The result is a very strong inflammatory response and tissue damage. The toxic effects of SEB are expected to occur due to secreted cytokines.

Symptoms of intoxication by SEB begin 3 to 12 hours after toxin inhalation and 4 to 10 hours after ingestion. Suddenly, fever, headache, chills, muscle pain, and dry cough appear. In severe cases, dyspnea and pain behind the sternum appear. When toxins are ingested, symptoms of the gastrointestinal tract usually appear, such as nausea, vomiting, and diarrhea. When toxins are inhaled, respiratory symptoms usually appear, such as dry cough, pain behind the sternum, and difficulty breathing.

As a respiratory pathology, inflammatory cytokines are generated in large quantities in the lungs, resulting in pulmonary edema and pulmonary edema. In severe cases, acute pulmonary edema and respiratory failure occur. Fever symptoms ranging from 39 to 41 degrees Celsius can last up to 5 days, coughing can last up to 4 weeks, and the patient is hampered for about 2 weeks. Physical examination findings are usually unclear. Orthostatic hypotension may occur due to conjunctival hyperemia or loss of fluid. Although chest scans have no specific findings, rare pulmonary edema may occur. In addition, in severe patients, interstitial shading increases, develops into atelectasis or pulmonary edema, or has adult respiratory distress syndrome.

Early diagnosis is difficult because SEB poisoning symptoms are similar to those caused by respiratory pathogens such as influenza, adenovirus, and mycoplasma. What is characteristic of inhalation poisoning by SEB is that multiple patients develop within a short period of time (24 hours). Naturally occurring pneumonia or influenza occurs over time.

Diagnosis can be made by detecting toxin antigens by ELISA. However, treatment of SEB addiction as of now is limited to conservative treatment. To relieve the symptoms of the patient, antipyretic acetaminophen and antitussives are administered.

There is currently no vaccine against SEB poisoning, and several vaccine candidates are under development. It is experimentally known that passive immunotherapy can reduce mortality when attempted within 4 to 8 hours after SEB inhalation. However, SEB experimentally binds to the MHC receptor within 5 minutes, so active immunotherapy should be considered for practical prevention.

Therefore, the development of a technology that can be quickly detected in the field in the event of a suspicion of a bioterrorism by the SEB is very important for promptly determining the existence of a terrorist situation and determining a response policy. To this end, the present inventor has developed a rapid detection kit based on immunochromatography technology for on-site detection of SEB toxin.

It is an object of the present invention to provide a SEB detection kit for on-site detection of Staphylococcal Enterotoxin B (SEB) for rapid response in a suspected bioterrorism situation. To this end, the present invention provides a SEB recognition antibody, a novel cell line (#C1, 2d 25-2) producing the antibody, and an immunochromatography kit for SEB detection using the same.

The present invention provides a monoclonal antibody characterized by specifically recognizing Staphylococcal Enterotoxin B (SEB).

The monoclonal antibody is generated by cell line #C1 deposited with accession number KCLRF-BP-00443 or cell line 2d 25-2 deposited with accession number KCLRF-BP-00444, specifically recognizing Staphylococcus aureus B, yellow It is characterized by.

The present invention relates to cell line #C1 deposited with accession number KCLRF-BP-00443 or cell line 2d 25-2 deposited with accession number KCLRF-BP-00444.

Specifically, the present invention comprises the steps of (i) obtaining a spleen of a mouse after immunization by injecting SEB into a mouse; (ii) fusing B lymphocytes and Myeloma cells obtained from the spleen; And (iii) relates to a cell line prepared by a method comprising the step of selecting the cells obtained above.

The present invention relates to a SEB detection kit comprising the monoclonal antibody. In the kit, the monoclonal antibody can be coated on the nitrocellulose membrane.

The kit may further include at least one of a sample pad and a moisture absorption pad.

The present invention can detect and detect SEB more quickly and accurately by providing an antibody with superior sensitivity and specificity than the antibody used in the existing Staphylococcal Enterotoxin B (SEB) detection kit. Specifically, the detection kit using the antibody of the present invention has superior sensitivity compared to conventional products, and has very good specificity since there is no cross reaction to other pathogens and toxins.

1 is a result of comparing and evaluating the sensitivity of a detection kit to which the novel antibody of the present invention is applied with an existing commercially available detection kit (product name: bioterrorism pathogen and toxin multi-detection kit 9, manufacturer: SD Co., Ltd.).
2 is a result of comparing and evaluating the presence or absence of a cross-reaction for 9 types of cells and toxins for specificity evaluation.
3 is a result of testing whether the detection kit is affected by the interference.
FIG. 4 shows the results of 20 sensitivity and specificity tests for each item to evaluate the reproducibility of the detection kit.

All terms used in the present invention, unless defined otherwise, are used in the sense as commonly understood by a person skilled in the art. In addition, although preferred methods, samples, and the like are described in this specification, similar or equivalent ones are included in the scope of the present invention.

