JP4614313B2 - Simple, quick and safe Q fever diagnostic method and diagnostic agent - Google Patents

Description

  The present invention relates to the production of a Q fever diagnostic agent containing an antigen of Bacillus subtilis or an antibody that binds to the antigen, and a method for diagnosing Q fever using them.

  Q fever (Cokesiosis) is a zoonotic disease caused by infection with the bacterium Koksiella Bernetti. It is called Q fever in humans and is suspected to be associated with chronic fatigue syndrome of unknown cause. In animals (including birds) it is called coccidiosis. Most of the animals are inapparent and are considered to be less lethal.

  The main causative agent of Q fever is a kind of bacteria, which is classified as Legionella sp. Like other Legionella bacteria, Bacillus subtilis is an animal cell parasite and divides and proliferates only in animal cells. This bacterium is Gram-negative, small gonococcus and large and small polymorphisms, and shows a phase mutation of phase I / II, similar to S / R (smooth / rough) mutation of E. coli. Phase I bacteria are found in freshly isolated bacteria and are highly toxic. The cell surface is covered with long-chain lipopolysaccharide (LPS). Phase II bacteria arise from subculture of phase I bacteria, are attenuated, and the cell surface is covered with short-chain LPS.

  The clinical picture of Q fever is diverse and can be divided into acute and chronic types. The acute form has many influenza virus-like infections characterized by fever, headache, muscle pain, joint pain, coughing, and the like. There is also a tendency to develop pneumonia and hepatitis. The prognosis is generally good, and fever and recovery usually takes about 2 weeks. On the other hand, the chronic symptom is often endocarditis, particularly with chronic hepatitis and myocarditis.

  Antibiotics (tetracyclines) can be used to treat Q fever. However, in the current situation where there is no useful and simple diagnostic agent, it takes time until the diagnosis of Q fever is made, and then antibiotics are administered gradually, so the effect of antibiotics spreads the infection It is not enough to prevent this.

  It is thought that mites are involved in the route of infection of humans with the body of Kokusiera. In addition, it is estimated that birds, such as outdoor animals and pigeons, and livestock include cattle, sheep, pets, etc., and humans are considered to be terminal hosts. The main route of infection to humans is inhalation, but some oral infections are also suspected. Infections of Coccoliella in humans are estimated to be tens of percent, but accurate statistical results have not yet been obtained. One reason for this is the lack of useful diagnostic agents.

  In general, it is difficult clinically to distinguish Q fever from other infections at an early stage, and it may be misdiagnosed as influenza infection. If a simple and highly sensitive Q fever diagnostic agent is developed, it is expected to contribute to the solution of this problem.

So far, three types of methods are known as Q heat detection methods.
1) Serological detection method 2) Genetic detection method 3) Detection method by pathogen isolation

  Although each of the known Q heat detection methods has advantages, the following problems have been pointed out for use in diagnostic agents.

  In serological (immunological) detection methods, the quantitative nature of fluorescent antibody method (FA) is not ensured, reaction time is relatively long, automation of measurement is difficult, or enzyme-labeled antibody method (ELISA) There was a problem that the amount of antibody used was large. When a microtiter plate is used as a carrier, an operation (B / F separation) is required to separate bound and free antigens or antibodies in the reaction solution, and the centrifuge required for this operation is expensive. . Further, since K. sierra bacteria divide and proliferate only in animal cells, serum has been used as an essential nutrient when culturing K. sierra bacteria in the production of antigens in this diagnostic method. This serum is contaminated with anti-Coxiella bacteria antibody, and it is considered that there is a possibility of forming a Coxiella bacteria-anti-Coxiella antibody complex, which is inappropriate as an antigen material for a diagnostic agent. In recent years, culture methods using a serum-free medium not containing an antibody corresponding to an antigen have been developed, but these culture methods have not been used for the culture of Coccoliella.

  In the genetic detection method (PCR method or the like), since necessary equipment and reagents are expensive, there are problems that facilities that can be used are limited and convenience is lacking. When used in a general medical facility, it is difficult to become a first-choice diagnostic method.

  The detection method by pathogen separation has a problem that it is not suitable for early diagnosis because of the long culture days. In addition, the facility had to be equipped with biohazard countermeasures and lacked simplicity.

  In other words, as a diagnostic method for Q fever, a method with high sensitivity, high selectivity, rapidity, and simplicity has not been developed so far, and a diagnostic method with less economic burden from the viewpoint of facilities and necessary equipment Did not exist. The development of a simple and rapid Q fever diagnostic method has been demanded all over the world.

Prior art document information related to the invention of the present application is shown below.
Sa, V. Et al., J.Clin. Microbiol. 1996. 34: 2947-2951

  The present invention has been made in view of such a situation, and an object of the present invention is to detect an antibody that binds to the antigen present in a test sample by using a diagnostic agent containing the antigen of Coccoliella. Or a method for detecting Q fever including a step of detecting an antigen that binds to the antibody present in a test sample using a diagnostic agent that includes an antibody that binds to an antigen of Coccoliella. There is to do. Furthermore, the present invention also provides a Q fever diagnostic agent comprising an antigen of Bacillus subtilis or an antibody that binds to the antigen.

  In order to solve the above-mentioned problems, the present inventors have developed an epidemiology of Q fever (Coxiellasis), a diagnostic method and a diagnostic agent. Since Kokusiella is a kind of intracellular parasite, the culture of Kokusiella usually uses animal cell culture technology. Since serum is usually added to the animal cell culture medium, the serum-added culture method has also been used in the culture of Coccoliella. When the culture obtained in this way is used as an antigen material for a diagnostic agent, there is a risk that a serum-derived anti-Coxiella antibody and a body of the B. coccella form a complex and interfere with detection sensitivity as a diagnostic agent.

  In order to solve the above problems, the inventors of the present invention have studied the culture conditions of bacterial cells. As a result, the present inventors have found for the first time that the culture of K. sierra can be performed by a serum-free culture method. It was clarified that it was possible to produce a diagnostic reagent for Q fever by serum-free culture method. In addition, using these diagnostic agents, an immunoassay method that can detect Cocciella with high sensitivity, for example, a latex agglutination method was established.

  That is, the present inventors have succeeded in developing a Q fever diagnostic agent comprising an antigen of K. sierra fungus or an antibody that binds to the antigen, and developing a method for diagnosing Q fever using them. It came to complete.

