KR100267752B1 - Polyclonal antibody and elisa system for detection and identification of pseudomonas tolaasii - Google Patents

Abstract

PURPOSE: A measurement of enzyme immunity to detect pseudomonas tolaasii in an agaric mushroom is provided to achieve a faster and more convenient detection of the degree or level of contamination by bacteria causing cercospora iythracearum. CONSTITUTION: The noncompetitive indirect enzyme immunity measurement to detect pseudomonas tolaasii is first carried out by coating a tissue and a sample having pseudomonas tolaasii with tris-HCl; cultivating pseudomonas tolaasii bacilli as bacterial brown blotch from agaric mushroom; adding an anti-pseudomonas tolaasii polyclonal antibody obtained from an animal; and adding an anti-rabbit enzyme zygous and substrate solution to cause an enzyme reaction.

Description

Enzyme-Immunosorbent Assay Using Anti-Pseudomonas Tolaci Antibodies for the Detection of Pseudomonas Tolaci, a Bacterial Reclamation Pathogen

The present invention produces a specific anti-Pseudomonas tolaci polyclonal antibody against Pseudomonas tolasi bacteria, Pleurotus eryngii bacteria, and quickly and easily detects and identifies Pseudomonas tolasi bacteria by using them, and contaminants in culture environments such as mushroom medium The present invention relates to an enzyme immunoassay that can be determined qualitatively and quantitatively.

Pseudomonas tolasi is a mushroom pathogen that causes bacterial brown blotch in edible mushrooms such as mushrooms, shiitake, shiitake, and tops, including oyster mushrooms. In particular, in artificial cultivation of oyster mushrooms, if it is not possible to determine whether the pathogen is infected at an early stage, proper control is not achieved, leading to enormous economic losses. The disease is misdiagnosed by symptoms such as excess water, lack of water, incomplete sterilization and growth inhibition due to fermentation, high temperature and dryness, poor ventilation, and similar environmental symptoms. In addition, contamination of other bacteria can misidentify pathogens. Therefore, in order to effectively prevent and control this disease, accurate identification of pathogens and the development of rapid diagnostic methods have been required.

Studies using monoclonal and polyclonal antibodies based on immunochemical properties are widely used in medical and food fields, such as immunodiagnoses, and immunoassays for the detection of residual substances such as toxins, heavy metals, and pesticides produced in cereals and foods. It has been used, and immunoassay in the field of agriculture is applied to the infection, transmission, classification and resistance evaluation methods of pathogenic viruses, fungi and bacteria. Recently, immunological studies have been actively carried out for the identification and detection of fungi of the genus Xanthomonas bacteria, TMB virus and Phytophthora among plant pathogens. No studies have yet been made on the production of anti-Pseudomonas tolasi-specific antibodies and the development of indirect enzyme-immunoassays using the same to detect, identify, and determine the presence of Pseudomonas tolasi.

Rapid and simple diagnostic methods, considering the identification of pathogenic bacteria, isolation of physiology, physiological and biochemical characteristics, and pathogenicity testing, are time consuming, complicated, and expensive. Is urgently needed.

Therefore, in order to effectively prevent and control bacterial gallbladder, it has been recognized that the rapid and accurate identification of pathogenic bacteria and the development of diagnostic methods are important tasks.

As a result of intensive studies to solve this problem, the present inventors have produced a highly selective polyclonal antibody against Pseudomonas tolasis by using the immunochemical properties of the antigen-antibody reaction. As a result, it was found that the detection and identification of Pseudomonas tolasi bacterium, which is the bacterial Streptococcus pathogen, as well as the qualitative and quantitative analysis of the contamination level in the mushroom extract infected with the bacterium, completed the present disease.

Figure 1 shows the standard curve according to the concentration of Pseudomonas tolasis by non-competitive indirect enzyme immunoassay in buffer conditions.

Figure 2 shows the standard curve according to the dilution ratio of the mushroom extract containing Pseudomonas tolasi bacteria by non-competitive indirect enzyme immunoassay under buffer conditions.

FIG. 3 shows the cross reactivity of Pseudomonas tolasis with other three anti-Pseudomonas tolasi antibodies by non-competitive indirect enzyme immunoassay.

Figure 4 shows the cross-reactivity of Pseudomonas tolasi with other bacteria's anti-Pseudomonas tolasi antibodies by competitive indirect enzyme immunoassay.