In the present invention, the term, "antibody (antibody)" is a protein that specifically binds the antigen, for the purpose of the present invention is a monoclonal antibody that specifically binds Staphylococcal Enterotoxin B (SEB). .

The present invention relates to a monoclonal antibody that specifically binds SEB, produced by cell line #C1 deposited with KCLRF-BP-00443 or cell line 2d 25-2 deposited with accession number KCLRF-BP-00444.

Antibodies produced in cell line #C1 deposited with KCLRF-BP-00443 or cell line 2d 25-2 deposited with accession number KCLRF-BP-00444 are more antigen-friendly than antibodies used in the staphylococcus enterotoxin B detection kit. It is a novel antibody with high degree. Hybridoma cell lines capable of producing antibodies specific for the antigen can be cultured in large quantities according to standard techniques. The monoclonal antibody produced by the above hybridoma cell line may be used without purification, but it is preferred to use it after purification in high purity according to methods well known in the art.

The present invention relates to a SEB detection kit comprising the aforementioned antibody. SEB can be rapidly detected in a bioterrorism suspected situation using the kit containing the above antibody.

Such a detection kit may include the monoclonal antibody of the present invention, as well as tools, reagents, and the like commonly used in the art for immunological analysis. Such tools and reagents include, but are not limited to, suitable carriers, labeling substances capable of generating detectable signals, solubilizers, detergents, buffers and stabilizers, and the like.

Suitable carriers include soluble carriers such as physiologically acceptable buffers known in the art, such as PBS, insoluble carriers such as polystyrene, polyethylene, polypropylene, polyester, polyacrylonitrile , Fluororesin, crosslinked dextran, polysaccharide, polymer such as magnetic fine particles plated with metal on latex, other paper, glass, metal, agarose, and combinations thereof, but are not limited thereto.

Formation of antigen-antibody complexes include tissue immunostaining, radioimmunoassay (RIA), enzyme-linked immunoassay, western blotting, immunoprecipitation assay, immunoprecipitation assay, immunodiffusion assay ), Complement Fixation Assay, FACS, and protein chip (protein chip).

Labels that enable qualitative or quantitative measurement of antigen-antibody complex formation include, but are not limited to, enzymes, fluorescent substances, ligands, luminescent substances, microparticles, redox molecules, and radioactive isotopes.

Enzymes available as detection labels include β-glucuronidase, β-D-glucosidase, β-D-galactosidase, urease, peroxidase, alkaline phosphatase, acetylcholinesterase, glucose oxy Multidose, hexokinase and GDPase, RNase, glucose oxidase and luciferase, phospho fructokinase, phosphoenolpyruvate carboxylase, aspartate aminotransferase, phosphenol pyruvate, decarboxylase, β- Ratamases, and the like, but is not limited thereto.

Fluorescent materials include, but are not limited to, fluorescein, isothiocyanate, rhodamine, picoerytherin, phycocyanin, allopicocyanine, o-phthalaldehyde, fluorescamine, and the like.

Ligands include biotin derivatives and the like, and emitters include, but are not limited to, acridinium esters, luciferin, luciferase, and the like.

The microparticles include, but are not limited to, colloidal gold, colored latex, and the like.

In the kit of the present invention, the monoclonal antibody may be coated on a membrane used as a material for a conventional detection kit, and the membrane may contain various synthetic polymers such as nitrocellulose, cellulose, cellulose acetate, and polyethylene. Can be used.

The present inventors conducted tests by membrane type (by manufacturer, by pore size) and by antibody concentration for test vessels to select membrane and antibody concentrations with high sensitivity and specificity. As a result, the kit of the present invention was coated with an antigen-specific monoclonal antibody and an antibody for the control line, respectively, on the nitrocellulose membrane at the site of the test line and the control line.

The present invention removes non-specific reactions through treatment of animal serum such as cow and mouse serum through a sample pad test, and pre-treatment with a stabilizer such as BSA, Casein, etc. to select a pad composition optimized for selection conditions.

Hereinafter, the present invention will be described in detail by examples. However, the following examples are only for illustrating the present invention and the scope of the present invention is not limited thereto.

[Example 1]-Preparation of monoclonal antibodies

1. Immunity and cell fusion

For the production of monoclonal antibodies, the Staphylococcal enterotoxin B (Sigma, S4881) antigen purchased from Sigma-Aldrich was immunized with mice after making an emulsion with Freund's adjuvant. The mouse immunity was intraperitoneally immunized 4 to 8 times at intervals of 1 to 2 weeks at a concentration of 200 to 400 µg/dose, and the antigen was inoculated with the intravenous vein 1 to 3 days before cell fusion.

One day before cell fusion, whole blood was collected through the vein and used for ELISA of the monoclonal antibody. Spleens to be used for cell fusion were collected under sterile conditions and induced cell fusion with myeloma cells using polyethylene glycol (PEG 1500 reagent), followed by cell culture in HAT medium and HT medium.