More specifically, the present invention provides the following (1) to (24).
(1) A Q fever diagnostic method comprising a step of detecting an antibody that binds to the antigen present in a test sample using a diagnostic agent containing an antigen of Coccoliella.
(2) The diagnostic method according to (1), wherein the antigen is an antigen comprising at least one selected from the following (a) and (b).
(a) Inactivated whole cell body coccyella obtained by incubating after incubating cocciella
(b) The inactivated antigen protein (3) obtained by inactivating one or a plurality of antigen proteins derived from Bacillus subtilis is an antigen obtained by the following steps (a) and (b): Diagnosis method.
(a) A process of culturing Kokusiella bacteria and producing a Kokusiella culture preparation
(b) A step of inactivating the whole cell body cocciella by adding an inactivating agent to the culture product of kokusiella (4) The antigen is an antigen obtained by the following steps (a) and (b) The diagnostic method according to (1).
(a) The process of culturing K. sierra
(b) a step of inactivating the antigen protein by adding an inactivating agent to one or a plurality of antigen proteins derived from K. sierra fungus (5) The diagnostic method according to any one of (2) to (4), comprising any one of the steps.
(a) Step of serum-free culture of K. sierra
(b) Step of culturing Kokusiella bacteria with serum (6) The Koxiella bacteria are wild strains, clinical isolates, or artificial mutants derived therefrom, or recombinant strains, or any one of (1) to (5) (7) The diagnosis method according to any one of (1) to (6), wherein the K. sierra fungus is K. sierra burnetti, and (8) using a diagnostic agent comprising an antibody that binds to the antigen of K. sierra A Q-fever diagnostic method comprising a step of detecting an antigen that binds to the antibody present in a test sample.
(9) The diagnostic method according to (8), wherein the antibody is a monoclonal antibody that recognizes a part of the body of the body of the body.
(10) The diagnostic method according to (9), wherein the monoclonal antibody is at least one selected from a monoclonal antibody that recognizes an outer membrane glycoprotein antigen of Bacillus subtilis and a monoclonal antibody that recognizes lipopolysaccharide.
(11) The Q fever diagnosis method according to any one of (8) to (10), wherein the antigen used in preparing the diagnostic antibody is one of the following (a) and (b):
(a) Inactivated whole cell body coccyella obtained by incubating after incubating cocciella
(b) The antigen used when preparing the antibody of the inactivated antigen protein (12) diagnostic agent in which one or a plurality of antigen proteins derived from Bacillus subtilis is inactivated. The following steps (a) and (b) The Q-fever diagnostic method according to any one of (8) to (10), which is an antigen obtained by:
(a) A process of culturing Kokusiella bacteria and producing a Kokusiella culture preparation
(b) a step of adding an inactivating agent to the kokusiella culture preparation to inactivate whole cell body cocciella (13) The antigen used in preparing the antibody of the diagnostic agent comprises the following (a) and The Q fever diagnosis method according to any one of (8) to (10), which is an antigen obtained by the step (b).
(a) The process of culturing K. sierra
(b) a step of inactivating the antigen protein by adding an inactivating agent to one or a plurality of antigen proteins derived from K. sierra bacteria (14) Cultivation of K. sierra bacteria is performed by the following (a) or (b) The Q fever diagnosis method according to any one of (11) to (13), which is any one of the steps.
(a) Step of serum-free culture of K. sierra
(b) The step of culturing Koshiera bacteria with serum (15) The diagnostic method according to any one of (1) to (14), wherein an immunoassay is used.
(16) The diagnostic method according to any one of (1) to (15), wherein a latex agglutination method is used.
(17) A Q fever diagnostic reagent comprising an antigen of K. sierra fungus or an antibody that binds to the antigen.
(18) The Q fever diagnostic agent according to (17), wherein the antigen is an antigen comprising at least one selected from the following (a) and (b):
(a) Inactivated whole cell body coccyella obtained by incubating after incubating cocciella
(b) An inactivated antigen protein (19) antigen obtained by inactivating one or a plurality of antigen proteins derived from Bacillus subtilis is an antigen obtained by the following steps (a) and (b): The described Q fever diagnostic agent.
(a) Step of culturing Kokusiella bacteria to produce Kokusiella culture preparations
(b) a step of adding an inactivating agent to the kokusiella culture preparation to inactivate the whole cell body cocciella (20) The antigen is an antigen obtained by the following steps (a) and (b) The Q fever diagnostic agent according to (17).
(a) The process of culturing Kokusiera bacteria
(b) a step of adding an inactivating agent to one or a plurality of antigen proteins derived from K. sierra fungus to inactivate the antigen protein (21) The Q fever diagnosis method according to any one of (18) to (20), which is any step.
(a) Step of serum-free culture of K. sierra
(b) A step of culturing Kojiella bacteria with serum (22) The Q fever diagnostic drug according to any one of (17) to (21), wherein the antibody is a monoclonal antibody that recognizes a part of Kokusiella cells.
(23) The Q fever diagnostic agent according to (22), wherein the monoclonal antibody is at least one selected from a monoclonal antibody recognizing an outer membrane glycoprotein antigen of Bacillus subtilis and a monoclonal antibody recognizing a lipopolysaccharide.
(24) A diagnostic kit comprising the diagnostic agent according to any one of (17) to (23).

  Previously, antigenic material produced by serum-added culture was present as a component of Q-fever diagnostics, but it was possible that anti-Coxiella bacteria antibodies and cocciella cells in the serum formed a complex There is a possibility that the accuracy as a diagnostic agent may include a problem. The Q-fever diagnostic agent of the present invention comprising an antigen obtained by serum-free culture of K. sierra fungus and an antibody that binds to the antigen is obtained by growing K. sierra fungus in a serum-free medium, thereby allowing anti-K. Sierra fungus contained in the serum. It is expected to be able to prevent antibody contamination and establish a diagnostic method with higher sensitivity than conventional Q fever diagnostic agents, and is very useful in the medical and pharmaceutical fields. By combining with a latex agglutination method or the like that has been used in other examinations in the past, it is expected that qualitative and quantitative determination of Cocciella bacteria can be carried out simply, quickly and inexpensively.

  In the present invention, it is also possible to use a monoclonal antibody or a polyclonal antibody for recognizing a part of the body of the body of Coxiella, for diagnosis. If a combination of these antibodies is used, the reactivity with the antigen is improved. Antigen detection sensitivity is expected to improve.

  Such high-sensitivity Q fever diagnostics can be applied not only to human Q fever diagnosis, but also to diagnosis of pets, domestic animals, outdoor animals and birds that are considered to be the source of infection to humans. It is also useful in the field of livestock industry.

  The present invention provides a Q fever diagnostic agent comprising an antigen of Bacillus subtilis or an antibody that binds to the antigen. The present invention also relates to a Q fever diagnostic method (test method) using the above-mentioned Q fever diagnostic agent.

  The diagnostic agent of the present invention provides a composition capable of simple, rapid and reliable diagnosis based on the Q fever detection method. The diagnostic agent of the present invention makes it possible to specifically detect an antigen or antibody of Q fever in a specimen sample by an immunological technique, thereby supporting clinical diagnosis of coccierella infection.

  In the present invention, various immunological techniques can be used to detect the antigen or antibody of Bacillus subtilis. In the diagnostic agent of the present invention, examples of immunological techniques that can be used for the detection of Coccoliella are enzyme immunization (ELISA), immunoelectrophoresis, latex agglutination, immunoturbidimetry, immunochromatography, radioimmunodiffusion, A fluorescent antibody method can be exemplified, but is not necessarily limited thereto.

  Among these, preferred immunodetection methods as diagnostic agents for Q fever include enzyme immunization (ELISA), immunoelectrophoresis, latex agglutination, and radioimmunodiffusion. These techniques allow qualitative as well as quantification of Cocciella. Among these, a latex agglomeration method is a more preferable method. According to the latex agglutination method, qualitative and quantitative determination of Coccella can be performed easily, rapidly, and inexpensively.

  As the antigen used for the diagnostic agent of the present invention, it is possible to use K. sierra fungus obtained from a conventional serum-added culture medium or an antigen derived therefrom, and a K. sierra fungus obtained from serum-free culture or an antigen derived therefrom. Although it is possible, more preferably, K. sierra bacteria obtained by serum-free culture or an antigen derived therefrom is used. When using K. sierra bacteria obtained by serum-free culture or an antigen derived therefrom, it is possible to prevent contamination of serum-derived anti-K. Sierra antibodies with the antigen as the diagnostic composition.

  Moreover, since the antibody used for the diagnostic agent of this invention can use a monoclonal antibody, its specificity is high.

