JP4107849B2 - Anthrax detection reagent - Google Patents

Description

【００２８】
【発明の効果】
本発明の炭疽菌抗体を用いることにより、簡便、安価かつ迅速に炭疽菌の検出ができる。さらに、莢膜を形成した炭疽菌、栄養型の炭疽菌及び芽胞を形成した炭疽菌のライフサイクル中のステージに拘わりなく、特異的に、簡便に、短時間に検出することができる。[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a method for detecting Bacillus anthracis using an antibody that recognizes the capsule, cells and spores possessed by Bacillus anthracis and a kit thereof. This reagent is effectively used for measures against infections caused by Bacillus anthracis and terrorism using Bacillus anthracis.
[0002]
[Prior art]
Bacillus athracis is a facultative anaerobic Gram-positive spore-forming bacterium belonging to the genus Bacillus, and forms a spore or capsule by the environment, or exists as a trophozoite that does not form a spore or capsule. This bacterium is known to cause anthrax, a common infectious disease of humans and livestock. It occurs in animals all over the world, and humans who handle livestock can be infected. In addition, this bacterium has been used as a biological weapon, and terrorism using the spore of this bacterium has also occurred.
[0003]
When humans are infected, they are classified into skin anthrax, intestinal anthrax, and lung anthrax depending on the route of infection, and lung anthrax is known to have a high mortality rate.
In the diagnosis of anthrax, the most reliable method is to isolate and identify anthrax. For identification of bacteria, capsular staining, immunoprecipitation test (Ascory thermal precipitation reaction), biochemical property test, PCR using genes, and the like have been used.
[0004]
However, it is not always easy to distinguish these methods from Bacillus cereus, which is an anthracnose species of Bacillus anthracis. In addition, since these methods require several hours to more than a day and special devices are required, a method for identifying in a shorter time and simply has been desired. In addition, there are cases where anthrax spores are sent by mail etc. due to terrorism, and it is necessary to quickly identify that the delivery is Bacillus anthracis, so a reagent that can be easily identified in a short time is desired. It was.
[0005]
[Problems to be solved by the invention]
The present invention relates to an antibody for detecting anthrax specifically, simply and in a short time regardless of the stage in its life cycle, and a method for specifically and simply detecting anthrax in a short time. And to provide a kit. Specifically, an object of the present invention is to provide an antibody that recognizes an anthrax capsule, fungus body and spore, an immunological detection method using the antibody, and a detection kit containing the antibody.
[0006]
[Means for Solving the Problems]
The present inventors immunized rabbits with antigens of the cells of each stage in the life cycle of anthrax that formed capsules, vegetative anthrax and anthrax that formed spores, and obtained antibodies against each. The present invention has been completed.
[0007]
That is, the present invention is as follows.
(1) An antibody against anthrax, an antibody selected from the group consisting of an antibody against an anthrax capsule, an antibody against an anthrax spore, and an antibody against an anthrax cell that does not contain the capsule and spore,
(2) An antibody composition comprising an antibody against anthrax, selected from the group consisting of an antibody against an anthrax capsule, an antibody against an anthrax spore, and an antibody against an anthrax cell that does not contain the capsule and spore. An antibody composition comprising two types of antibodies;
(3) An antibody composition comprising an antibody against anthrax, comprising an antibody against an anthrax capsule, an antibody against an anthrax spore, and an antibody against an anthrax cell that does not contain the capsule and spore Composition,
(4) An anthrax detection method characterized by detecting anthrax using the antibody or antibody composition of any one of (1) to (3), and (5) of (1) to (3) A kit for detecting Bacillus anthracis comprising any antibody or antibody composition.
[0008]
DETAILED DESCRIPTION OF THE INVENTION
Hereinafter, the present invention will be described in detail.
The antibody or antibody composition of the present invention comprises at least one antibody selected from the group consisting of an antibody against an anthrax capsule, an antibody against an anthrax spore, and an antibody against an anthrax cell that does not contain the capsule and spore. An antibody or antibody composition comprising. Here, the “antibody composition” refers to an antibody mixture containing one or more antibodies. In the present specification, the term “antibody” includes not only one kind of antibody but also an antibody composition.
