JPH09178750A - Production of q fever antigen - Google Patents

Abstract

PROBLEM TO BE SOLVED: To produce a high purity Q fever antigen by heat treating an aqueous suspension, where Q fever rickettsia (Coxiellaburnetii) somatic cells are dispersed uniformly into an aqueous solution, under alkaline conditions, bringing the heat treated aqueous suspension into contact with an organic solvent to extract the components soluble to organic solvent, and employing the components dissolved into residual water layer as an antigen. SOLUTION: An aqueous suspension, where Q fever rickettsia (Coxiellaburnetii) somatic cells are dispersed uniformly into an aqueous solution, is heat treated under alkaline conditions. Consequently, antiqenic components elute into the aqueous solution from the Q fever rickettsia somatic cells or the surface layer thereof is destroyed partially and the antigenic components elutes into a water layer tram the surface layer of Q fever rickettsia somatic cells in a process for touching an organic solvent. When the temperature of heat treatment is 50 deg. or above, the antigenic components are extracted efficiently. Subsequently, it is brought into contact with the organic solvent and the soluble components are extracted. Finally, only the dissolved components in the water layer are collected thus collecting high purity Q fever antigen useful as a reagent toe inspecting Q fever infective disease.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a method for producing an antigen used for a Q fever infection test reagent. More specifically, the present invention relates to a method for producing an antigen for a Q fever infection test reagent, which has high specificity and high sensitivity, and particularly can detect an initial infection.

[0002]

2. Description of the Related Art Q heat is a type of rickettsia, Cox.
A zoonotic disease caused by iella burnetii infection in Australia in 1935, a febrile disease of unknown cause, derived from Query fever and widely distributed worldwide. Clinical symptoms are divided into acute and chronic. Incubation period for acute cases is 14 to 26 days
Days and short. Acute and febrile rickettsemia that resembles influenza is present, with varying degrees of severity and duration,
Bronchitis, pneumonia, hepatitis due to fever, severe headache, chest pain, myalgia, joint pain, sweating, chills, loss of appetite, vomiting, etc.
Causes diseases such as meningitis. Patients have a variety of medical conditions and may exhibit a few symptoms at the same time. Rarely, skin rash, meningoencephalitis, optic neuritis, and thyroiditis. The prognosis is generally good, with many recovering from underheating in about 12 days. Delayed treatment leads to death. In the case of chronic disease, the recovery period after acute infection shifts to endocarditis. Incidence of endocarditis ranges from 2% to 20
%, With a higher mortality rate. Other types of diseases such as chronic hepatitis, myocarditis and epicarditis are known.

Q heat has been increasing year by year around the world.
This is said to be due to an increase in cocciosis of domestic animals and companion animals, but the source of infection has not been identified. In foreign countries, as a causative bacterium of febrile respiratory disease, Coxiella
burnetii is widely recognized. Recently, data has been accumulated that can estimate the existence of many Q fever in Japan. In the case of febrile respiratory disease, it is important to suspect Q fever and list it as one of the differential diagnoses. It seems that the examination for Q fever infection will become more important in the future.

However, as a test method for Q fever infection, a specific antigen-antibody reaction between the Q fever rickettsiae cells immobilized on a glass plate and the anti-Q fever rickettsiae antibody in the patient serum and plasma is treated with a fluorescent substance. The fluorescent antibody method (hereinafter abbreviated as IFA method) for detecting with a labeled secondary antibody is only performed at the experimental level of researchers. Although this IFA method has excellent specificity, it is not suitable as an inspection method for a large number of patients because the operation is complicated. In order to perform a large amount of tests as a clinical test, it is necessary to use a simple test reagent such as a microtiter method that is simpler than the IFA method, a carrier agglutination reaction reagent by a latex agglutination method, and a reagent (EIA reagent) by an enzyme immunoassay method. is important. In order to prepare a simple test reagent such as a microtiter method or a latex agglutination reagent, unlike the IFA method in which Q heat rickettsiae cells are directly used as an antigen, a Q heat antigen containing an antigen component is prepared from Q heat rickettsiae cells. Method development is required.

