JP5202367B2 - Simple assay for enterohemorrhagic Escherichia coli - Google Patents

Description

  The present invention relates to a method for detecting enterohemorrhagic E. coli. Specifically, the present invention relates to a method of detecting verotoxin in E. coli cells by reacting carrier particles sensitized with an anti-verotoxin antibody with E. coli.

  Enterohemorrhagic Escherichia coli is characterized by producing verotoxin, causing hemorrhagic colitis, acute renal failure due to hemolytic uremic syndrome (HUS), thrombotic thrombocytopenic purpura (TTP), etc. To death. Therefore, the presence or absence of enterohemorrhagic Escherichia coli is detected in a short time and accurately in order to diagnose the presence or absence of enterohemorrhagic Escherichia coli early and to take appropriate treatment to prevent the transition to severe symptoms. There is a need.

  Escherichia coli is classified into approximately 170 types according to the serotype of the O antigen of the surface LPS antigen, of which approximately 90 types produce verotoxin. Serotypes of enterohemorrhagic E. coli that produce verotoxin include O157: H7, O157: NM, O26: H11, O91: H21, O111: NM, O113: H21, O145: NM, and the like. Conventionally, there has been a method for measuring the serotype of E. coli O antigen and diagnosing that enterohemorrhagic E. coli is infected when E. coli of the above serotype is detected (Patent Document 1, etc.). However, not all cells of Escherichia coli that have enterohemorrhagic Escherichia coli serotypes produce verotoxin, but enterohemorrhagic Escherichia coli that has a certain serotype does not produce cells that produce verotoxin. There are fungus bodies. For example, about 95% of E. coli O157 produces verotoxin, and other serotypes of E. coli produce a lower percentage of verotoxin. Therefore, it cannot be determined from the serotype only whether it is verotoxin-producing Escherichia coli, that is, enterohemorrhagic Escherichia coli.

  In contrast, there has also been a method of indirectly diagnosing enterohemorrhagic Escherichia coli infection by detecting the production of verotoxin using an antibody against verotoxin or the like. In this method, Escherichia coli is cultured, and free verotoxin in the culture supernatant or bacterial cell extract is detected. However, in order to produce and secrete verotoxin with a small amount of verotoxin that can be detected in the culture supernatant, it took 1 to several days of culture, and extraction took time and effort. . Since E. coli capable of producing verotoxin is only a part of the total E. coli, the method for detecting verotoxin is to first detect and serotype E. coli from a sample such as feces, and then detect verotoxin. Only E. coli that could produce was further cultured to detect verotoxin. According to such a two-step method, there is an advantage that it is not necessary to detect verotoxin for all E. coli, but it requires two more cultures because it requires two cultures. In this method, since verotoxin is once released and isolated from the bacterial cells, the bacterial cells producing verotoxin were not directly detected. In particular, since verotoxin is toxic only when 6 kinds of two proteins are assembled, immunochemical methods for detecting released verotoxin react with individual proteins. Give incorrect measurement results. In addition, there is a problem that the accuracy of detection may be reduced due to sample misconception. As a method for detecting verotoxin released from bacterial cells, there were a reverse passive latex agglutination method, an EIA method, an immunochromatography method and the like.

  There is also a method of amplifying the verotoxin gene by gene amplification such as PCR and detecting the verotoxin gene (Patent Document 2, Patent Document 3, Patent Document 4, and Patent Document 5). However, this method requires a pretreatment of the specimen in order to prevent false positives, so that the operation is complicated.

JP 10-339731 A JP 07-008280 A JP 11-009281 A Japanese Patent Laid-Open No. 11-332599 Japanese Patent Laid-Open No. 2001-095576

  The present invention relates to the detection of verotoxin-producing E. coli, ie enterohemorrhagic E. coli. Specifically, the present invention relates to a method for directly detecting enterohemorrhagic Escherichia coli producing verotoxin by a latex agglutination method.