[0009]
Anthrax forms capsules and spores depending on the environment. The anthrax that formed the capsule, the anthrax that formed the spore, and the anthrax that did not form the capsule or spore can be inactivated with formalin and used as an immunogen. At this time, the anthrax that formed the capsule, the anthrax that formed the spore, and these anthrax that did not form the capsule or spore may be used separately as an immunogen, or mixed and used as an immunogen. May be. An immunogen can be prepared by suspending the inactivated strain in a buffer solution such as PBS at a concentration of 0.5 to 1.5 mg / ml, preferably 1 mg / ml. The immunogen is desirably administered with a suitable adjuvant such as commercially available Freund's complete adjuvant, Freund's incomplete adjuvant, BCG, aluminum hydroxide gel, pertussis vaccine and the like. Rabbits, sheep, horses, pigs and the like can be used as immunized animals, but rabbits are generally used. For rabbits, use about 3 kg. The prepared immunogen can be administered through any route such as subcutaneous, intraperitoneal, intravenous, intramuscular, or intradermal routes of animals. The interval between immunizations is not particularly limited. For example, immunization is preferably performed 2 to 5 times at intervals of 1 to 2 weeks. The dose per administration is not limited, but for example, the immunogen prepared as described above may be mixed with an appropriate adjuvant and administered in an amount of 1 ml to several ml. Three to ten days after the final immunization, blood is collected from the immunized animal, left in a refrigerator, etc., and a blood clot is generated. Then, an antibody is obtained by separating the serum part by centrifugation or the like. Can do.
[0010]
Detecting anthrax, regardless of stage, by mixing antibodies obtained from immunogens of anthrax that formed capsules, anthrax that formed spores, and anthrax that did not form these capsules or spores Can be used as an antibody composition. Further, an antibody composition may be used by mixing two kinds of antibodies against an anthrax capsule, an antibody against an anthrax spore, and an antibody against an anthrax bacterium containing no capsule and spore as necessary. Alternatively, anthrax that forms capsules, anthrax that forms spores, and antibodies obtained as an immunogen by mixing these anthrax that do not form capsules or spores remain as they are regardless of stage. Can be used as an antibody composition capable of detecting. Furthermore, two types of Bacillus anthracis having formed a capsule, Bacillus anthracis forming spores, and Bacillus anthracis not forming these capsules and spores may be mixed and used as an immunogen.
[0011]
Moreover, as an antibody used in the present invention, a monoclonal antibody obtained by a known cell fusion method after immunizing a mouse with the above immunogen can also be used. The monoclonal antibody can be prepared by a known method such as the method of Kohler and Milstein (Kohler, G. and Milstein, C., Nature, 256, 495-497, 1975). A hybridoma obtained by cell fusion of a spleen cell or lymph node cell of a mouse immunized with the above anthrax immunogen and a myeloma cell of a mouse was prepared, and a culture supernatant of the hybridoma or a mouse administered with the hybridoma intraperitoneally It can be prepared from ascites. Immunized animals are not limited to mice, and rats, guinea pigs, and the like can also be used. Myeloma cells are generally obtained from the same species as the immunized animal, but may be possible between different species. Sensitized cells can also be obtained by immunizing in vitro spleen cells or lymph nodes of non-immunized animals. Hybridoma screening can be performed by various immunochemical methods such as ELISA and Western blotting.
[0012]
In this case, monoclonal antibody that reacts specifically only with anthrax spores, monoclonal antibody that reacts specifically only with anthrax capsule, and only cells that exclude the anthrax capsule and spores. If a monoclonal antibody is used separately, the anthrax of each stage in a life cycle can be detected, and if these are used as a mixed antibody composition, anthrax can be detected regardless of the stage. Moreover, you may mix and use two types of these monoclonal antibodies as needed.
[0013]
When antibody purification is required, known methods such as ammonium sulfate salting-out, ion exchange chromatography using anion exchangers such as DEAE cellulose, molecular sieve chromatography based on molecular weight and structure, hydroxyapatite chromatography, etc. The method can be appropriately selected, or can be purified by combining them.
[0014]
It is possible to detect Bacillus anthracis using the thus obtained anti-Anthrax antibody. Methods known to those skilled in the art such as immunoblotting, enzyme immunoassay (EIA, ELISA), radioimmunoassay (RIA), fluorescent antibody method, method using agglutination, immunochromatography, etc. Can be performed. At this time, as the sample, for example, bacteria separated and cultured from a patient specimen, a powdery substance suspected to be anthrax, and the like can be used, but are not limited thereto.
[0015]
For example, in a method using an agglutination reaction, anthrax can be detected using a carrier sensitized with an anthrax antibody. As a carrier for sensitizing the anthrax antibody, any carrier may be used as long as it is insoluble, does not cause a nonspecific reaction, and is stable. For example, latex particles, bentonite, collodion, kaolin, fixed sheep erythrocytes, gold colloid particles, selenium colloid particles and the like can be used, but it is preferable to use latex particles. As latex particles, for example, polystyrene latex particles, styrene-butadiene copolymer latex particles, polyvinyltoluene latex particles, and the like can be used, and polystyrene latex particles are preferably used. When latex particles are used, the antibody can be easily sensitized to the carrier without any special treatment, and the aggregated image generated by the reaction between the target strain and the carrier becomes clear, and the reactivity of the target strain to the carrier is easy. Further, it is further advantageous in that it can be discriminated accurately.
