KR102092466B1 - Yersinia pestis diagnostic detection kit using rapid immunochromatography, its specific antibody and antibody-producing cell lines - Google Patents

Abstract

The present invention relates to Fest F1 capsule protein-specific monoclonal antibody, a novel cell line SDV2 Pest 2a 40-2 (Accession No. KCLRF-BP-00427) producing the antibody, and an immunochromatography kit for detecting pest bacteria using the same . The kit exhibits excellent sensitivity to plague bacteria and no cross-reactivity to other pathogens and interfering substances.

Description

Yersinia pestis diagnostic detection kit using rapid immunochromatography, its specific antibody and antibody-producing cell lines}

The present invention includes a novel cell line SDV2 Pest 2a 40-2 (Accession No. KCLRF-BP-00427), which is a cognitive antibody of the F1 capsule protein of Fest bacteria, and the antibody, and an improved sensitivity and specificity plague. It relates to an immunochromatography kit for detecting bacteria.

The causative agent of the Black Death (Fest) is Yersinia pestis, and the main symptom of the plague is swollen lymph nodes of the infected patient and accompanied by high fever. The plague typically includes 'granular plague' that infects lymph nodes, 'septic plague' that infects blood vessels, and 'pulmonary plague' that occurs when plague bacteria infect the lungs.

Vaccines against plague have been developed, but have not been used because they have not been proven effective, and can be treated with antibiotics in the early detection. When pesticides are aerosolized and sprayed in the air, they are highly likely to be used in bioterrorism because they can infect through the lungs and spread rapidly through saliva or cough.

In addition, the US Centers for Disease Control and Prevention recently reported that they found a pesticide resistant bacteria. The antibiotic-resistant pestilities were resistant to all antibiotics used in the plague. Treatment with other types of antibiotics, trimetroprin, was possible, but even with some genetic mutations, it is possible that mutants such as plague, which caused the plague of the Middle Ages.

The pest is a Gram-negative, anaerobic bacterium that does not form spores and does not have blunt motility, and can survive for weeks in water, wet grains, and flour. It can also survive for months or years, even at extremely low temperatures, but is inactivated or killed within hours if exposed to sunlight or conditions above 55 degrees Celsius.

Sun Fest is a fever, chills, boredom, muscle aches, nausea, nausea, sore throat, and headaches that occur in the lymph nodes. Flea bites (90% of the inguinal region) are swollen and swollen on the same or next day. Progress and become an abscess. Subsequently, bacteremia develops and spreads throughout the body, and severe patients die from sepsis in 2-4 days. The mortality rate of untreated patients reaches 50-60%, but when treated, the mortality rate decreases to 5%. Sun fest does not occur between humans unless it comes into direct contact with the pus of lymph node fest patients.

Lung plague is divided into primary pulmonary plague caused by inhaling pathogens and secondary pulmonary plague in which the pathogen causing sepsis spreads to the lungs through the blood. Lung plague can penetrate lung tissue, cause pneumonia, pleural effusion, and mediastinitis, which can lead to death in one day.

FA test, antigen capture ELISA test, and PHA method using Y. pestis Fraction I antigen are commonly used as methods for detecting and diagnosing pests.

Streptomycin, tetracycline, chloramphenicol, and gentamicin are very effective antibiotics for pesticides. It is especially effective when used within 24 hours of symptoms. However, if treatment is not started within 24 hours after the symptoms of pneumonia caused by pulmonary plague, the mortality rate increases.

Therefore, the development of a technology that can be quickly detected in the field in the event of a suspected bioterrorism situation is very important for promptly determining the existence of a terrorist situation and determining a response policy. To this end, the inventor tried to develop a rapid detection kit based on immunochromatography technology for on-site detection of plague bacteria.

An object of the present invention is to provide a pest detection kit for on-site detection of pest bacteria for rapid response in suspicion of bioterrorism. To this end, the present invention provides an F1 capsule protein recognition antibody of Fest bacteria, a new cell line SDV2 Pest 2a 40-2 (Accession No. KCLRF-BP-00427) for producing the antibody, and an immunochromatography kit for detecting Fest bacteria using the same do.

The present invention provides a monoclonal antibody characterized by specifically recognizing the F1 capsule protein of Fest bacteria.

The monoclonal antibody is produced by the cell line SDV2 Pest 2a 40-2 deposited with accession number KCLRF-BP-00427, and is characterized by specifically recognizing the F1 capsule protein of Pest bacteria.

The invention also relates to the cell line SDV2 Pest 2a 40-2 deposited with accession number KCLRF-BP-00427.