The present invention provides an anti-Pseudomonas tolasi polyclonal antibody against Pseudomonas tolasi bacteria, which is a bacterium of Streptococcus pneumoniae, and provides an enzyme immunoassay method using the same.

Pseudomonas tolasi used in the present invention was isolated from the diseased body of Pleurotus eryngii and deposited it in the Biotechnology Research Institute (Accession No .: KCTC 8863P).

The characteristics of this strain are as follows.

Table scan

Pseudomonas tolaci was inoculated into the medium and cultured, and then the cells were recovered by means of centrifugation or the like, and the recovered longitudinal was adjusted to an appropriate concentration, for example, 10 ⁸ cfu / ml, and then killed (30 minutes, 60 ° C.). The cells are left in a freezer, and then lyophilized to prepare an immunogen for the production of anti-Pseudomonas tolaci antibody.

Various animals may be used as experimental animals to produce specific antibodies against Pseudomonas tolasi, but rabbits are preferred among them. The rabbit may be a New Zealand white rabbit or a domestic hybrid rabbit. The lyophilized Pseudomonas tolasis strain obtained above was dissolved in sterile buffer (phosphate buffer) to make an emulsion in the same ratio (1; 1, v / v) with Freund's adjuvant and subcutaneously injected. Then, three additional immunizations with subcutaneous injection with Freund's adjuvant at intervals of two weeks, and blood drawn from ear vein one week after each immunization. After collection, the blood is allowed to coagulate at room temperature for about 3 hours and then centrifuged to produce anti-Pseudomonas tolaci polyclonal antibody.

This polyclonal antibody is used in the enzyme immunoassay for the detection and identification of Pseudomonas tolasi bacteria, the smallest mushroom Streptococcus pathogen obtained by using Pseudomonas tolasis bacteria as an immunogen in the laboratory of the present inventors, and its characteristics are as follows.

1) As shown in Example 2, looking at the standard curve by non-competitive indirect enzyme immunoassay (FIGS. 1, 2 and 3), the titer of the antibody shows that the absorbance tends to increase as the concentration of the bacterium increases. In addition, the antibody is an antibody very excellent in detection sensitivity if the search is available at a concentration of 10 ^3, a very high recognition of the bacteria Pseudomonas toll Lashio the navigation concentration of bacteria the buffer state for 10 ^second, mushroom extract.

On the other hand, as shown in Example (Fig. 4), the results of the competitive indirect enzyme immunoassay shows that, unlike the non-competitive method, the titer of the antibody decreases the absorbance as the concentration of the bacteria increases.

2) Since the secondary antibody is an anti-rabbit IgG antibody, it can be seen that the antibody is an IgG type antibody that binds thereto.

3) It shows very high selectivity and specificity because it is not responsive to some bacteria of the same genus and bacteria and fungi of other genera.

First, a non-competitive indirect enzyme immunoassay for the detection of Pseudomonas tolasi is performed by, for example, the process shown in the following table.

TABLE 1 Competitive Indirect Enzyme Immunoassay for Detection of Standard Pseudomonas Tolasis.

[Table 2] Non-competitive Indirect Enzyme Immunoassay for Detection of Pseudomonas Tolasis in Mushroom Extracts

The present invention will be described in more detail with reference to the following Examples. However, the scope of the present invention is not limited to these Examples.

Example 1 Anti-Pseudomonas Tolasi Polyclonal Specific Antibody and its Use for Enzyme Immunoassay for Detection and Quantitative Analysis of Pseudomonas Tolasi Bacillus

Pseudomonas tolacy bacteria used in the present invention were isolated from the pathogen of Pleurotus eryngii and identified as P-1 strain. Pseudomonas tolasi was inoculated in PAF medium, shaken and incubated at 25 ° C. at 100 rpm for 24 hours, followed by centrifugation (10 minutes, 5,000 rpm) to recover the cells. The recovered cells were adjusted to 10 ⁸ cfu / ml concentration, then killed (30 minutes, 60 ° C.), centrifuged again (10 minutes, 5,000 rpm), and the cells were placed in a defreezer (-65 ° C.). After standing for 1 day, lyophilization (-50 ° C., 24 hours) was prepared as an immunogen for the production of anti-Pseudomonas tolaci antibody.