  The diagnostic procedure for detecting the antigen or antibody of the present invention is described below. 1. production of antigens; 2. production of antibodies; The antigen detection measurement method and the 4 antibody detection measurement method will be described in this order.

First, the method for producing an antigen in the present invention will be described.
The antigenic material of C. sierra is used as one of the diagnostic agent compositions for antibody detection. For this reason, phase II bacteria are preferred for safety reasons, but phase I bacteria can also be used after being appropriately inactivated. In addition, the antigenic material of Coccoliella is used as a standard antigen in the antigen detection method. The antigen in this case is preferably a phase I bacterium with strong pathogenicity, but a phase II bacterium can also be used.

  In the present invention, as a strain used for culture, it can be used after being purified from field strains or clinical isolates of Cocciella, or artificial mutants of these strains can also be used. . As the bacterial species, Kokusiella Bernetti is preferable, and among these bacterial species, the Nine Mile strain is more preferable. The Coxiella Bernetti Nine Mile strain can be obtained as the VR615 strain from ATCC, a strain preservation organization. In the case of using Cokesiella Bernetti Nine Mile strain II phase bacteria, clinically isolated phase II bacteria can also be used if they are purified and purified. The present inventors used a strain distributed as a yolk sac emulsion by Dr. Kazar, J. (Slovakia) and then stored at the Kitasato Institute.

  As for the antigen of the body sierra germ, the form which uses the whole body body of body sierra germ, the form which uses the antigenic protein which has part of the body component, for example, the antigenicity by crushing body sierra germ, the antigen protein which is produced in the cell culture solution It can be obtained and used either in a form to be used or in a form in which an antigen is collected from a DNA recombinant microorganism encoding the antigen protein.

  The antigenic protein used in the present invention is not particularly limited, but materials containing known proteins can be used. For example, the following three articles Zhang, GQ, et al. J. Clin. Microbiol. 42: 423-428 (1998), Nguyen, Sa V., et al. FEMES Microbiol. Lett. 175: 101-106 (1999) , Nguyen, Sa V., et al, Microbiol. Immunol. 43: 743-749 (1999) can be used.

  Further, an antigen obtained in the vaccine production process can be used as the antigen material.

  When cultivating K. sierra fungus and using microbial cells or a crushed product thereof as an antigen material, a serum-free medium is more preferably used for cell culture. However, if the reactivity between the antigens and antibodies of the resulting K. sierra is good, the K. sierra cells obtained by serum-added culture can be used.

  The reason why serum-free culture is desired or necessary in some cases in the production of an antigen used for a diagnostic agent is as follows. Commercially available serum used for culturing may be contaminated with anti-Coxiella antibodies. Commercially available sera are often derived from cattle, pigs, sheep, horses, etc., and these animals may generally be infected with coccyella. This is because the presence or absence of antibodies is not always examined.

  If C. sierra is cell-cultured using serum mixed with anti-C. Sierra antibody, the C. sierra product may be contaminated with serum-derived C. sierra antibody. In other words, the body of the body sierra may contain a body body of the body body-anti-body body antibody complex. This is inappropriate as an antigen material for a diagnostic agent. However, such a situation cannot occur in the body of Kokusiella obtained by serum-free culture. That is, it is desirable to use whole cells obtained by serum-free culture or antigen proteins derived therefrom as an antigen used as a diagnostic agent composition.

  If serum is used for cell culture of Coccoliella, it is desirable to use the serum after checking beforehand that the serum does not contain anti-Coxiella antibody. In addition, if whole cell antigen or antigen protein obtained by serum-added culture is used as an antigen of a diagnostic agent, it should be used after checking in advance that anti-Coxiella antibody is not mixed in the antigen material. desirable.

  In these cases, Kokusiella cells obtained by serum-free culture or antigen proteins derived therefrom are necessary as standard substances. Needless to say, it would be convenient and useful to have a diagnostic or diagnostic agent using this.

Examples of antigen acquisition methods are given below.
In the antigen acquisition of the present invention, the process of cultivating K. sierra fungus and producing a K. sierra fungus culture preparation will be described.

  In the present invention, when cultivating K. sierra fungus as a bacterial species, animal cells are used as a substrate, the cells are grown, and K. sierra fungus is grown therein. Examples of animal cells to be used include BGM (buffalo green monkey) cells, Vero cells, L-929 cells, and CHO cells in the case of recombinants.

  The medium for animal cell culture used for the production of the vaccine of the present invention is more preferably a serum-free medium. The serum-free medium can be used in any composition that is suitable for the growth of the cells used. The medium can contain a carbon source, nitrogen source, phosphate source, trace metals, etc., but serum is not used. In addition, a commercially available product can be used as the serum-free medium. For example, VP-SFM, Opti-Pro SFM, CD-CHO, and the like are more preferable as examples of commercially available serum-free media that can be used for culturing K. sierra bacteria, but are not limited thereto.

  In the culture of bacterial cells in the present invention, a glass culture vessel, a stainless steel vessel, a plastic vessel, a commercially available fermenter, or the like can be used as a cell culture vessel.

  In the cell culture of the present invention, the culture temperature is usually 35 to 37 ° C., but the temperature is not particularly limited as long as it is a temperature at which growth is possible.

  In the cell culture of the present invention, the pH range is usually from 6.8 to 7.6, but is not particularly limited to this pH, and the culture can be performed under acidic or alkaline conditions where the cells do not die.

  As the culture method of the present invention, any known method such as stationary culture, roller bottle culture, microcarrier suspension culture, microchip culture, and floating cell culture can be used. The optimal culture period and culture conditions can be selected depending on the cells used.

When culture is performed using a serum-free medium or serum medium, the number of coccyella bacteria can be measured as necessary to confirm the progress of the culture. In this measurement, the inclusions are also measured and totaled. The number of bacteria can be measured after staining for a short period of time, after staining and measuring with a microscope, or with a turbidity of 650 nm. Recently, it can also be measured as a DNA copy number using quantitative PCR. As a method for measuring the number (titer) of the body sierra germ, the following method can be specifically used according to a known method (Schneider, W. Zentralbl. Bakteriol. 271: 77-84, 1989). First, BGM cells are cultured in 24-well plates with coverslips. After inoculating a single layer sheet, inoculate 0.5 ml of each live suspension of C. sierra Bernetti, approximately 100 times, adsorb for 30 minutes, and then centrifuge at 1,500 rpm. After centrifugation, the supernatant is removed, 1 ml of MEM is added to each well, and cultured at 37 ° C. (CO ₂ 5%). On day 4 of culture, cover slips are collected and fixed with cold acetone. The number of inclusion bodies can be measured by the fluorescent antibody method (FA) or the enzyme antibody method. In the fluorescent antibody method, the serum of a mouse infected with C. sierra Bernetti is diluted with the primary antibody using phosphate buffered saline (PBS) and reacted at 37 ° C. for 60 minutes in a wet box. After washing 3 times with PBS for 5 minutes, FITC-labeled anti-mouse IgG sheep serum (manufactured by CAPPEL) is adjusted with PBS and reacted in the same manner. After washing, adjust to 1,200 times with propidium iodide and react for 5 minutes. After washing, the cells are sealed with Slow Fade antifade Kits (Moleculer Probes), and the number of sealed pairs showing strong color development under a fluorescence microscope (BX-50-FLA, OLYMPUS) is measured.

  As the bacterial cell culture method of the present invention, a recombinant microorganism can be cultured, and in this case, it can be cultured by a culture method suitable for each condition.