Specifically, the present invention comprises the steps of (i) introducing the nucleotide sequence of the F1 capsule protein, synthesizing an E. coli expression vector, and transforming it with E. coli (XL1-Blue) to express the antigen; (ii) obtaining the spleen of the mouse after immunization by injecting the antigen into the mouse; (iii) fusing B lymphocytes and Myeloma cells obtained from the spleen; And (iv) relates to a cell line prepared by a method comprising the step of selecting the cells obtained above.

The present invention relates to a pest 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 provide faster and more accurate detection and detection of pesticides by providing antibodies with superior sensitivity and specificity than those used in conventional pesticide detection kits. Specifically, the detection kit using the antibody of the present invention has superior sensitivity compared to conventional products, and has very good specificity because 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 multiple detection kit 9, manufacturer: SD).
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. Also, although preferred methods and samples 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, and for the purposes of the present invention is a monoclonal antibody that specifically binds to the F1 capsule protein of Pest bacteria. F1 capsules act to inhibit phagocytosis by immune cells

In the present invention, "recombinant vector" is a vector capable of expressing a target gene in a suitable host cell, and refers to a gene construct comprising essential regulatory elements operably linked to express a gene insert. The operative linkage with a recombinant vector can be made using genetic recombination techniques well known in the art, and site-specific DNA cleavage and linkage can use enzymes generally known in the art.

The "vector" of the present invention includes a plasmid vector, a cosmid vector, a bacteriophage vector, a viral vector, and the like, preferably a plasmid vector, but is not limited thereto. Suitable expression vectors may include signal sequence or leader sequences for membrane targeting or secretion in addition to expression control elements such as promoters, operators, start codons, termination codons, polyadenylation signals and enhancers. The promoter of the vector can be constitutive or inducible. In addition, the expression vector includes a selection marker for selecting a host cell containing the vector, and in the case of a replicable expression vector, the origin of replication.

The present invention relates to a monoclonal antibody that specifically binds to the Fest capsule F1 capsule protein produced by the hybridoma cell line SDV2 Pest 2a 40-2 with accession number KCLRF-BP-00427.

The antibody produced in the hybridoma cell line SDV2 Pest 2a 40-2 of the deposit number KCLRF-BP-00427 of the present invention is a novel antibody with a higher affinity for antigen than the antibody used in the conventional pest detection kit. 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 detection kit for plague bacteria comprising the above-described antibody. By using the kit containing the above-described antibody, the on-site detection of the pest bacteria in the bioterrorism suspected situation can be quickly performed.

Such detection kits may include monoclonal antibodies 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 immunosorbent assay, western blotting, immunoprecipitation assay, immunoprecipitation assay, immunodiffusion assay ), Complement Fixation Assay, FACS, and 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.

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, at the site of the test line and the control line on the nitrocellulose membrane.

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 pad compositions optimized for the 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 Fest F1 recombinant antigen

In order to investigate and prepare immunogens for antibody development, the Fest (Recognition No. 15-RDM-014) is approved through the national approval screening for experiments and development of genetically modified organisms in accordance with the Act on the Movement of Genetically Modified Organisms Across Countries. All experiments were conducted in compliance with the biosafety operating guidelines at the reported biosafety research facility.

In order to prepare F1 capsule protein antigen expression vectors of the genetically recombined Fest bacteria (Yersinia pestis), chromosomal DNA in the heat inactivated Fest bacteria was used as template DNA.

In order to prepare expression vectors capable of expressing the F1 capsule antigen protein of Fest bacteria, the gene corresponding to the protein part to be expressed using template DNA was amplified by polymerase chain reaction (PCR). The fragments obtained by cutting the amplified gene with restriction enzymes were conjugated to the E. coli expression vector, thereby obtaining a vector expressing the F1 capsule antigen protein of Fest bacteria. Then, the vector was transformed with XL1-Blue to obtain E. coli expressing the above antigen.

The oligonucleotide primers used in PCR are as follows.

Introduced F1 capsule protein coding gene F1 (caf1) is composed of 513 bp nucleotides, but BamHI and SalI restriction enzyme sites were added to 5 'and 3' for cloning, respectively, amplified by PCR, and used for protein expression. Codons and termination codons were replaced with those present in the vector.

[Example 2]-Fest F1 recombinant antigen production and purification

The transformant with cloning confirmed was incubated at 37 ° C and 220 rpm in LB medium, and when the absorbance value was 0.5 at OD600, it was induction with IPTG (Isopropyl β-D-1-thiogalactopyranoside). For purification, pellets excluding the supernatant from the culture were crushed by sonication. After centrifugation, the insoluble protein was dissolved in 6M guanidine (guanidine), and Ni-affinity purification was performed. The purified protein was dialyzed against 20 mM Carbonate buffer (pH 9.6). After crushing the culture medium in which the recombinant protein was overexpressed, Ni-affinity purification was performed to secure the purified single-band recombinant F1 (about 19 kDa).