Q containing the antigen component from heat rickettsia
As a method for preparing a heat antigen, Infection and Immuity (Infection and Immu) is used.
night, vol. 48, 359-365, 1985
Year)), phenol-water solution was added to the Q heat rickettsia fungus body, suspended and heated to recover the antigen in the aqueous layer,
Further treatment with DNA degrading enzyme and RNA degrading enzyme, Q
Method for recovering heat antigen, dimethyl sulfoxide (DM
SO) Q heat rickettsia cells were treated at 50 ° C for 24 hours, and the centrifugation supernatant was used as Q heat antigen. 10% trichloroacetic acid (TCA) was treated at 4 ° C for 4 hours, and the centrifugation supernatant was treated with Q heat. The method of making an antigen etc. are mentioned. Neither method is sufficient as a method for preparing a highly pure Q fever antigen.

[0006]

DISCLOSURE OF THE INVENTION An object of the present invention is to provide a simple method for producing a high-purity antigen in a method for producing a Q fever antigen used for a Q fever infection test reagent.

[0007]

[Means for Solving the Problems] The present inventors have conducted extensive research to solve the above technical problems. As a result, after heat-treating an aqueous suspension of Q. thermophilic cells under alkaline conditions, contacting the aqueous suspension with an organic solvent, and using the dissolved component in the remaining aqueous layer as an antigen The inventors have found that the above objects can be achieved and completed the present invention.

That is, Q heat rickettsia (Coxiella)
burnetii) After heat-treating the aqueous suspension in which the bacterial cells are dispersed under alkaline conditions, the aqueous suspension is contacted with an organic solvent to extract the organic solvent-soluble component, and the dissolved component in the remaining aqueous layer is removed. A method for producing a Q fever antigen, which comprises using an antigen.

[0009]

BEST MODE FOR CARRYING OUT THE INVENTION The present invention will be described in detail below.

The Q. rickettsiae bacterium referred to in the present invention is a kind of rickettsiae, which is a pathogen of Q fever.
a burnetii. The shape of the Q heat rickettsia bacterium is a rod-like shape and polymorphic. Size is 0.2 ~
It is 0.4 μm. Small cells with spore-like structure (SC
V) and large cells of the mother cells (LCV), both types are infectious. The Q fever rickettsiae, like other rickettsiae, showed a phase mutation similar to the SR mutation, I and II.
There are competing bacteria. Phase I bacteria are highly virulent fresh isolates, but they can be cultured in vitro and are weakly virulent when cultured for a long time.
Becomes phase II bacteria. Antibodies against phase I bacteria appear in the late stage of convalescence of patients with Q fever infection (4 weeks after the onset) and disappear in a short period of time. On the other hand, antibodies against phase II bacteria appear early in the recovery period (1 to 2 weeks after onset) and persist for a long time. Therefore, phase II bacteria are preferably used as the antigen for the Q fever infection test reagent. The Q fever rickettsia cells referred to in the present invention can be applied to both phase I and phase II bacteria, but as a raw material for the antigen of the Q fever infection test reagent, they are weakly toxic because of early diagnosis and safety during culture. Phase II bacteria are preferably used.

In the present invention, the Q heat rickettsiae cells are uniformly dispersed in an aqueous solvent to prepare an aqueous suspension. In order to efficiently carry out the heat treatment under alkaline conditions, it is preferable that the Q heat rickettsia cells are uniformly dispersed in the aqueous suspension. In the aqueous suspension, the dispersion concentration of the Q heat rickettsia cells may be arbitrary, but about 10 ² cells / ml to 10 ⁹ cells / ml is preferably used. Further, as the aqueous solvent used for the aqueous suspension, pure water having no buffer action is preferably used. However, the salt concentration in the aqueous solvent is not particularly limited.