[0016]
The method for sensitizing the anti-anthrax antibody to the carrier is not particularly limited. For example, the anthrax antibody may be physically adsorbed on a carrier or chemically bound. More specifically, for example, after mixing an anthrax antibody and a carrier, the antibody can be sensitized to the carrier by heating and shaking at 30 to 37 ° C. for 1 to 2 hours. The amount of the antibody sensitized to the carrier can be appropriately set according to the particle size of the single substance used. After sensitizing the antibody to the carrier, the non-sensitized portion on the carrier surface is preferably blocked with bovine serum albumin, human serum albumin, rabbit serum albumin, ovalbumin or the like. The carrier sensitized with the anthrax antibody is preferably retained as a medium dispersion until it is reacted with the target strain. At this time, as the medium, for example, a phosphate buffer, a glycine buffer, or the like can be used. The content of the carrier sensitized with the anti-anthrax antibody can usually be 0.2 to 0.5% by weight, preferably 0.25 to 0.3% by weight, based on the medium dispersion. Bovine serum albumin, gelatin, gum arabic and the like may be added to the medium as necessary. The anti-anthrax antibody-sensitized carrier thus prepared can be reacted with the target strain, and the reactivity between the target strain and the anti-anthrax antibody can be determined based on the presence or absence of the aggregation or the degree thereof, thereby detecting anthrax.
[0017]
In enzyme immunoassay (EIA), an anthrax antibody is immobilized on a carrier such as a microplate, resin beads, or magnetic beads by physical adsorption or chemical bonding. The amount of the solid phase is not particularly limited, but when the carrier is a microplate, it is preferably several ng to several tens of μg per well. The immobilization can be performed by dissolving the antibody to be immobilized in an appropriate buffer and bringing it into contact with a carrier. For example, in the case of using a microtiter well, the antibody solution can be immobilized by dispensing into a well of a microtiter plate and placing it for a certain period of time. After immobilizing the antibody, it is preferable to perform blocking using a blocking solution containing bovine serum albumin, human serum albumin, rabbit serum albumin, ovalbumin and the like in order to prevent non-specific binding during the assay. Next, the immobilized carrier and the sample are reacted, and after washing, a labeled antibody that recognizes anthrax is reacted. The labeling can be performed using an enzyme such as β-D-galactosidase, peroxidase, alkaline phosphatase or glucose oxidase. For example, in enzyme immunoassay (EIA), a large amount of measurement can be performed at once by immobilizing an antibody on a microtiter plate having a large number of wells (eg, 96 wells) and causing an antigen-antibody reaction in the well. It becomes possible. It is also possible to reduce the amount of antibody used and the amount of sample cells used. Furthermore, it is possible to use an automatic measuring device such as a fully automatic EIA measuring device.
[0018]
The present invention is also aimed at providing a kit that enables detection of anthrax, but the kit contains at least an anthrax antibody. When the kit is based on an enzyme immunoassay, an antibody-immobilized carrier may be included, and the antibody may be bound to the carrier in advance. When the kit is based on an agglutination method using a carrier such as latex, the kit may contain a carrier on which an antibody is adsorbed. The kit may appropriately contain a blocking solution, a reaction solution, a reaction stop solution, a reagent for treating the sample, and the like.
[0019]
As described above, the anti-anthrax antibody of the present invention contains anthrax capsular, bacterial and spore antibodies and reacts with almost all anthrax, so that the isolated bacteria can be quickly distinguished from anthrax. Thus, anthrax can be detected, and the time and labor required for examining the biochemical properties can be largely omitted. In addition, unlike PCR using genes, sample pretreatment and special equipment are not required, leading to low costs.
Hereinafter, the present invention will be described specifically by way of examples. However, the present invention is not limited to these examples.
[0020]
【Example】
Example 1 Preparation of immunizing antigen A single colony of 2 seedlings of Bacillus anthracis Pasteur was inoculated into 5 ml of tryptic soy broth and cultured at 37 ° C. to prepare a preculture.
Preparation of vegetative cells was carried out by adding 1 ml of precultured bacteria to 100 ml of tryptosoy broth and shaking culture at 37 ° C. for 4 hours. The culture solution is centrifuged at 7000 rpm for 15 minutes, the sediment is washed twice with 20 ml of physiological saline, resuspended in 100 ml of physiological saline, and then added with formalin to a final concentration of 0.5%. It was activated. After confirming that the bacteria were inactivated, the cells were washed twice with physiological saline to obtain an antigen solution.