The present inventors solved the above-mentioned problems of the conventional method, and as a result of earnestly examining the method for detecting enterohemorrhagic Escherichia coli accurately in a short time, as a result of the slide aggregation method using an anti-verotoxin antibody, This book can detect enterohemorrhagic Escherichia coli quickly and accurately by detecting verotoxin directly on the surface of enterohemorrhagic Escherichia coli using the microorganism itself as a specimen without releasing verotoxin from the organism. The invention has been completed.
As mentioned above, conventionally, when trying to specifically detect verotoxin, latex particles in which enterohemorrhagic Escherichia coli verotoxin is released and separated from cells and sensitized with anti-verotoxin antibody have been used. It was detected by the reverse passive latex agglutination method.

  The present inventors examined a method for detecting verotoxin present on the surface of E. coli using the Escherichia coli cell itself as a specimen without releasing verotoxin. Verotoxin produced by enterohemorrhagic E. coli is stored in the periplasmic space between the cell wall and cell membrane of E. coli. Since verotoxin stored in the periplasmic space is difficult to be exposed on the surface, it was difficult to directly detect verotoxin on bacterial cells using an antibody. The present inventors have found that verotoxin can be exposed by partially breaking the cell wall of E. coli and can be measured with an anti-verotoxin antibody, and the present invention has been completed.

That is, the present invention is as follows.
(1) A method for detecting enterohemorrhagic Escherichia coli comprising reacting anti-verotoxin antibody-sensitized particles with Escherichia coli cells and measuring the degree of aggregation of the reaction mixture,
(2) The method for detecting enterohemorrhagic Escherichia coli according to (1), comprising treating E. coli cells with polymyxin B,
(3) The method for detecting enterohemorrhagic Escherichia coli according to (1) or (2), wherein the particles are latex particles,
(4) The method for detecting enterohemorrhagic E. coli according to (3), wherein the latex particles have a diameter of 0.1 to 1 μm,
(5) The method for detecting enterohemorrhagic Escherichia coli according to (3) or (4), which is a slide agglutination method,
(6) An enterohemorrhagic Escherichia coli detection kit for detecting verotoxin on E. coli cells, comprising anti-verotoxin antibody-sensitized particles, and (7) the sensitized particles are sensitized latex particles. Enterohemorrhagic Escherichia coli detection kit.
The present invention is as follows.
[1] Detection of enterohemorrhagic Escherichia coli characterized by reacting anti-verotoxin antibody-sensitized particles with Escherichia coli cells having verotoxin bound to the surface of the cells and measuring the degree of aggregation of the reaction mixture Method.
[2] The method for detecting enterohemorrhagic Escherichia coli according to [1], comprising measuring the degree of aggregation of the reaction mixture by a change in absorbance or a change in intensity of scattered light.
[3] The method for detecting enterohemorrhagic Escherichia coli according to [1] or [2], which comprises a step of destroying the cell wall of Escherichia coli cells.
[4] The method for detecting enterohemorrhagic Escherichia coli according to any one of [1] to [3], wherein measurement is performed using an analytical measurement device.
[5] Treating E. coli cells with polymyxin B, reacting anti-verotoxin antibody-sensitized particles with E. coli cells in which verotoxin is bound to the surface of the cells, and measuring the degree of aggregation of the reaction mixture. A method for detecting enterohemorrhagic Escherichia coli according to any one of [1] to [4].
[6] The method for detecting enterohemorrhagic Escherichia coli according to any one of [1] to [5], wherein the polymyxin B concentration in the reaction mixture is 1,000 to 10,000 units / mL.
[7] The method for detecting enterohemorrhagic Escherichia coli according to any one of [1] to [6], wherein the measurement is performed in a plastic cell or a glass cell.
[8] The method for detecting enterohemorrhagic Escherichia coli according to any one of [1] to [7], wherein the carrier of the anti-verotoxin antibody-sensitized particles is latex.
[9] The method for detecting enterohemorrhagic Escherichia coli according to [8], wherein the latex particles used for the anti-verotoxin antibody-sensitized particles are polystyrene latex particles having a diameter of 0.1 to 1 μm.

  According to the method of the present invention, as shown in Example 2, the verotoxin on the surface can be detected using the microbial cells of enterohemorrhagic Escherichia coli itself as a sample, and no extra culturing operation is required. Enterohemorrhagic E. coli can be detected.