  The recombinant antigen can also be produced by a recombinant cell organism, and the following known methods can be applied mutatis mutandis. As the microbial cells, Kokusiera bacteria isolated from patients is used. Usually, clinical isolates have both a protein antigen derived from Coccoliella and a protein antigen derived from heat-resistant Cocciella. First, a protein antigen derived from K. sierra is isolated. A gene encoding a protein antigen derived from K. sierra fungus (hereinafter referred to as “koku sierra antigen gene”) (6.7 kbp) is excised from the plasmid by restriction enzyme treatment or the like and ligated to a plasmid such as pBR322. For example, the following paper (Zhang, GQ, et al. J. Clin. Microbiol. 42: 423-428 (1998), Nguyen, Sa V., et al. FEMES Microbiol. Lett. 175: 101-106 (1999), Nguyen, Sa V., et al, Microbiol. Immunol. 43: 743-749 (1999)) can be used.

  Next, after the K. sierra antigen gene is amplified by PCR or the like, the K. sierra antigen gene is ligated to another expression vector. Next, position-directed mutagenesis is performed on this vector, and then amplified by PCR using selective primers to produce a recombinant Escherichia coli strain that produces a protein antigen derived from the recombinant mutant Koksiella. . This recombinant Escherichia coli can be cultured according to a conventional method, and a protein antigen derived from Coccoliella can be obtained from the culture solution.

  Recombination Escherichia coli producing a protein antigen mutant derived from Coccoliella can be obtained by performing position-directed mutagenesis during the above gene recombination operation. If this is cultured and purified, a protein antigen mutant derived from Coccella can be obtained. For example, as described above, by applying a position-specific mutagenesis method, it is possible to change an amino acid residue at any site to another residue in the amino acid sequence of a protein antigen derived from Bacillus subtilis. . Conversely, it is possible to retain a specific amino acid residue at a specific site, for example, a glutamic acid residue without change. The same applies to sugar residues. Among the obtained mutants, those having immunogenic activity are selected and used as protein antigen mutants derived from the body sierra fungus of the present invention. It should be noted that the mutant sicillus antigen activity of this mutant can be at an arbitrary level. In order to obtain a mutant, some trial and error experiments are required. However, the obtained recombinant mutant cocceria antigen generally has an advantage that it is difficult to return to a natural coccella antigen.

  In the process of producing the Kokusiella culture preparation, the whole cell antigen is washed, purified and concentrated after being cultured and produced in a serum-free medium. Hereinafter, cleaning, purification, and concentration methods will be described.

  After completion of the culture in the serum-free medium, the cells are collected, disrupted by freezing and thawing, sonication, etc., and then Cokesiella is collected from them. Centrifugation, gel filtration, salting out, etc. can be used for this method. Usually, after that, the cells are washed and purified with a phosphate buffer solution added with salt. A purification method, a centrifugal method, a stirring method, a concentration method, a column method, and the like can be used for purification, but the purification method is not limited to these. Cultivation, collection, washing, and purification of Coccella must be performed in an environment that allows for strict containment. Usually, a Biosafety Regulation Level 3 (BSL3) environment is required.

  In the present invention, one or a plurality of antigenic proteins derived from Coccellaella cultured in serum-free culture may be collected. The antigenic protein can be purified and collected by performing a treatment for partially destroying or completely destroying the body of the body sierra germ.

  In the case of producing a subunit vaccine, an effective antigen fraction may be collected by performing a treatment for partially destroying or destroying the body of the body sierra germ. Furthermore, when using the antigen protein produced in the culture solution, the antigen protein may be concentrated and purified by a combination of an effective centrifugation method, concentration method, column method and the like.

  The period of washing, purification, and concentration is preferably before inactivation, but can be performed after inactivation. You may implement both before and after inactivation.

  In the antigen acquisition of the present invention, the step of adding an inactivating agent to the coccoliella culture preparation and the like to inactivate the coccierella culture preparation and the like will be described below.

  Various methods can be used to inactivate the antigen. In general, the optimum inactivation method varies depending on whether the antigen is a whole cell or a protein. Inactivation can be carried out by various chemical treatment and physicochemical treatment methods. Examples thereof include, but are not limited to, the following.

  The following substances can be mentioned as chemical treatment substances used for treating bacterial cells or antigen-containing proteins for the purpose of inactivation. Although the concentration which can be used is shown together, it is not limited to these.

Formalin (0.1-10 v / v%), phenol (0.1-5 v / v%), chloroform
(10-60 v / v%), acetone (10-80 v / v%), SH reagent (1-100 mM),
Hydrogen peroxide (0.1-5%), peracetic acid (0.5-10 w / v%), carbon dioxide (5-90 v / v%),
Ozone (0.1-10 v / v%), surfactant (0.01 -5 w / v%),

  Moreover, the following means can be used for the physicochemical processing method for inactivation. Although an example of the conditions which can be used is shown below, it is not limited to these.

Heating treatment (temperature: 30-70 ° C; heating time: 10-120 minutes), gamma irradiation (radiation source: cobalt 60; 5-50 kGy (kilo gray)), laser light irradiation (light source: various laser irradiation devices) Wavelength 500-700 mn; light quantity: 0.01-1 J (joule) / cm ² ), electron beam irradiation (microwave oven), ultrasonic irradiation, and the like.

  The treatment conditions are not fixed, and suitable conditions can be set by changing the amount of cells, temperature, pH of the buffer solution, treatment time, and the like. It is usually operated under aseptic conditions.

  These inactivation methods may be used alone or in combination of a plurality of methods, regardless of whether they are whole cells or cells derived from cells (including antigenic proteins derived from culture broth). Also good. In the case of inactivation, coexistence of amino acids and amines may improve the stability of quality after inactivation, and these substances can be added as necessary. Moreover, in addition to formalin inactivation, by carrying out organic solvent treatment, heating treatment, and γ-ray irradiation treatment, it is possible to obtain stable inactivated whole cells with a quality that hardly causes lysis.

An example of inactivation is as follows. First, 1% (v / v) formalin is added to a live suspension of K. sierra fungus and left at room temperature for 5 days to inactivate. Thereafter, the mixture is centrifuged at 13,000 rpm for 15 minutes. After removing the supernatant, the suspension is suspended in an equal amount of physiological saline-added phosphate buffer (0.1 M, pH 6.8) to obtain an inactivated Coxiella burnetti suspension. Confirmation of inactivation can be confirmed by the fact that after inactivation treatment, a part of the sample is taken and cultured, the bacteria do not grow. After the inactivation treatment, the chemicals used for inactivation are removed by washing with a phosphate buffer or the like. Thereafter, the number of bacteria is adjusted so that the turbidity at 650 nm is about 1-10 (about 1-10 × 10 ⁹ cells / mL). Alternatively, the concentration of the antigen protein is adjusted.

Next, a method for producing the antibody of the present invention will be described.
The antibody material is used as a composition of an antigen detection type diagnostic agent. The antibody material is used as a standard antibody in the antibody detection type diagnostic method. The “antibody” of the present invention uses a gene recombination technique to highly express the cocciella antigen on the cell surface (natural cells, established cells, tumor cells, etc.) expressing the cocciella antigen of the present invention. The transformant prepared by using the polypeptide, the polypeptide constituting the coccyellae antigen, the cocciella polypeptide, or the fusion polypeptide containing the extracellular region of the cocciellae antigen is used as an antigen, and the antigen is used in mice, rats , Natural antibodies obtained by immunizing mammals such as hamsters, guinea pigs, and rabbits, chimeric antibodies and human antibodies (CDR-grafted antibodies) that can be produced using genetic recombination techniques, and human antibody-producing transgenic animals And the like, and human antibodies that can be produced using the above.