2. Screening, multiple production and antibody purification

Fusion cells cultured in 96 wells were screened for positive clones without cross-reactivity through primary and secondary screening by ELISA. The resulting cell line was deposited with the Korea Cell Line Bank as of August 1, 2018 (Accession Nos. KCLRF-BP-00443, KCLRF-BP-00444). These cell lines were inoculated intraperitoneally into mice to produce ascites. When intraperitoneal ascites were produced 10-20 days after intraperitoneal inoculation, about 2-4 ml per ascites were collected per mouse. The collected ascites were centrifuged to remove blood cells, fibrin, etc. and used for purification. For the mouse multiple purification, mouse IgG, an antibody specific for protein G, was purified using Protein G chromatography gel. The mouse IgM was used for formulation by purifying IgM by dialysis and DEAE gel purification without using a specific gel affinity chromatography method. The purified monoclonal antibody candidate group was selected by screening using gold.

[Example 2]-Preparation of Staphylococcus aureus enterotoxin B detection kit

Antigen-specific antibodies and control antibodies selected at the test and control positions of the nitrocellulose membrane were coated, respectively. The gold conjugate to which the selected antibody was conjugated was dispensed on the pad, dried and cut to prepare a gold pad. A kit was prepared by attaching a membrane, a gold pad, a sample pad, and a moisture absorption pad to a plastic card using a laminator.

[Test Example 1]-Sensitivity (detection limit) test

To test the detection limit of Staphylococcal Enterotoxin B (SEB), Sensitivity was evaluated while diluting the concentration. Existing commercially available SD detection kit (product name: bioterrorism pathogen and toxin multi-detection kit 9, manufacturer: SD Co., Ltd.) and the sensitivity of the detection kit prepared in Example 2 were evaluated and compared, and the results are shown in FIG. 1.

As shown in FIG. 1, as a result of 10 times of repeated tests, the detection limit was measured to be 0.6 ng/ml, and it was confirmed that the sensitivity was improved by 8 times compared to 5 ng/ml of the existing product.

[Test Example 2]-Specificity test

For specificity evaluation, cross reactivity of 10 strains and strains such as fest, lysine and brucella was evaluated, and the results are shown in Tables 1 and 2 below.

As shown in Table 1 and FIG. 2, it was confirmed that no cross reaction with other samples occurred.

[Test Example 3]-Interference test

Ten predicted interferers, including bovine serum, human serum, sugar, wheat flour, soil, etc., which can affect the performance of the detection kit, were selected and specificity tests were conducted to confirm the presence or absence of interference.

The experimental results are shown in Table 2 and FIG. 3 below.

As shown in Table 2 and Fig. 3, it was confirmed that 10 kinds of interfering substances were not affected.

[Test Example 4]-Reproducibility test

A total of 20 sensitivity and specificity tests were conducted to confirm that the detection kit prepared in Example 2 was reproducible. The sensitivity was tested at the positive detection limit concentration, and for the specificity test, the sample dilution was tested as a negative sample.

The experimental results are shown in FIG. 4, and it was confirmed that all of them were reproducible.

[Test Example 5]-accelerated stability test

In order to set the expiration date of the detection kit prepared in Example 2, the strip was stored at 55°C for 7 weeks, and then taken out at intervals of 1 week, and a sensitivity and specificity comparison test was performed with the strip stored at room temperature.

The experiment was conducted with a limit of positive detection for the sensitivity test and a negative sample for the specificity test. As a result of the experiment, it was confirmed that there was no abnormality in both sensitivity and specificity after the 7-week accelerated stability test.

[Test Example 6]-Cross-reaction test

In order to confirm that the detection kit has a cross-reaction with other strains or toxins, a cross-test with 24 strains or toxins was performed. As shown in Table 3 below, it was confirmed that there was no crossing for the cross-reaction test strain and 24 kinds of substances.

Korea Cell Line Research Foundation KCLRFBP00443 20180801 Korea Cell Line Research Foundation KCLRFBP00444 20180801

Claims (8)

A monoclonal antibody produced by cell line #C1 deposited with accession number KCLRF-BP-00443 and specifically recognizing Staphylococcus aureus B.
Monoclonal antibody produced by cell line 2d 25-2 deposited with accession number KCLRF-BP-00444 and characterized by specifically recognizing Staphylococcus aureus B.
Cell line #C1 deposited with accession number KCLRF-BP-00443.
Cell line 2d 25-2 deposited with accession number KCLRF-BP-00444.
The cell line according to claim 3 or 4, which is produced by the following steps:
(i) injecting Staphylococcus aureus B into the mouse to proceed with immunization and obtaining a spleen of the mouse;
(ii) fusing B lymphocytes and Myeloma cells obtained from the spleen; And
(iii) selecting the cells obtained above.
A yellow staphylococcal enterotoxin B detection kit comprising the monoclonal antibodies of claim 1 and 2.
According to claim 6, The monoclonal antibody is a Staphylococcus aureus toxin B detection kit, characterized in that coated on the nitrocellulose membrane.
According to claim 6, Staphylococcus aureus toxin B detection kit, characterized in that it further comprises at least one of a sample pad and a hygroscopic pad.

Images