KR100270930B1 - Hybrid cell producing monoclonal antibodies for escherichia coli 0157 and monoclonal antibodies produced by them - Google Patents

Abstract

PURPOSE: A fusion cell line producing a monoclonal antibody specifically binding to E. coli O157 is provided to rapidly and exactly detect the infection of E. coli O157. CONSTITUTION: The microorganism NVRIO157(KCTC 0429BP) producing a monoclonal antibody specifically binding to E. coli O157 is produced by the steps of: immunizing a mouse with E. coli O157:H7 by injecting E. coli O157:H7 as an antigen into the mouse; separating immune cells that produce an antibody; and fusing the immune cells with myeloma cells to produce NVRIO157(KCTC 0429BP). The monoclonal antibody MNVRIO157 specifically binding to E. coli O157:H7 is produced from NVRIO157(KCTC 0429BP). The specificity of monoclonal antibody MNVRIO157 is analyzed by ELISA.

Description

Hybrid cell lines producing monoclonal antibodies specific for Escherichia coli O157 and monoclonal antibodies produced by the same strain

The present invention is to prepare a monoclonal antibody for use in a method that can quickly and accurately detect the contamination of pathogenic E. coli O157 in meat and food.

Hemorrhagic E. coli, which causes hemorrhagic diarrhea and hemolytic uremic disease, is known to be the causative agent of O157: H7 serotype among Escherichia coli. Food poisoning caused by Escherichia coli O157: H7 has been occurring worldwide in North America, Europe, Asia, and Africa since it was first introduced in the United States in 1982, and E. coli O157: H7 is mainly present in the intestine of cattle and is contaminated during slaughter. It can be transmitted through livestock products, and in recent years, the occurrence of food poisoning through contaminated water, crude oil, vegetables and apple juice, as well as ground beef, has been reported to quickly and accurately detect the pathogenic E. coli O157: H7 in food. Development is urgently needed.

Isolation and identification of pathogenic E. coli O157: H7 in food is isolated from the sample, followed by specific biochemical test and serologically confirmed, and finally identified by toxin acid activity and several pathogenic factors of the isolated bacteria. Implementing these standard methods can be tricky and time consuming. First, a special enrichment medium and selective medium are used to separate the bacteria from the sample, which makes it difficult to isolate the causative organism depending on the composition of the sample and the competitive action of other bacteria included in the sample. In particular, E. coli O157: H7 can cause food poisoning in very small amounts (about 100), and it is very difficult to isolate bacteria by standard separation methods because of the small number of common samples.

On the other hand, after identification of the bacteria, a characteristic biochemical test is performed for identification and identification, and the serotype of the bacteria using antisera is determined. In this process, the antiserum used also cross-reacts with other enterobacteriaceae and other serotypes of E. coli. Therefore, final identification of Escherichia coli O157: H7 requires the identification of pathogenic factors of the isolated bacteria. Verification of verotoxin among pathogenic factors is carried out by cell culture method using verocell, and other pathogenic factors using enzyme probe (PCR) method or DNA hybridization method using gene probe. . Since it is possible to identify four pathogenic factors in the recently developed multiplex PCR, it is very useful for the final identification of bacteria. However, the multiplex PCR technique is difficult to apply directly to a sample, is cumbersome because it has to go through DNA extraction process, and there is a problem in searching many samples at once.

The standard bacteriological identification method is a matter of time consuming and detection sensitivity, and has been studied around the world for the development of faster and more sensitive methods.

The present invention immunized with E. coli O157: H7 cell antigens in mice, isolating immune cells that produce antibodies, fused with tumor cells (myeloma cells), hybrid cells NVRIO157 producing specific antibodies against E. coli O157 (1998.1.22. Korea Institute of Science and Technology Biotechnology Research Institute Strain Accession No. KCTC 0429BP) was prepared to detect E. coli O157: H7 in food using monoclonal antibodies produced by two cells.

In general, many immunological methods have been developed for increasing the degree of isolation of bacteria and for the rapid detection of bacteria, and are currently being developed and utilized as products. These methods apply immunoidnabead method and monoclonal antibody to chromatographic principle to increase the isolation rate of bacteria by applying monoclonal antibody to magnetic beads. Immunochromatography, in particular, using the Capture-ELISA technique, which performs rapid screening of bacteria with or without the production of microorganisms, is a method that can commercialize even automated machines and efficiently search for many samples in a short time.

Conventional methods using immunological principles are very useful for the rapid retrieval of many samples, but the sensitivity and specificity of each method depend on the characteristics of the monoclonal antibody, which is the basic condition used. Many studies have been conducted to produce monoclonal antibodies specific for Escherichia coli O157: H7, but production of monoclonal antibodies specific for Escherichia coli O157 was very difficult because the LPS structure of Escherichia coli O157 has a structure very similar to that of several other bacteria. Therefore, the monoclonal antibodies produced so far are Salmonella N group (O30), Citroacter freundii, Yersinia enterocolitica O9, Escherichia hermanii, Brucella spp. It has been reported to cause cross-reaction with bacteria, and the diagnostic method using the same acts as a cause of a false-positive reaction when searching a sample, which is problematic for the specificity of the diagnostic method.