Next, in the present invention, the aqueous suspension is heat-treated under alkaline conditions. As a result, the component having the antigenicity from the Q heat rickettsia fungus body is eluted into the aqueous solvent, or the surface layer of the Q heat rickettsia fungus is partially destroyed, and the surface layer of the Q heat rickettsia fungus body is contacted with the subsequent organic solvent. A component having an antigenicity can be eluted in a more aqueous layer. In the present invention, the alkaline condition may be pH 7.1 or higher, and preferably pH in consideration of treatment efficiency.
The pH used is 9.0 or higher, and more preferably pH 11.0 or higher. Alkaline conditions may be achieved by adding alkali to the aqueous solvent in advance, or may be achieved by adding alkali to the aqueous suspension. Any kind of alkali may be used as long as the pH is alkaline, and sodium hydroxide, potassium hydroxide, ammonia and the like are preferably used. More preferably, sodium hydroxide may be used. When the alkali concentration is 10 mM or more, the component having antigenicity is efficiently extracted. When it is 100 mM or more, the extraction efficiency of the component having antigenicity is further improved.

When the temperature of the heat treatment in the present invention is 50 ° C. or higher, the extraction efficiency of the component having antigenicity is good, and the temperature is 80 ° C.
As described above, the extraction efficiency of the component having antigenicity is further improved. The heat treatment temperature is preferably 120 ° C. or lower.
The time required for the heat treatment is effective if it is 1 minute or longer, and works properly if it is 10 minutes or longer. Heat treatment was performed by placing an aqueous suspension of Q heat rickettsia cells in a reaction container such as glass or stainless steel,
It may be performed in a water bath or oil bath. The pressure during the heat treatment is atmospheric pressure (1 atm), or sufficient effects can be obtained even under pressure. At this point, most of the antigenic components are dissolved in the aqueous solvent, and some remain on the surface layer of the Q heat rickettsiae cells.

Even after the heat treatment under alkaline conditions,
The aqueous suspension containing the Q heat rickettsia cells is in alkaline condition. Before the subsequent contact with an organic solvent, the aqueous suspension may be subjected to alkali removal by dialysis or the like, neutralization or the like. Further, contact with an organic solvent that continues under alkaline conditions may be carried out.

By contacting the aqueous suspension thus obtained with an organic solvent, the antigenicity remaining on the surface layer of the Q heat rickettsiae cells partially destroyed by heat treatment under alkaline conditions. Can be eluted in an aqueous solvent, and the organic solvent-soluble component, which is a contaminant present in the aqueous solvent, can be extracted into the organic solvent by heat treatment under alkaline conditions. By this operation, the component having antigenicity is fractionated into the aqueous layer, and the component having no antigenicity is efficiently fractionated into the organic solvent. Therefore, it is possible to easily remove the non-antigenic component by the operation. If the above procedure is not added, the extraction efficiency of the components having antigenicity as a whole is lowered, and the organic solvent-soluble components having no antigenicity are mixed as contaminants into the finally obtained Q heat antigen. , The purity is significantly reduced. The components extracted in the organic solvent are presumed to be hydrophobic components such as hydrophobic proteins, lipoproteins and lipids. The organic solvent that can be used in the present invention is not particularly limited as long as it can dissolve hydrophobic components such as hydrophobic proteins, lipoproteins and lipids, but the dissolved component in the subsequent aqueous layer is recovered as an antigen. A straight-chain or aromatic hydrocarbon having a specific gravity of 1.05 or more or 0.95 or less and a substituted product thereof, an ether, a ketone, or the like is preferably used in the step of carrying out fractionation with an aqueous solvent. For example, pentane, hexane, heptane,
Chloroform, carbon tetrachloride, methylene chloride, dichloroethane, trichloroethane, tetrachloroethane, pentachloroethane, hexachloroethane, benzene, diethyl ether, acetone, methyl ethyl ketone and the like are used. Chloroform is preferably used.

The operation may be carried out by thoroughly mixing an aqueous suspension containing heat-treated Q. rickettsiae cells heat-treated under alkaline conditions and an organic solvent. The conditions for mixing are not particularly limited as long as the aqueous suspension containing the heat-treated Q. rickettsia cells heat-treated under alkaline conditions and the organic solvent can be completely mixed. An effective effect is obtained when the mixing ratio of the aqueous suspension containing the heat-treated Q. rickettsiae cells heat-treated under alkaline conditions and the organic solvent is in the range of 50: 1 to 1:50.