In order to produce specific antibodies against Pseudomonas tolasi, male rats weighing more than 2.5Kg and New Zealand white rabbits were used as experimental animals. Lyophilized Pseudomonas tolasi strain (5 × 10 ⁷ cfu / ml) was dissolved in 0.5 ml of sterile phosphate buffer (phosphate buffered saline, PBS: 1.9 mM sodium phosphate, 154 mM sodium chloride, pH 7.4) in equivalent ratio with Freund's adjuvant. (1: 1, v / v) made an emulsion and injected subcutaneously. Thereafter, further immunization three times with subcutaneous injection with Freund's adjuvant at intervals of two weeks, blood was drawn from the ear vein one week after each immunization. Blood samples were left at room temperature for about 3 hours to coagulate and then centrifuged (10 minutes, 3,000 rpm) to produce anti-Pseudomonas tolaci polyclonal antibody.

This antibody has a very high immunological specificity that specifically reacts to Pseudomonas tolasi, the Pleurotus erythematophyte bacterium, by which it can be used for rapid detection of Pseudomonas tolaci, as well as qualitative infection or contamination and its level. It can be usefully used in immunochemical assays such as enzyme immunoassay, which can be analyzed quantitatively.

Example 2 Addition of Pseudomonas Tolasis to Qualify Contamination Levels, including Identification, and Non-Competitive Indirect Enzyme Immunoassay for Quantitative Analysis, pH 5.0, and Hydrogen Peroxide to a Final Concentration of 0.02%]

Fill 100 μl of the mushroom cell extract obtained by washing Pseudomonas tolassi bacteria and Pleurotus erythritis infected or contaminated with the bacteria with surface adsorption buffer (0.02M Tris, 0.15M sodium chloride, pH 9.0). After standing overnight at 4 ° C. for surface adsorption, washing with 150 μl of washing buffer (0.02M Tris, 0.15M sodium chloride, 0.05% Tween20, pH 7.4) three times, the anti-Pseudomonas tolasi antibody was added to the washing buffer in 1 / After diluting 10,000 times, 100 μl was added and the antigen and antibody reaction was performed at room temperature for 1 hour. After washing three times with washing buffer again, the Hosradish Peroxidase Conjugated Goat Anti_Rabbit IgG (Sigma), a secondary antibody-enzyme conjugate that binds to an anti-Pseudomonas tolasi antibody, was prepared. Diluted 1 / 15,000-fold diluted in the same washing buffer and 100 ㎕ each one and a half hour was washed three times with the wash buffer. Subsequently, 0.01% TEMB (3,3 ', 5,5'-teramethy1 benzidine <dihydrochloride), 0.05% phosphate cirate buffer, pH 5.0, and hydrogen peroxide before use were added to the final concentration of 0.02%. 100 μl per well was added and allowed to stand for 30 minutes to develop color. Then, 50 μl of a reaction stopper (2M sulfuric acid) was added, and the absorbance of each well was measured at a wavelength of 450 nm using a mitroplate reader. Qualitative and quantitative analysis of Pseudomonas tolasi bacteria in Pleurotus erythropoietic tissues and other contaminated samples was performed on the basis of the absorbance of the standard Pseudomonas tolacy strain. This standard curve is shown in FIGS. 1 and 2.

Also, P. solanacearum, Pseudomonas spp., Other genus bacteria, Erwinia chrysanthemi, P. reactans, and Pseudomonas agarishi (P. solanacearum) Agarici strains were tested with Pseudomonas tolaci by non-competitive indirect method to investigate cross-reactivity of the antibody against Pseudomonas tolasi. The result is shown in FIG.

Example 3 Competitive Indirect Enzyme Immunoassay for Qualitative and Quantitative Analysis Including Detection and Identification of Pseudomonas Tolaxi

100 μl of standard Pseudomonas tolasi bacteria dissolved in the surface adsorption buffer solution was added to the wells, and allowed to stand overnight at 4 ° C., followed by surface adsorption for 3 times with 150 μl of the cleaning buffer solution. And 100 μl of a 1: 1 mixture of anti-Pseudomonas tolaci antibody (1 / 10,000-fold dilution) and contaminated antigen-antibody reaction at room temperature for 1 hour. After washing three times with washing buffer, and then a second antibody, anti-rabbit IgG-enzyme conjugate, horseradishperoxidase conjugate sheep anti-rabbit IgG (Horseradish Peroxidase Conjugated Goat Anti-Rabbit IgG, manufactured by Sigma) (1 / 15,000-fold dilution) was added to 100 µl each and reacted at room temperature for 1 hour, followed by washing three times with a washing buffer. The substrate solution was treated and developed as in Example 3, and then absorbance was measured at a wavelength of 450 nm with a microplate reader to obtain a standard curve therefrom. P. solanacearum, Pseudomonas spp., Other genus bacteria, Erwinia chrysanthemi, P. reactans and Pseudomonas agaricis. (P. agarici) was subjected to a competitive indirect method with Pseudomonas tolaci bacteria to investigate the cross-reactivity of the antibody against Pseudomonas tolasi bacteria. The result is shown in FIG.