  Monoclonal antibodies include monoclonal antibodies having any isotype such as IgG, IgM, IgA, IgD or IgE. Preferably, it is IgG or IgM.

  A polyclonal antibody (antiserum) or a monoclonal antibody can be produced by an existing general production method. That is, for example, an antigen as described above, together with Freund's Adjuvant if necessary, a mammal, preferably a mouse, rat, hamster, guinea pig, rabbit, cat, dog, pig, goat, horse or cow. More preferably, mice, rats, hamsters, guinea pigs or rabbits are immunized.

  Polyclonal antibodies can be obtained from serum obtained from the immunized animal. Monoclonal antibodies are prepared from the antibody-producing cells obtained from the immunized animal and myeloma cells (myeloma cells) having no autoantibody-producing ability, and the hybridomas are cloned and used for immunization of mammals. It is produced by selecting clones that produce monoclonal antibodies that show specific affinity for the antigen.

  Specifically, the monoclonal antibody can be produced as follows. That is, an antigen as described above is used as an immunogen, and the immunogen is optionally combined with Freund's Adjuvant, and a non-human mammal, specifically a mouse, rat, hamster, guinea pig or rabbit, preferably Is subcutaneous, intramuscular, intravenous, or Hoodpad of mouse, rat or hamster (including transgenic animals produced to produce antibodies from other animals such as human antibody-producing transgenic mice described below) Immunization is performed by injecting into the abdominal cavity or abdominal cavity one to several times or transplanting. Usually, immunization is performed 1 to 4 times every about 1 to 14 days from the initial immunization, and antibody-producing cells are obtained from the immunized mammal about 1 to 5 days after the final immunization. The number of times of immunization and the time interval can be appropriately changed depending on the nature of the immunogen used.

  A hybridoma secreting a monoclonal antibody can be prepared according to the method of Kohler and Milstein et al. (Nature, 256, 495-497, 1975) and a modification method according thereto. That is, antibody-producing cells contained in spleen, lymph nodes, bone marrow, tonsils, etc., preferably spleen, obtained from a non-human mammal immunized as described above, and preferably mouse, rat, guinea pig, hamster, rabbit Alternatively, it is prepared by cell fusion with a myeloma cell having no autoantibody-producing ability derived from a mammal such as a human, more preferably a mouse, rat or human.

  For screening of hybridoma clones producing monoclonal antibodies, the hybridomas are cultured in, for example, a microtiter plate, and the reactivity of the culture supernatant of wells in which proliferation has been observed to the immunizing antigen used in the immunization described above is determined, for example. It can be carried out by measuring by an enzyme immunoassay such as RIA or ELISA.

  Production of monoclonal antibodies from hybridomas is carried out in vitro or in vivo in mice, rats, guinea pigs, hamsters or rabbits, preferably mice or rats, more preferably mice ascites, and the resulting culture supernatant. Or by isolation from ascites of mammals.

  When cultivating in vitro, in order to grow, maintain and preserve the hybridoma according to various conditions such as the characteristics of the cell type to be cultured, the purpose of the research and the culture method, etc., to produce a monoclonal antibody in the culture supernatant It is possible to carry out using any nutrient medium that is derived from a known nutrient medium or a known basic medium as used in the above.

  Isolation and purification of the monoclonal antibody can be carried out by using the above-mentioned culture supernatant or ascites, saturated ammonium sulfate, euglobulin precipitation method, caproic acid method, caprylic acid method, ion exchange chromatography (DEAE or DE52 etc.), anti-immunoglobulin column or It can be carried out by subjecting it to affinity column chromatography such as a protein A column.

  A “recombinant chimeric monoclonal antibody” is a monoclonal antibody produced by genetic engineering. Specifically, the variable region thereof is variable derived from an immunoglobulin of a non-human mammal (mouse, rat, hamster, etc.). It means a chimeric monoclonal antibody such as a mouse / human chimeric monoclonal antibody, characterized in that it is a region and the constant region is a constant region derived from human immunoglobulin.

  For example, transgenic mice producing human antibodies can be obtained from Nature Genetics, Vol. 7, p. 13-21, 1994; Nature Genetics, Vol. 15, p. 146-156, (1997), WO94 / 25585, Nature, Vol.368, p.856-859, (1994) and the like.

Next, the antigen detection type measurement method or antibody detection type measurement method of the present invention will be described.
There are two methods for diagnosing Cocciella by an immunological detection method: (1) a method using an antigen detection type diagnostic agent for detecting an antigen and (2) a method using an antibody detection type diagnostic agent for detecting an antibody. Can be mentioned. The optimum method is used in consideration of the detection sensitivity, selectivity, speediness of the detection operation, simplicity, and economy of the body sierra germ.

The latex agglutination method and others will be described below as an example of the antigen detection type measurement method.
The principle of the latex agglutination method is as follows.

  The antibody-sensitized latex in the latex reagent and the antigen in the specimen, or the antigen-sensitized latex in the latex reagent and the antibody in the specimen are bound and aggregated by the antigen-antibody reaction. This agglomerate increases with time, and the latex agglutination method is a method for quantifying the concentration of antigen or antibody from the change in absorbance per unit time obtained by irradiating the agglomerate with near infrared light.

  Antigen-antibody reaction aggregates generated by immunoturbidimetry are very small, and it is difficult to optically detect the degree of aggregation in a low concentration region with a small amount of antigen. In the latex agglutination method in which antibodies are sensitized (bound), the antigen-antibody reaction appears in the form of latex agglutination, and even when the amount of antigen in the low concentration range is small, it appears as a large agglomeration and a slight amount. Changes in agglomerates can also be captured optically.

  In order to carry out the latex agglutination reaction, the antibody-bound latex is mixed with the body sierra germ or antigen-containing sample. Incubate for several hours to around 20 hours, observe the resulting aggregated image visually or under a microscope, measure the limiting dilution, and quantify the amount of antigen.

  Various commercially available products can be used as the latex. Latex particles used in the present invention are not particularly limited as long as they can support an antigen or antibody, but it is usually preferable to use an organic polymer substance that is insoluble in the sample solution. Examples of the fine particles made of the organic polymer material include latex particles made of a synthetic polymer of carboxylic acid. Further, the latex particles having a diameter of 0.1-50 mcm (micrometers) can be used, but usually within the range of 0.1-20 mcm (micrometers).

  The antigen or antibody obtained by the method described above can be diluted with an appropriate buffer or physiological saline and used for latex sensitization. Examples of the buffer used include phosphate buffer, Tris buffer, and MOPS.

  As a method for sensitizing the above-mentioned antigen or antibody to latex particles, latex particles may be obtained by a physical adsorption method in which physical adsorption is caused by mixing latex particles and an antigen or antibody solution, or by a coupling agent. Examples include a chemical bonding method in which a surface carboxyl group or amino group is chemically bonded to an antigen or antibody. Further, antigens or antibodies may be bound to latex particles through spacer molecules. There is no particular restriction on the sensitization amount of the antigen or antibody to the latex particles, but in the case of latex particles, 0.01 to 0.5 mg is usually suitable for 1 ml of the latex suspension, but with the antigen or antibody of the present invention, The amount is preferably 0.01 to 0.15 mg per 1 ml of the latex suspension.

  As a suspension of latex particles sensitized with the above antigen or antibody, pure water or an appropriate buffer such as phosphate buffer, Tris buffer, MOPS or the like is used. Further, a dispersion stabilizer or an antioxidant surfactant may be added to the latex particle suspension.