Therefore, in order to solve this problem, the present invention provides a development of monoclonal antibodies specific for Escherichia coli O157 for use in developing a rapid and accurate detection method of Escherichia coli O157: H7 in food.

1 shows the sensitivity of the monoclonal antibody of the present invention.

E. coli O157: H7 cells were used as antigens for the development of the monoclonal antibodies of the present invention, and the monoclonal antibodies were searched by ELISA. The developed monoclonal antibody did not cross-react with 9 strains of 15 strains, such as Salmonella N group, Escherichia hermanii, Brucella spp, and Yersinia enterocolitica, which are problems of antibodies developed overseas.E. Coli O157: H7 standard All 12 strains can be detected.

Example 1

* Antigen preparation: E coli O157: H7 used in this experiment was incubated for 12-18 hours in TSB (Tryptic Soy Broth) from bacteria purchased from American type culture collection, and then allowed to stand overnight after adding 1% formalin. In phosphate buffer (PBS, Phosphate Buffered Saline), centrifuged at 2,500 rpm for 20 minutes, washed three times, and then used as an immunogen.

Hybrid cell production

Immunizing antigen was diluted with phosphate buffer solution, homogenously mixed with Incomplete Freund's Adjuvant at a ratio of 1: 1, and then inoculated in a foot pad of 7-week old Balb / c mouse by 0.05 ml each. .

After 14 days of immunization for fusion, both mice were aseptically harvested and crushed from two mice, and only lymphocytes were recovered with serum free media (SFM). SFM was used by adding antibiotics and glutamine to DMEM (Dulbecco's Mudified Eagle Media). SP2 / 0 myeloma cells were used for cell fusion, and the cells were fused with PEG 4,000 by measuring the number of lymphocytes and SP2 / 0 cells at a ratio of 5: 1. The fused cells were suspended in proliferation medium containing HAT (containing 10% FBS in SFM), aliquoted in 96-well microplates at 0.1 ml /, and cultured in a 37 ° C. carbon dioxide incubator. Feeder cells (macrophage) were harvested as a growth medium in the mouse and the abdominal cavity and cultured one day before fusion.

Hybrid cell selection

The culture supernatant from which the hybrid cells have been grown is collected, and the cells producing the antibody against E coli O157 are collected by ELISA and diluted to include 1-0.5 cells per well and cultured for 2 weeks. Cells which formed a single clone while forming an antibody against E coli O157 were selected and selected as hybrid cells. The selected hybrid cells were cultured in a culture flask and used as a supernatant. . The fusion cell formation in this experiment, specific antibody-producing cell lines are shown in Table 1, and the immunoglobulin M heavy chain, IgM heavy chain The monoclonal antibody MNVRIO157 (KCTC 0429BP), which was invented by the characteristics of the prepared monoclonal antibody, was incomplete freund's adjuvant after inactivating the E. coli O157: H7 standard strain with formalin. And immunized with the footpad of BALB / c mouse after 14 days of immunization and aseptically harvested lymph nodes of mice and observed after fusion with cells. It was as Table 1.

Example 2

As a result of the sensitivity test of the monoclonal antibody prepared as in Example 1, the E. coli O157: H7 standard strain was diluted to 10 ¹ -10 ⁹ CFU / ml, coated on an ELISA plate, and then subjected to conventional ELISA. ^The absorbance was found to be 0.3 or more up to ⁵ CFU / ml. This result is similar to the result of sensitivity test of monoclonal antibody developed in foreign countries (1.85 × 10 ⁵ CFU / ml, 10 ⁴ -10 ⁵ CFU / ml).

[Test Example]

Specificity test of the monoclonal antibody prepared as in Example 1 was carried out by ELISA method, the monoclonal antibody developed as shown in Table 3 to detect all 14 strains of E. coli O157 including 12 E. coli O157: H7 standard strains In addition, other monoclonal antibodies developed previously showed high specificity that did not respond to 10 strains of 32 strains including Salmonella.

By using the monoclonal antibody developed in the present invention, the development of an immunological method for the rapid detection of Escherichia coli O157: H7 in food, ensuring the safety of food by effective rapid detection of pathogenic E. coli O157: H7 in food and animals, It is expected to contribute greatly to improving public health. In addition, since the specificity is higher than that of foreign products, excellent domestic diagnostic kits are expected to be developed.

Claims (2)

Hybrid cell line NVRIO157 (KCTC 0429BP), which produces monoclonal antibodies specific for Escherichia coli O157. MNVRIO157, a monoclonal antibody produced by the hybrid cell line NVRIO157 (KCTC 0429BP).