In the present invention, an organic solvent-soluble component is contacted with an organic solvent to extract the organic solvent-soluble component, and only the dissolved component in the remaining aqueous layer is recovered to thereby react with the anti-Q heat rickettsiae antibody in the blood of the patient. The component possessed, that is, a high-purity antigen useful for a Q fever infection test reagent is recovered. It can be expected that the main components are water-soluble components such as proteins and glycolipids.

The fractionation operation of the organic solvent layer and the aqueous layer containing the component having antigenicity is carried out by utilizing the difference in specific gravity. For example, the fractionation is achieved by a centrifugation operation with a weak centrifugal force of about 2,000 × g. After such a fractionation operation, the soluble component in the remaining aqueous layer can be recovered as the Q heat antigen by removing the organic solvent layer.

The Q fever antigen obtained in the present invention is highly pure and shows high antigenicity to anti-Q fever rickettsiae antibody. The Q fever antigen can be suitably used as an antigen for a Q fever infection test reagent.

The Q fever antigen produced by the present invention can be used, for example, as a microtiter reagent, latex nephelometric agglutination reagent, immunonephelometric reagent, enzyme immunoreagent and the like. The Q fever antigen of the present invention is effective as an antigen for an agglutination reagent, such as a microtiter reagent or a latex agglutination reagent, which captures an antigen-antibody reaction by an agglutination reaction.

Usually, this solution state is used as a Q heat antigen, and if necessary, diluted or concentrated with physiological saline, physiological saline containing 10 mM phosphate buffer, pH 7.2 (hereinafter also abbreviated as PBS) or the like. Then, it is subjected to the sensitization operation of the insoluble carrier described later.

The Q fever antigen obtained by the production method of the present invention can be carried on an insoluble carrier to produce a Q fever infection test reagent. As the insoluble carrier particles, carriers that can be used for a diagnostic reagent of a known immunological agglutination method can be used without limitation. For example, high specific gravity composite particles obtained by coating an inorganic compound serving as a core with a dye (JP-A-62-1153).
66), sheep red blood cells, polystyrene particles, gelatin particles, polyamino acid particles and the like. Among them, high specific gravity composite particles and erythrocytes have a large adsorption amount of the Q heat antigen and are preferably used.

The particle size of the insoluble carrier is not particularly limited as long as it is within a known range that can be used as a diagnostic reagent for immunological agglutination method, but usually the particle size is 0.01 μm to 3 μm.
Those up to are preferably used.

The specific gravity of the insoluble carrier may be 1.0 or more. Especially, the larger the specific gravity of the carrier particles for microtiter reagent utilizing the precipitation reaction, the faster the sedimentation speed and the shorter the determination time. The above is preferably used.

As a specific supporting method, the insoluble carrier is dispersed and mixed in a buffer solution in which the sensitizing substance is dissolved, and the sensitizing substance is supported on the insoluble carrier, and then the excess sensitizing substance is removed by centrifugal washing. Then, a method of supporting a blocking agent in the same manner is used.

As a method for supporting the sensitizing substance on the insoluble carrier, a known method such as a physical adsorption method such as a hydrophobic adsorption method or a chemical adsorption method such as a chromium chloride method is adopted. Since the sensitizing substance is supported on a carrier and then binds to the target substance in the analyte by the antigen-antibody reaction, the sensitizing substance is preferably supported under as mild a condition as possible. Therefore, the hydrophobic adsorption method is particularly preferably used.