  The latex agglomeration method of the present invention can be carried out by the following method. Latex particles sensitized with an antigen or antibody are mixed with a test sample such as serum or plasma collected from a specimen, and the target antibody or antigen in the sample and the antigen or antibody sensitized with latex particles And the degree of aggregation of latex particles produced by the reaction is measured, and the presence of antibody or antigen in the sample is detected or the concentration is calculated. Aggregation can be measured visually as a simple method. More preferably, the turbidity can be measured optically.

  In the case of using latex sensitized with the K. sierra antigen of the present invention, the anti-K. Sierra antibody concentration is calculated based on the degree of latex particle aggregation due to the reaction with the anti-K. Sierra antibody in the test sample. Can also be determined. The reaction is carried out in a microplate well or in a plastic cell or 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 intensity change of scattered light is detected to measure the degree of latex particle aggregation. At this time, it is possible to calculate the concentration of the antigen or antibody in the sample using a calibration curve prepared in advance.

  As an apparatus for performing such agglutination measurement, a photoelectric colorimeter, a microplate reader, and the like can be given. The agglutination reaction of the latex particles is usually performed in a solution such as physiological saline and a suitable buffer solution having a pH of about 5 to 10, such as a phosphate buffer solution. (In addition, in order to suppress non-specific binding between latex particles and protein in the sample, an appropriate surfactant, for example, an adsorption inhibitor such as Tritone 100 or Tween 20 may be added to the reaction solution. .) A diagnostic agent for use in an immunological test including the above reaction and measurement steps can be prepared by a known method that has been conventionally used. These diagnostic agents are provided by the same configuration as that of a diagnostic agent using a normal immunoaggregation reaction. The Q-fever diagnostic agent of the present invention includes at least a lyophilized latex reagent sensitized with an antibody derived from Cocciella or an antibody derived from Cocciella, and further includes a sample diluent, a condensate, a standard substance, and the like. Also good.

  The latex agglutination reaction of the present invention can be combined with a slide agglutination method for antigen detection. At this time, a 24 hole, 48 hole, or 96 hole microplate can be used as the microplate. In consideration of reactivity, flat bottom, round bottom, etc. are prepared. When the aggregation reaction is performed using these microplates, there is a possibility that the detection can be automated. In addition, the latex agglutination method of the present invention can be used for antigen detection in combination with a microtiter method.

  EXAMPLES Hereinafter, the present invention will be described more specifically with reference to examples, but the present invention is not limited to these examples.

[Example 1] Serum-free culture of K. sierra fungus As a strain, K. sierra fungus Nine Mile strain (ATCC VR 615) was used. Vero cells (ATCC CCL-81) were used for cell culture of Coccoliella, and VP-SFM, which is a serum-free medium, was used for the cell growth medium.

A large flask with Vero cells in serum-free culture in a single layer, or Vero cells cultured with microcarriers was inoculated with K. sierra fungus and cytopathic effect (CPE) appeared after culturing at 37 ° C for 5-7 days After confirming this, the culture solution was recovered. The culture solution was filtered using a filter having a pore size of 0.45 μm.
All subsequent operations were performed in a facility environment where microbial containment was possible.

  The collected K. sierra fungus culture solution was centrifuged at 4500C for 7,500 rpm for 60 minutes, and the precipitate was suspended in PBS. The suspension was centrifuged at 1,500 rpm for 10 minutes at 4 ° C., and the supernatant was collected. The sedimentation was suspended in an appropriate amount of PBS, centrifuged at 1,800 rpm for 5 minutes at 4 ° C., and the supernatant was collected. The above operation was repeated again, and all the collected supernatants were centrifuged at 7,500 rpm for 60 minutes at 4 ° C. The obtained precipitate was suspended in a small amount of PBS, layered on PBS containing 7.6% urografin (Nihon Schering) containing 30% sucrose, and centrifuged at 4 ° C. and 10,000 rpm for 60 minutes using an RPD40T rotor (Hitachi). After removing the supernatant, the cloudy layer formed at the bottom of the tube was collected. The collected cloudy layer was layered on PBS, and centrifuged at 14,000 rpm for 60 minutes at 4 ° C. using the same rotor. After removing the supernatant, the precipitate was suspended in PBS to obtain a crudely purified bacterium.

Next, density gradient centrifugation of the roughly purified bacteria was performed. 76% urografin was diluted to 25, 30, 35 and 40% with PBS and layered gently. After standing for 2 hours, the crudely purified bacteria were overlaid, and density gradient centrifugation was carried out at 24,000 rpm for 60 minutes at 4 ° C. using the same rotor. After centrifugation, the layers formed between 25-30%, 30-35% and 35-40% were recovered. The collected bacteria were layered on PBS and centrifuged at 14,000 rpm for 60 minutes at 4 ° C. using the same rotator. After removing the supernatant, the precipitate was suspended in a small amount of PBS to obtain purified bacteria.
The concentration of the purified cells was adjusted to 1 mg / mL (protein concentration).

  When the properties of the cells in the culture supernatant and the cells after purification were compared with those cultured in serum, no substantial difference was observed when examined by electrophoresis.

[Example 2] Production of solubilized antigen of K. sierra fungus The solubilizing antigen of K. sierra bacteria is used as a purified antigen after crushing purified bacteria, obtaining antigen components from the crushed material, and purifying as necessary. In the present invention, as an example, solubilization was performed by an alkali treatment method.

  Purified bacteria were solubilized with NaOH (detailed literature: N. S. V. Otsuka et al. J. Clin. Microbiol, 34, 2947-51, 1996). The purified bacteria were centrifuged at 13,000 rpm for 15 minutes. After removing the supernatant, the precipitate was suspended in 0,2N NaOH. After the tolerization treatment by boiling for 30 minutes, it was dialyzed in PBS for 24 hours. After dialysis, the mixture was centrifuged at 13,000 rpm for 30 minutes, and the supernatant was recovered to make the permissible antigen.

[Example 3] Acquisition of monoclonal antibody against K. sierra cells Monoclonal antibodies recognizing the proteins constituting K. sierra were obtained by a known monoclonal antibody collection method. Spleen of Balb / c mice immunized with Nine Mile strain and myeloma cell line P3-X63-Ag8.653 were fused by a known method, and LPS-specific and outer membrane proteins of Coccoliella were cloned several times. Multiple specific clones were selected. As a result, monoclonal antibodies H5A and H36 recognizing lipopolysaccharide of a phase I bacterium of K. sierra and H106 recognizing outer membrane glycoprotein were obtained (Hotta, A., et al Infect. Immun. 70: 4747-4749, 2002). These show reactivity comparable to polyclonal antibodies in latex agglutination reactions.

Monoclonal antibody was obtained by a known cell culture method.
First, 8-week-old BALB / c mice: females (SLC) were inoculated intraperitoneally with pristane (2, 6, 10, 14-tetramethylpentadecane, nacalai) per mouse. Two weeks later, 1.0 × 10 ⁷ hybridomas per mouse were inoculated intraperitoneally. Chest fullness was confirmed, and ascites was collected using an 18 gauge needle.