Hereinafter, the present invention will be described in detail.
1. Preparation of anti-verotoxin antibody-sensitized particles The present invention sensitizes anti-verotoxin antibodies to carrier particles, that is, binds or adsorbs them, mixes the sensitized particles with enterohemorrhagic Escherichia coli cells, reacts them, and aggregates. And verotoxin present in the wall of E. coli is directly detected based on the presence or absence of the aggregate.

  As the carrier particles for sensitizing the anti-verotoxin 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 and the like can be used, but it is preferable to use latex particles. Examples of latex particles include homopolymer and / or copolymer particles of vinyl monomers such as vinyl chloride, acrylonitrile, vinyl acetate, acrylic acid esters, and methacrylic acid esters, polystyrene latex particles, and styrene-butadiene copolymers. Butadiene copolymer latex particles such as methyl methacrylate-butadiene copolymer, and polyvinyl toluene latex particles. Among these, polystyrene-based latex particles are preferable because they are excellent in adsorptivity for various proteins or polypeptides, and can retain biological activity stably for a long period of time. The particle size of the latex particles is preferably 0.01 to 1 μm, more preferably 0.1 to 1 μm. If the particle size is less than 0.01 μm, microaggregation occurs frequently, the apparent particle size becomes non-uniform, and the simultaneous reproducibility may be adversely affected, and sufficient aggregation is obtained for the number of antibodies. It may not be possible. When the particle size exceeds 1 μm, self-aggregation proceeds and the dispersibility decreases. 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 cell and the carrier becomes clear, and the reaction of the target cell to the carrier It is further advantageous in that the characteristics can be easily and accurately discriminated.

  The anti-verotoxin antibody can be obtained by purifying verotoxin from enterohemorrhagic Escherichia coli and using the purified verotoxin as an immunogen by a known method. Verotoxin can be purified by methods such as Ito et al., Microbial Pathogenesis 1988; 5: 189-195, Noda et al., Microbial Pathogenesis 1987; 2: 339-349. A recombinant verotoxin prepared by a genetic engineering technique can also be used. There are two types of verotoxin, verotoxin type 1 (VT1) and verotoxin type 2 (VT2). Since enterohemorrhagic Escherichia coli that produces only VT1 or VT2 exists, antibodies that recognize VT1 and VT2 Both of the recognized antibodies need to be prepared. Antibodies can be prepared by using VT1 and VT2 separately as immunogens and mixed, or both can be mixed to prepare both as immunogens at once. Alternatively, anti-VT1 antibody and anti-VT2 antibody may be prepared separately, and latex particles may be separately sensitized and used. In this case, VT1 and VT2 can be detected separately by each sensitized latex particle. VT1 and VT2 can also be detected simultaneously using a mixture of VT1-sensitized latex particles and VT2-sensitized latex particles. Moreover, since VT1 and VT2 are composed of subunits A and B, subunits may be used as immunogens. The antibody may be a polyclonal antibody or a monoclonal antibody. Polyclonal antibodies use purified verotoxin as an immunogen to immunize animals such as goats and rabbits, obtain antiserum, and use antiserum to deposit ammonium sulfate, and ion exchange chromatography using anion exchangers such as DEAE cellulose. It can be purified by appropriately selecting a known method such as molecular sieve chromatography, hydroxyapatite chromatography, affinity chromatography, etc., which is classified according to chromatography, molecular weight and structure, or a combination thereof. 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). At this time, purified verotoxin may be used as an immunogen, or an anti-verotoxin monoclonal antibody can be obtained by using an enterohemorrhagic Escherichia coli that produces verotoxin as it is as an immunogen. A hybridoma obtained by cell fusion of a spleen cell or lymph node cell of a mouse immunized with the above immunogen and a myeloma cell of a mouse is prepared, and the culture supernatant of the hybridoma or the ascites of a mouse administered the hybridoma intraperitoneally Can be prepared. 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. When antibody purification is required, it can be purified by the method described above. A commercially available verotoxin antibody may also be used. As a commercially available anti-verotoxin antibody, for example, one manufactured by Virostat is available.