[0021]
The spore cells were prepared by adding 1 ml of precultured bacteria to 100 ml of tryptic soy broth, followed by shaking culture at 37 ° C. for 24 hours. The culture solution is centrifuged at 7000 rpm for 15 minutes, and the sediment is washed twice with 20 ml of physiological saline, resuspended in 100 ml of physiological saline, and statically cultured at 37 ° C. for 7 days to a final concentration of 0.5%. It was inactivated by adding formalin. After confirming that the bacteria were inactivated, the cells were washed twice with physiological saline to obtain an antigen solution.
[0022]
For preparation of capsular cells, 0.1 ml of the preculture was smeared on 5 NBY agar media and cultured in a 20% carbon dioxide incubator for 24 hours. The cells were collected, suspended in 10 ml of physiological saline, and inactivated by adding formalin to a final concentration of 0.5%. After confirming that the bacteria were inactivated, the cells were washed twice with physiological saline to obtain an antigen solution.
[0023]
Example 2 Preparation of Antibody An immunogen was prepared by adding an equal volume of incomplete Freund's adjuvant to 1 ml of antigen solution. Immunization was performed by inoculating several places on the back of the rabbit subcutaneously. The inoculation interval was inoculated four times every other week, and a part of blood was collected one week after the final inoculation, and the increase in antibody titer was confirmed by Western blotting. After confirmation, a booster was performed twice every other week, 50 ml of blood was collected in the first week, a booster was performed again in the first week, and blood was collected in the first week. After repeating this twice, the whole blood was collected and pooled to prepare antibodies.
[0024]
Salting out was performed by adding 10 ml of a saturated ammonium sulfate solution to 5 ml of the antibody thus prepared and adding 5 ml of a phosphate buffer solution to dilute. The precipitate was dissolved in 10 ml of phosphate buffer, 10 ml of saturated ammonium sulfate solution was added again for salting out, and the precipitate was dissolved in 2 ml of phosphate buffer. It was desalted using a Sephadex G-25 column to obtain a purified antibody.
[0025]
Example 3 Detection of Bacillus anthracis by latex agglutination method (1) Preparation of latex sensitized with Bacillus anthracis antibody Polystyrene spherical particles (latex: commercially available) having a diameter of 0.3 μm were used. The anthrax antibody and latex particles obtained in Example 2 are each dissolved in phosphate buffered saline (pH 7.0), and the antibody is immobilized on a carrier so that the weight ratio of antibody to latex particles is 1:10. did. This was washed with phosphate buffered saline and blocked with bovine serum albumin to obtain antibody-sensitized latex.
(2) Culture of specimen Anthrax was cultured on a blood agar medium to prepare a specimen.
(3) 25 μl of the antibody-sensitized latex prepared in (1) was dropped into a circle of a slide plate made of slide agglutination reaction paper, and a small amount of the sample cultured in (2) was added and mixed. After stirring for 1 minute, the presence or absence of agglomeration was visually observed, and those in which agglomeration was observed were positive and those in which agglomeration was not recognized were negative.
[0026]
Example 4 Specificity of antibody-sensitized latex reagent In addition to Bacillus anthracis, Bacillus natto, Bacillus subtilis and Bacillus cereus belonging to the same genus Bacillus were cultured in a blood agar medium. The test was conducted as in Example 3 (3) using a trace amount of cells.
The results are shown in Table 1. All anthrax were positive in this sensitized latex. On the other hand, natto, Bacillus subtilis, and Bacillus cereus other than Bacillus anthracis were all determined to be negative.
[0027]
[Table 1]

[0028]
【The invention's effect】
By using the anthrax antibody of the present invention, anthrax can be detected simply, inexpensively and rapidly. Furthermore, regardless of the stage in the life cycle of the anthrax that formed the capsule, the vegetative anthrax, and the anthrax that formed the spore, it can be detected specifically, simply, and in a short time.

Claims (3)

Using an antibody-sensitized latex that sensitizes latex particles with antibodies against anthrax capsule, antibodies against anthrax spores, and antibodies against anthrax and spore-free cells. Latex agglutination method that detects Bacillus anthracis regardless of the stage in the life cycle and distinguishes it from Bacillus cereus and Bacillus subtilis. The latex agglutination method for detecting anthrax according to claim 1, wherein the latex particles are selected from the group consisting of polystyrene latex particles, styrene-butadiene copolymer latex particles, and polyvinyltoluene latex particles. At the stage in the life cycle of Bacillus anthracis, the antibody against the anthrax capsule, the antibody against the anthrax spore, and the antibody against the anthrax without capsular and spore are sensitized to latex particles. A latex agglutination method kit containing an antibody-sensitized latex for detecting Bacillus anthracis, which is distinguished from Bacillus cereus and Bacillus subtilis, regardless of the stage in the life cycle of Bacillus anthracis, and Bacillus cereus And a latex agglutination method kit for detecting Bacillus anthracis discriminated from Bacillus subtilis.