  Monoclonal antibody was purified from mouse ascites containing the monoclonal antibody, and polyclonal antibody was purified from mouse immune serum. Ascites fluid and serum were inactivated at 56 ° C. for 30 minutes, and then centrifuged at 4 ° C. and 15,000 rpm for 25 minutes, and the supernatant was collected. After adding an equal amount of PBS, saturated ammonium sulfate was added dropwise to 45%. After standing on ice for 1 hour, the mixture was centrifuged at 4 ° C. and 10,000 rpm for 10 minutes, and the supernatant was removed. The precipitate was suspended in a small amount of PBS, and saturated ammonium sulfate was added dropwise to 33%. After standing on ice for 1 hour, the mixture was centrifuged at 4 ° C. and 10,000 rpm for 10 minutes, and the supernatant was removed. The precipitate was suspended in a small amount of PBS and dialyzed in PBS for 24 hours. Thereafter, centrifugation was performed at 4 ° C. and 15,000 rpm for 30 minutes, and the supernatant was collected. Four times the amount of the supernatant, 0.02 M sodium phosphate buffer, was added, followed by filtration using a filter having a pore size of 0.45 μm. After filtration, purification was performed according to the protocol using a monoclonal antibody Trap Kit (Amersham Biosciences). The purified antibody was concentrated using MICROCON YM-10 (MILLIPORE), and the amount of antibody was measured using BIO-RAD Protein Assey Kit (BIO-Rad).

  The purity of the monoclonal antibody was confirmed by SAS-PAGE. The reactivity between the monoclonal antibody and Cocciella was confirmed by a sandwich ELISA method or the like.

  When using SDS-PAGE, the separation gel was 12.5% and the concentration gel was 4.5% (w / v) acrylamide. 6 μg of the purified antibody is suspended in 15 μl of PBS, and an equal amount of sample buffer (20% (v / v) glycerin, 5% (v / v) 2-mercaptoethanol, 0.1 M diostreitol, 4% (w / v) SDS, 0.01% (w / v) 0.125 MTris-HCl containing bromophenol blue, pH 8.0) was mixed, boiled for 5 minutes, and used for electrophoresis. The electrophoresis was carried out using a running buffer containing 25 mM Tris-HCl, 192 mM glycine and 1% (w / v) SDS. The gel after electrophoresis was stained for 1 hour in Coomassie Blue staining solution (0.25% (w / v) Coomassie brilliant blue, 25% (v / v) methanol, 7.5% (v / v) acetic acid-containing DW), Decolorization was carried out for 5 hours in a decolorizing solution (25% (v / v) methanol, 7.5% (v / v) acetic acid-containing DW), and an electrophoretic image was confirmed (FIG. 1).

[Example 4] Analysis of change in reactivity due to mixing of monoclonal antibodies When a plurality of monoclonal antibodies are used in combination, the reactivity with an antigen can be improved, and a test example confirming this is shown. Changes in reactivity due to the use of a mixture of monoclonal antibodies were analyzed by ELISA. As the primary antibody, the total antibody amount was 10 μg / ml, and three types of monoclonal antibodies (H5A, H36, H106) were mixed at various ratios.

  On the antigen plate, 240 μl of purified bacteria was suspended in 10 ml of 0.05M bicarbonate buffer (pH 9.6) and adsorbed. The results are shown in Table 1.

  From this result, it was found that by combining a plurality of monoclonal antibodies, the reactivity with the antigen was improved and the detection sensitivity of the antigen was improved.

[Example 5] Antigen detection by latex agglutination using a polyclonal antibody In order to carry out a latex agglutination reaction, antibody-bound latex was mixed with cocciella or an antigen-containing sample. The mixture was kept warm for several hours to around 20 hours, and the resulting aggregated image was observed visually or under a microscope, the limiting dilution rate was measured, and the amount of antigen was quantified.

  In this example, first, antibody-sensitized latex (antibody-bound latex) was prepared. Latex (antibody-conjugated latex) was prepared according to the method of March et al. (March et al., J. Clin. Microbiol., 38, 4152-59, 2000). The standard latex particle suspension was washed 3 times by centrifugation at 15,000 rpm for 10 m minutes using PBS. After the final wash, the sediment was adjusted to 1.0% (w / v) with PBS. 150 μg / ml of purified antibody was added thereto, and the antibody was bound by stirring with a rotator for 4 hours at room temperature. This was followed by 3 washes as before. After the final washing, the sediment was adjusted to 1.0% (w / v) with a preservation solution (PBS containing 1% BSA, 5% glycerol, 0.05% NaN3). For blocking (to stabilize antibody binding), the mixture was allowed to stand at 4 ° C. for 24 hours or longer and used for the agglutination reaction.

  Next, antigen detection was performed by combining the antibody-sensitized latex agglutination reaction of the latex prepared in this Example and the slide agglutination method.

A hydrophobic circle having a diameter of 1 cm was drawn on a micro slide glass (manufactured by Matsunami) using DAKO PEN RED (DAKO). As an antigen, inactivated C. burnetii was used by diluting 2-fold serially with 0.5 M Tris buffer (Tris-HCl, pH 6.8) from 7.8 × 10 ³ to 1.0 × 10 ⁶ IFU / ml. As the latex, a standard antibody and a carboxylic acid-modified latex were sensitized with a polyclonal antibody (Pab) and used. 15 μl of antigen and 5 μl of sensitized latex were mixed on a slide glass. Aggregated images were judged at 15, 60 and 120 minutes after mixing.
Furthermore, antigen detection was performed by a microtiter method using antibody-sensitized latex agglutination reaction.

In this example, six types of 96-well plates were used as microplates. These plates were used after blocking for 2 hours at room temperature with blocking solution (PBS containing 3% skim milk). As the antigen, inactivated K. sierra was used in a 2-fold serial dilution with Tris-HCl from 6.25 × 10 ⁴ to 1.0 × 10 ⁶ IFU / ml. As the latex, a carboxylic acid-modified latex having a particle size of 0.35 and 0.9 m was used after sensitizing the polyclonal antibody. 45 μl of antigen and 5 μl of sensitized latex were mixed on a 96-well plate. Aggregated images were judged at 24 and 48 hours after mixing.

Example 6 Antigen Detection by Latex Aggregation Reaction Using Monoclonal Antibody In order to detect an antigen by latex agglutination reaction, carboxyl group-modified latex was first fixed and sensitized.

  Carboxyl group fixation and sensitization of carboxylic acid-modified latex were performed according to the method of Inzana et al. (Inzana, T., J. Clin. Microbiol., 33: 2297-2303, 1995). The carboxylic acid-modified latex particle suspension was washed three times with 0.1 M bicarbonate buffer (pH 9.6) by centrifugation at 15,000 rpm for 10 minutes. Next, it was similarly washed 3 times with 0.02 M phosphate buffer (pH 4.7). After the final wash, the sediment was adjusted to 0.375% (w / v) with phosphate buffer. Then, a phosphate buffer solution containing 2% WSC (1-ethyle-3- (3-dimethylaminopropyl) carbodiimide) equivalent to the phosphate buffer solution was dropped, and the mixture was stirred with a rotator at room temperature for 4 hours to fix the carboxyl group. . After fixation, the plate was washed 3 times with 0.01M borate buffer (pH 8.0) as before. After the final wash, the sediment was adjusted to 1.0% (w / v) with borate buffer. 200 μg / ml of purified antibody and 10 mM of NHS (N-hydroxysuccinimide) were added thereto, and the mixture was sensitized by stirring with a rotator for 18 hours at room temperature. After sensitization, ethanolamine was added to 4 mM, and the mixture was further stirred for 30 minutes. After stirring, the plate was washed 3 times with a borate buffer containing 1% BSA as before. After the final washing, the precipitate was adjusted to 1% (w / v) with a preservation solution, and used for the agglutination reaction after standing at 4 ° C. for 24 hours or more for blocking.