The specificity of the anti-Pseudomonas tolaci antibody was confirmed from the above Example. In other words, using the anti-Pseudomonas tolaci polyclonal antibody produced according to Example 1 to obtain a standard curve for each concentration of standard Pseudomonas tolasi (P-1) by non-competitive indirect enzyme immunoassay method by the method of Example 2 It was. As a result , the average absorbance ( A ₄₅₀ ) of Pseudomonas tolassis bacteria was 0.070, 0.243, 0.639, 0.778, 0.830, and 0.868 (10 ² , 10 ³ , 10 ⁴ , 10 ⁵ , 10 ⁶ , 10 ⁷ cfu / mL, respectively). The average absorbance of the untreated bacteria was 0.05). The limiting concentrations of Pseudomonas tolasi bacteria in buffer and mushroom extracts were 10 ² and 10 ³ cfu / mL, respectively.

In addition, the anti-Pseudomonas tolasi polyclonal antibody is P. solanacearum, non-pathogenic Pseudomonas sp. Cross-reactivity with Erwinia chrysanthemi, Xanthomonas citri, Streptococcus mutans, and fungus Fusarium oxysporum was examined. Turned out not to. P. reactans and P. agarici of the same genus showed weak reactions with anti-Pseudomonas antibodies at high concentrations, but there was no problem in detecting Pseudomonas tolaci.

On the other hand, as a result of examining the cross-reactivity with the bacteria by the competitive indirect enzyme immunoassay of Example 3, no cross-reactivity was found.

These results confirm that the present anti-Pseudomonas tolasi antibody is highly selective and specific for Pseudomonas tolasi bacteria despite being a polyclonal antibody.

As described above, the characteristics of the present invention are anti-Pseudomonas showing a selective response only to Pseudomonas tolasi bacteria in a situation in which there is no rapid and simple immunological detection method of Pseudomonas tolasi bacteria, which is bacterial bacillus pathogen, at home and abroad. Indirect enzyme-immunoassay using the immunochemical characteristics of Tolasi specific antibody was used to inoculate not only the fruiting bodies of the mushrooms with crab disease at the mushroom cultivation site, but also the inoculation of the mycelium by inoculating the medium before and after sterilization, the medium under fermentation and the seed It is an agriculturally beneficial invention in that it can qualitatively, quantitatively and quickly analyze whether or not Pseudomonas tolasis is infected from the medium, the groundwater, the water stored in the storage tank, or the fermentation site of the medium.

Therefore, the present invention clearly distinguishes bacterial plaque disease of mushrooms caused by Pseudomonas tolasi bacteria and the damage caused by other causes, even if the fungus is infected or contaminated with Pseudomonas tolasi bacteria at very low concentrations. By detecting before the outbreak of the disease caused by bacteria, it is expected to be effectively used in the mushroom disease prediction system for preventing or preventing the occurrence of bacterial crab disease.

Claims (2)

After coating the tissue and the sample with the branch disease with tris-HCl, After culturing Pseudomonas tolaci, a bacterium of the Pleurotus erythematosus bacterium, and adding an anti-Pseudomonas tolaci polyclonal antibody obtained by immunizing the animal, A non-competitive indirect enzyme immunoassay for detecting and diagnosing Pseudomonas tolasi, characterized by adding an anti-rabbit IgG enzyme conjugate followed by adding a substrate solution. After coating Pseudomonas tolaci with tris-HCl, After culturing Pseudomonas tolasi bacteria, the Pleurotus eryngii bacterium, and adding an anti-Pseudomonas tolaci polyclonal antibody and a sample obtained by immunizing an animal, Competitive indirect enzyme immunoassay for the detection and diagnosis of Pseudomonas tolasi, characterized by the addition of an anti-rabbit IgG enzyme conjugate followed by the addition of a substrate solution.