The loading by the above-mentioned hydrophobic adsorption method is carried out in a state in which the insoluble carrier and the adsorbing substance are dispersed in a buffer solution having a buffering action. The buffer solution can be used without limitation as long as it has a buffering action such as a phosphate buffer solution, a glycine buffer solution, a Tris buffer solution, an acetate buffer solution and the like. Regarding the pH of the buffer solution, an optimum range is selected and adopted depending on the adsorbing substance to be supported, but generally, the neutral range pH 6.0 to pH 8.
0 is desirable. The time for supporting the adsorbent on the insoluble carrier may be a time sufficient to allow the adsorbent to be uniformly supported on the surface of the insoluble carrier. For example, when the Q heat antigen is subjected to the hydrophobic adsorption method for high specific gravity composite particles. 30 minutes or more is sufficient. Further, regarding the temperature, a range in which the adsorbed material to be supported is not subjected to thermal denaturation, that is, 1 ° C. or more and 80 ° C. or less is adopted.

Further, the blocking agent may be loaded on the insoluble carrier by the same method as that for loading the sensitizing substance.

[0029]

The present invention will be described below in detail with reference to examples. However, the technical scope of the present invention is not limited by these examples.

Example 1 Production of Q fever antigen (1) 1-1. Cultivation of Q heat rickettsiae microbial cells Q heat rickettsiae cells were obtained by the method of Hirai et al. (Microbio).
l. Immunol, vol. 39, 663-671
(1995). It should be noted that Q heat rickettsiae phase I and phase II bacteria are American Type C
The Ultra Collection (ATCC) strain was used.

That is, the Q heat rickettsia cells for inoculation were aseptically cultured in embryonated chicken eggs. Q heat rickettsia fungus body 3
Inoculate 0.5 ml of egg yolk preheated at 7 ° C for 5 days,
Culture was performed at 35 ° C. for 7 days in a humidified incubator.

On the other hand, Buffalo Green Monkey was used as a host cell for infecting Q fever rickettsia.
(BGM) cells were cultured. BGM cells were inoculated with the Q heat rickettsiae cells for inoculation that had been cultured in egg yolk, and the cells were placed in a humidified incubator for 3
The cells were cultured at 7 ° C, and the culture supernatant was collected every 5 to 7 days. The culture supernatant contained Q heat rickettsiae cells, and the culture supernatant was stored to obtain a culture solution containing Q heat rickettsiae cells.

1-2. Recovery of Q heat rickettsia fungus body Put the Q heat rickettsia fungus containing culture solution into a centrifuge tube,
Centrifugation was performed at 000 × g for 1 hour at 4 ° C. To the precipitated Q heat rickettsiae cells, an equal amount of PBS to the used Q heat rickettsiae cell-containing culture solution was added, and centrifugation was performed again. By performing the above centrifugal washing operation three times in total, washed Q heat rickettsiae cells were obtained.

The washed Q heat Rickettsia cells were layered on a 30% sucrose solution and centrifuged at 25,000 × g for 1 hour at 4 ° C. PBS containing 0.25 mol / l sucrose was added to and suspended in the Q heat rickettsial cells as a precipitate.

The Q heat rickettsia cells were recovered by a centrifugation method. The sucrose density gradient centrifugation method was performed by superimposing layers on a stepwise sucrose density gradient of 30, 40, 50, 60, 70% prepared in advance, and centrifugation at 87,000 xg for 2 hours at 4 ° C. went.

After centrifugation, the band of Q heat rickettsiae cells was collected with a pipette and centrifuged at 2,000 × g for 1 hour, and the Q heat rickettsiae cells were collected in the precipitate. The Q
The heat rickettsiae cells were suspended in pure water at 2 × 10 ³ to obtain an aqueous suspension of Q heat rickettsiae cells.

1-3 Heat treatment under alkaline conditions After adding sodium hydroxide to an aqueous suspension of Q heat rickettsia cells to a final concentration of 0.2 M, the mixture was placed in a plastic container and the liquid temperature was adjusted to 100 ° C. Then, the heat treatment under alkaline conditions was completed by reacting for 30 minutes.

The aqueous suspension containing the Q heat rickettsiae cells was placed in a dialysis tube and dialyzed against PBS to remove the alkali to obtain a hot alkali treated Q heat rickettsiae cell suspension.

1-4 Solvent extraction The heat-treated Q. rickettsiae cells heat-treated under alkaline conditions were then subjected to solvent extraction with chloroform.