Next, antigen detection by an agglutination reaction using a monoclonal antibody-sensitized latex was performed.
Mix 1: 1 ratio of monoclonal antibody H5A: H106, 1: 1 of monoclonal antibody H36: H106, and 1: 1 of monoclonal antibody H5: AH36: H106 in carboxylic acid-modified latex with a particle size of 1.0 μm. I was sensitized. As the antigen, the C. burnetti phase I viable bacteria used in Example 1 was used after diluting twice with Tris-HCl from 9.75 × 10 ² to 6.25 × 10 ⁴ IFU / ml. Polyclonal antibodies were used as positive controls for sensitized latex, and antibodies (negative polyclonal antibodies) purified from rabbit serum immunized with Newcastle disease virus (NDV, Kitasato Laboratories) were used as negative controls. Used to sensitize. In addition, an agglutination reaction was performed using a microtiter method, and an aggregated image was determined after 24 hours.

When using latex mixed with monoclonal antibody H5A: H106 1: 1 and monoclonal antibody H5A: H36: H106 in a 1: 1: 1 ratio, and polyclonal antibody-sensitized latex, 7.8 × 10 ³ IFU / ml Aggregation was observed until. When latex was sensitized by mixing H36: H106 at a ratio of 2: 1, aggregation was observed up to 3.9 × 10 ³ IFU / ml.

  On the other hand, no aggregation was observed in the serum-sensitized latex used as a latex negative control. Aggregation was not observed when Yersinia enterocolitica and Legionella pneumophila used as negative antigen controls were treated in the same manner. From this result, it was found that the sensitivity of the detection of Cocciella was improved when a plurality of monoclonal antibodies were used in combination and when a polyclonal antibody was used.

[Example 7] Detection of serum antibody by latex agglutination method and diagnosis of clinical serum sample First, antigen-bound latex was prepared. A standard latex with a particle size of 1.1 mcm was sensitized with the solubilized Coccoliella antigen. Antigen-sensitized latex was prepared in the same manner as antibody sensitization using 150 mcg of solubilized Coccoliella antigen in 1 mL of 1% latex suspension.

  The test serum was 106 small animal clinical veterinary sera, 25 cases from the Kitasato Institute Biopharmaceutical Research Laboratories, which had an IgG antibody titer of 64 times or more, and FA among the sera from patients visiting the Gifu University Hospital. The LA and FA antibody titers in a total of 211 cases were compared among 80 cases showing a multivalent antibody titer of 16 times or more.

  Next, antibodies in serum were detected by a microtiter method using an antigen-sensitized latex agglutination reaction. Serum was used as a 2-fold serial dilution with DW from 128-fold to 2,048-fold. In each well of the microtiter plate, 5 μl of antigen-sensitized latex and 45 μl of serum dilution were mixed. Aggregated images were judged after 14-18 hours.

  The sensitivity and specificity of LA for FA in the detection of coccyella antibodies in serum were evaluated by setting two positive limits. First, when the positive limit of LA antibody titer was 256 times, 128 out of 142 cases positive for FA antibody titer showed LA antibody titer positive, and the sensitivity was 90.8%. Of 69 cases of FA antibody negative, 6 cases showed negative LA antibody titer and specificity was 91.3% (Table 2).

It is a figure which shows the confirmation of the monoclonal antibody which recognizes the Cocciella microbe by SDS-PAGE.

Claims (16)

A method for diagnosing Q fever of a non-human animal, comprising a step of detecting an antibody that binds to the antigen present in a test sample using a diagnostic agent containing an antigen of Coccoliella, wherein the antigen comprises the following ( A diagnostic method which is an antigen comprising at least one selected from a) and (b).
(a) inactivated whole cell body coccyella obtained by performing inactivation treatment after serum-free culture of cocciella,
(b) Inactivated antigen protein obtained by inactivating one or a plurality of antigen proteins derived from K. sierra bacteria cultured in serum-free culture The diagnostic method according to claim 1, wherein the antigen according to claim 1 (a) is an antigen obtained by the following steps (a) and (b).
(a) A step of culturing Kojiella bacteria serum-free to produce Kokusiella culture preparations
(b) A step of inactivating all coccoliella bacteria by adding an inactivation agent to the coccierella culture preparation The diagnostic method according to claim 1, wherein the antigen according to claim 1 (b) is an antigen obtained by the following steps (a) and (b).
(a) Step of serum-free culture of K. sierra
(b) A step of inactivating the antigen protein by adding an inactivating agent to one or a plurality of antigen proteins derived from K. sierra bacteria cultured in a serum-free manner   The diagnostic method according to any one of claims 1 to 3, wherein the coccierella is a wild strain, a clinical isolate, an artificial mutant derived therefrom, or a recombinant strain.   The diagnostic method according to any one of claims 1 to 4, wherein the K. sierra fungus is K. sierra burnetti. A method for diagnosing Q fever of a non-human animal comprising a step of detecting an antigen that binds to an antibody present in a test sample using a diagnostic agent that includes an antibody that binds to an antigen of Coccoliella, comprising: A diagnostic method, wherein the antigen used in preparing the antibody is one of the following (a) or (b).
(a) inactivated whole cell body coccyella obtained by performing inactivation treatment after serum-free culture of cocciella,
(b) Inactivated antigen protein obtained by inactivating one or a plurality of antigen proteins derived from Coccellaella cultured in serum-free culture   The diagnostic method according to claim 6, wherein the antibody is a monoclonal antibody that recognizes a part of the body of the body.   The diagnostic method according to claim 7, wherein the monoclonal antibody is at least one selected from a monoclonal antibody that recognizes an outer membrane glycoprotein antigen of Bacillus subtilis and a monoclonal antibody that recognizes lipopolysaccharide. The antigen according to claim 6 (a) used in preparing the antibody of the diagnostic agent is an antigen obtained by the following steps (a) and (b): Q fever diagnostic method as described in.
(a) A step of culturing Kokusiella bacteria without serum to produce a Koxiella bacteria culture preparation
(b) a step of inactivating whole body cocciella by adding an inactivation agent to the culture preparation of cocciella The antigen according to claim 6 (b) used in preparing the antibody of the diagnostic agent is an antigen obtained by the following steps (a) and (b): Q fever diagnostic method as described in.
(a) Step of serum-free culture of K. sierra
(b) A step of inactivating the antigen protein by adding an inactivating agent to one or a plurality of antigen proteins derived from K. sierra bacteria cultured in a serum-free manner   The diagnostic method according to any one of claims 1 to 10, wherein an immunoassay is used. The diagnostic method according to claim 1, wherein a latex agglutination method is used. A diagnostic reagent for Q fever comprising an antigen of Coccoliella, wherein the antigen is an antigen comprising at least one selected from the following (a) and (b).
(a) inactivated whole cell body coccyella obtained by performing inactivation treatment after serum-free culture of cocciella,
(b) Inactivated antigen protein obtained by inactivating one or a plurality of antigen proteins derived from Coccellaella cultured in serum-free culture The Q fever diagnostic agent according to claim 13, wherein the antigen according to claim 13 (a) is an antigen obtained by the following steps (a) and (b).
(a) A step of culturing Kojiella bacteria serum-free to produce Kokusiella culture preparations
(b) A step of inactivating all coccoliella bacteria by adding an inactivation agent to the coccierella culture preparation The Q fever diagnostic agent according to claim 13, wherein the antigen according to claim 13 (b) is an antigen obtained by the following steps (a) and (b).
(a) Step of serum-free culture of K. sierra
(b) a step of inactivating the antigenic protein by adding an inactivating agent to one or a plurality of antigenic proteins derived from K. sierra bacteria cultured in serum-free culture Diagnostic kit comprising a diagnostic agent according to any of claims 13-15.