[Example 3]-Preparation of monoclonal antibodies

1. Immunity and cell fusion

For the production of monoclonal antibodies, the recombinant antigen was made with Freund's adjuvant and immunized with mice. 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 from 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-reaction through primary and secondary screening by ELISA. The resulting cell line was deposited with the Korea Cell Line Bank on April 17, 2018 (Accession No. KCLRF-BP-00427). 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. As for mouse multiple purification, mouse IgG, an antibody specific for protein G, was purified using Protein G chromatography gel. The mouse IgM did not use a specific gel affinity chromatography method, but was purified and purified by dialysis and DEAE gel purification method. The purified monoclonal antibody candidate group was selected by screening using gold.

[Example 4]-Preparation of plague bacteria detection kit

The antigen-specific antibody and the antibody for the control line selected at the test line and the control line position 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

In order to test the detection limit of the paste, the sensitivity was evaluated while diluting the concentration of the paste. 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 4 were evaluated and compared, and the results are shown in FIG. 1.

As shown in FIG. 1, the detection limit was measured as ⁶ × 10 ⁴ cfu / ml as a result of 10 repeated tests, and it was confirmed that the sensitivity was improved by about 8 times compared to the existing product ( ⁵ × 10 ⁵ cfu / ml).

[Test Example 2]-Specificity test 1

For specificity evaluation, cross reactivity was evaluated for 9 strains and strains such as lysine and brucella, and the results are shown in Tables 1 and 2 below.

Item density Fest bacteria detection kit reaction One Sample dilution N / A voice 2 Fest 1X10 ⁸ cfu / ml positivity 3 Lee Sin 10uf / ml voice 4 Botulinum toxin A 200ng / ml voice 5 Night soil 1X10 ⁸ cfu / ml voice 6 Cholera O139 1X10 ⁸ cfu / ml voice 7 Cholera O1 1X10 ⁸ cfu / ml voice 8 Ubiquitous bacteria 1X10 ⁸ cfu / ml voice 9 Brucellella 1X10 ⁸ cfu / ml voice 10 SEB 100ug / ml voice

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

[Test Example 3]-Specificity test 2

27 strains (> 1x10 ⁸ cfu / ml) or toxins (> 100 ng /) disclosed in Table 2 below to confirm whether cross-reaction with toxins and strains other than 9 strains and toxins of Test Example 2 ml). As shown in Table 2 below, it was confirmed that the cross-reaction was not shown for the cross-reaction test strain and 27 kinds of substances.

[Test Example 4]-Interference test

Predicted inhibitory substances that could affect the performance of detection kits such as bovine serum, human serum, sugar, flour, house dust, etc. were selected, and specificity tests were conducted to confirm the presence or absence of interference.

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

Item Detection result Obstruction 5X10 ⁶ cfu / ml 2.5x10 ⁵ cfu / ml Bovine serum (NCS 1%) voice positivity positivity Human serum (diluted 5 times NHS) voice positivity positivity Bovine albumin (BSA 1%) voice positivity positivity Sugar (Sucrose 10%) voice positivity positivity Baby milk (Skim milk 10%) voice positivity positivity Baking powder (saturated suspension) voice positivity positivity Wheat flour (saturated milk) voice positivity positivity Detergent (saturated suspension) voice positivity positivity House dust (saturated floating liquid) voice positivity positivity Soil (saturated suspension) voice positivity positivity

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

[Test Example 5]-Reproducibility test

A total of 20 sensitivity and specificity tests were conducted to confirm that the detection kit prepared in Example 4 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 they were all reproducible.

[Test Example 6]-accelerated stability test

In order to set the expiration date of the detection kit prepared in Example 4, 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.

Depository Name: Korea Cell Line Research Foundation

Accession number: KCLRFBP00427

Date of accession: 20180417

Claims (8)

delete delete Monoclonal antibody produced by cell line SDV2 Pest 2a 40-2 deposited with accession number KCLRF-BP-00427, and characterized by specifically recognizing Fest F1 capsule protein.
Cell line SDV2 Pest 2a 40-2 deposited with accession number KCLRF-BP-00427.
delete A kit for detecting plague bacteria comprising the monoclonal antibody of claim 3.
The method of claim 6, wherein the monoclonal antibody is a pesticide detection kit, characterized in that coated on the nitrocellulose membrane.
According to claim 6, Paste bacteria detection kit, characterized in that it further comprises at least one of a sample pad and a hygroscopic pad.

Images