  The method for sensitizing the antibody to the carrier is not particularly limited. For example, the antibody may be physically adsorbed on a carrier or chemically bound. More specifically, for example, after mixing an 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. Furthermore, it is desirable to heat at 30 to 37 ° C. for 1 to 2 hours, followed by heating at 50 ° C. for 30 minutes. The amount of antibody sensitized to the carrier can be appropriately set according to the particle size of the carrier used. For example, latex particles can be sensitized by mixing an equal amount of 0.1 to several mg / mL antibody solution into a latex particle suspension prepared to 1% by weight. 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 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 to the monoclonal antibody can usually be 0.1 to 1.0% by weight, preferably 0.2 to 0.5% by weight, based on the medium dispersion. Bovine serum albumin, gelatin, gum arabic and the like may be added to the medium as necessary.

2. Latex aggregation method As a specimen of the enterohemorrhagic Escherichia coli detection method of the present invention, a sample contaminated with Escherichia coli can be used, for example, feces, urine, blood, tissue homogenate and the like. Moreover, you may use food material. A part of these specimens is collected and cultured on an agar medium. At this time, DHL agar or McConkey agar can be used as the separation medium.

  After culturing for 6 to 20 hours, about 1 to 3 platinum ears are collected from the colony appearing on the agar medium with platinum ears or the like. The collected E. coli is treated with a reagent capable of lysing the cell wall of E. coli to destroy the cell wall. Examples of reagents that can dissolve the cell wall of E. coli include enzymes, alkaline solutions, and polymyxin B solutions. When polymyxin B is used, it is used at a concentration of 1,000 to 10,000 unit / mL, preferably 3,000 to 7,000 unit / mL, particularly preferably 4,000 to 6,000 unit / mL. The collected E. coli may be suspended in the polymyxin B solution. At this time, several tens of μL, preferably 10 to 50 μL of polymyxin may be dropped on a slide glass or the like to be reacted, and the collected E. coli may be suspended therein.

  The aggregation reaction between E. coli and anti-verotoxin-sensitized particles is performed on a glass slide. In this case, the degree of aggregation of the carrier particles can be measured visually. Moreover, it can also carry out in a plastic cell or a glass cell. In this case, visible light to near-infrared light is irradiated from the outside of the cell, and a change in absorbance or an intensity change of scattered light is detected to measure the degree of aggregation of carrier particles. Aggregation is measured using, for example, a fully automatic LPIA-S500 latex agglomeration measuring instrument manufactured by Mitsubishi Chemical Corporation, a COBAS FARA apparatus and a COBAS MIRA apparatus manufactured by Roche Diagnostics Systems, and a Hitachi 7070 analyzer manufactured by Hitachi, Ltd. It can be carried out.

  The agglutination reaction may be performed in a physiological saline solution or an appropriate buffer solution having a pH of 5.0 to 10, for example, a phosphate buffer solution, borate buffer solution, Tris buffer solution or the like. At this time, an appropriate surfactant such as Triton X-100, Tween 20, or Tween 80 may be added to suppress non-specific binding between the sensitized particles and E. coli.

(Example)
Hereinafter, the present invention will be described specifically by way of examples. However, the present invention is not limited to these examples.

Preparation of sensitized particles As latex particles, polystyrene latex particles having a diameter of 0.34 μm were used (PROLABO, estapor K035). As a sensitizing antibody to latex particles, a purified antibody obtained by purifying an anti-verotoxin rabbit serum using an affinity column bound with purified verotoxin as follows was used.

A) Method for producing anti-verotoxin serum Purified verotoxin type 1 or type 2 was inoculated into rabbits according to the following schedule, and the collected blood was allowed to stand overnight, and the centrifuged supernatant was used as antiserum.
Weeks 1 to 4: Purified verotoxin type 1 or type 2 is mixed with an adjuvant and inoculated weekly into rabbit limbs.
Type 5-8 weeks: Rabbit ear vein is inoculated weekly with purified verotoxin type 1 or type 2.

B) Purified antibody production method
Anti-verotoxin serum diluted 5 times with 0.075M phosphate buffer solution (pH 7.2) containing 0.5M sodium chloride is poured into an affinity gel prepared by binding purified verotoxin. After binding to the gel, anti-verotoxin antibody was eluted with 2M aqueous sodium thiocyanate. Next, this eluate is added to Sephadex G-25 equilibrated with PBS, and eluted with PBS to obtain a purified antibody.

The purified antibody was sensitized to latex particles by the following method.
Equal volume of purified antibody solution (1mg / mL) is mixed with latex suspension adjusted to 1% by weight with PBS, kept at 37 ° C for 1 hour, and further heated at 50 ° C for 30 minutes. Thereafter, PBS containing 0.5% by weight bovine serum albumin is added and allowed to stand for 16 hours. Latex particles are collected by centrifugation and resuspended in PBS containing 0.5% by weight albumin to obtain a sensitized latex solution.

Detection of enterohemorrhagic Escherichia coli using anti-verotoxin antibody-sensitized latex particles Brain heart infusion of E. coli O26, O1, O18, O157, and OUT strains The cells were cultured on an agar medium (manufactured by DIFCO).
Divide the slide glass into several sections with a glass pencil, drop 25 μL of physiological saline containing 5,000 unit / mL polymyxin B (manufactured by Pfizer) into each section, and then use the platinum loop to put the above E. coli on the agar medium. Ears were collected and suspended in polymyxin B solution on a slide glass. Next, 25 μL of the anti-verotoxin antibody-sensitized latex particle suspension prepared in Example 1 was added, stirred for 5 minutes with a slide mixer, and the formation of aggregates was visually observed and judged. Table 1 shows the results.

As shown in Table 1, Escherichia coli producing verotoxin became positive, and Escherichia coli not producing verotoxin became negative.
After veromixin-producing Escherichia coli was treated with polymyxin B, it was centrifuged and the supernatant was confirmed to contain verotoxin by reverse passive latex agglutination (RPLA) (using a Denka Seiken kit). Thereafter, when mixed with the anti-verotoxin-sensitized latex particles of the present invention, no aggregation was observed. However, when the same amount of Escherichia coli cells of the same strain were reacted with the anti-verotoxin antibody-sensitized latex particles of Example 1, aggregation was observed. Further, after the E. coli was treated with physiological saline instead of polymyxin B, the E. coli cells were reacted with the anti-verotoxin antibody-sensitized latex of Example 1, and no aggregation was observed. This result shows that, by polymyxin B treatment, verotoxin is liberated, and verotoxin bound to the cell surface is exposed so that the cell itself can be detected by the aggregation method of the present invention.

Claims (8)

A method for detecting enterohemorrhagic Escherichia coli, comprising reacting anti-verotoxin antibody-sensitized particles with Escherichia coli cells having verotoxin bound to the surface of the cells, and measuring the degree of aggregation of the reaction mixture. And detecting the cell wall of Escherichia coli cells .   The method for detecting enterohemorrhagic Escherichia coli according to claim 1, comprising measuring the degree of aggregation of the reaction mixture by a change in absorbance or a change in intensity of scattered light. 3. The method for detecting enterohemorrhagic Escherichia coli according to claim 1 or 2 , wherein the measurement is performed using an analytical measurement device. Treating E. coli cells with polymyxin B, reacting anti-verotoxin antibody-sensitized particles with E. coli cells having verotoxin bound to the surface of the cells, and measuring the degree of aggregation of the reaction mixture Item 4. The method for detecting enterohemorrhagic Escherichia coli according to any one of Items 1 to 3 . The method for detecting enterohemorrhagic Escherichia coli according to any one of claims 1 to 4 , wherein the concentration of polymyxin B in the reaction mixture is 1,000 to 10,000 units / mL. The method for detecting enterohemorrhagic Escherichia coli according to any one of claims 1 to 5 , wherein the measurement is performed in a plastic cell or a glass cell. The method for detecting enterohemorrhagic Escherichia coli according to any one of claims 1 to 6 , wherein the carrier of the anti-verotoxin antibody-sensitized particles is latex. The method for detecting enterohemorrhagic Escherichia coli according to claim 7 , wherein the latex particles used for the anti-verotoxin antibody-sensitized particles are polystyrene latex particles having a diameter of 0.1 to 1 µm.