Chloroform was added in an amount equal to that of the Q heat rickettsiae cell suspension treated with hot alkali, and the mixture was shaken for 20 minutes. After shaking, centrifugation was performed at 500 × g for 5 minutes at room temperature to fractionate a PBS-containing aqueous layer (upper layer) and a chloroform layer (lower layer). The upper aqueous layer was collected and used as the Q heat antigen. Ultraviolet light (wavelength: bovine serum albumin as standard substance)
(280 nm) absorption was measured to determine the protein concentration.
The protein concentration was 200 μg / ml.

1-5 Evaluation by Q-Fever Infectious Diseases Testing Reagent (Sensitization) High specific gravity composite particles (Tokuyama Co., Ltd.) with a diameter of 1.8 μm were suspended in PBS to a concentration of 5 (w / w)%. A high specific gravity composite particle suspension was prepared. A Q heat antigen solution was prepared at 50, 100, and 200 μg / ml of the prepared Q heat antigen. High specific gravity composite particle suspension 1 ml and Q heat antigen liquid 1 ml
Were mixed in a test tube and allowed to stand at room temperature for 1 hour to be hydrophobically supported on the surface of the high specific gravity composite particles (hereinafter, this adsorption operation is also referred to as sensitization).

(Washing operation) After sensitization, in order to remove the surplus Q heat antigen, the mixed solution of the Q heat antigen solution and the high specific gravity composite particle suspension was centrifuged at 2,000 × g for 5 minutes. The centrifugation supernatant was removed. PBS2 was added to the centrifugal precipitate for washing.
After adding and suspending ml, the mixture was centrifuged at 2,000 xg for 5 minutes and the supernatant was removed. Q The high specific gravity composite particles adsorbing the heat antigen are
The particles were heat-sensitized particles.

(Blocking Operation) 5 ml of 1% casein was added to the Q heat-antigen-sensitized particles (50 mg), and the mixture was incubated at 37 ° C.
Blocking operation was carried out for 3 hours. After the blocking operation, a suspension of 1% casein-containing Q-heat antigen-sensitized particles in a suspension of 2,0
Centrifugation was performed at 00 × g for 5 minutes, and the centrifugation supernatant was removed. The blocking operation was completed by a series of operations to prepare a Q fever infection test reagent. Q fever infectious disease inspection reagent 3 (vo
1 / vol)% normal rabbit serum-containing PBS (hereinafter also abbreviated as A solution) was suspended at 0.5 (w / vol)% to prepare a Q fever infection test reagent solution, and the following measurements It was subjected to operation.

(Measurement operation) On the other hand, the sample used for the test is A
The solution was serially diluted from 8 times. Next, add A solution of the sample to 9
25 μl of each was dropped onto 6-well microtiter plates from 1 to 8 holes. Then, 25 μl of the Q fever infection test reagent solution was dropped into each hole. After dropping, the mixture was shaken with a plate mixer and allowed to stand for 30 minutes, and then a tube bottom aggregation image was observed.

(Results) The performance evaluation of the Q fever infection test reagent was carried out by using 1 sample of a healthy subject and 1 sample of a patient with Q fever infection. The results are shown in Table 1. Sensitization concentration 200 μg / m
The highest performance was shown by l.

Regarding the reagent performance, a positive image could not be detected even when the sample of healthy subjects was diluted 8 times or less, whereas a positive image could be detected in the sample of Q fever infection patient when diluted 512 times. A non-specific agglutination reaction did not occur in a healthy subject sample, and a good Q fever antigen test reagent having high sensitivity to a sample of a Q fever infection patient could be prepared.

Example 2 Production of Q fever antigen (2) Cultivation of Q fever rickettsia cells and recovery of Q fever rickettsia cells were carried out in the same manner as in 1-1 and 1-2 of Example 1.

(Heat Treatment under Alkaline Condition) After adding potassium hydroxide to an aqueous suspension of Q heat rickettsia fungus to a final concentration of 1 M, it was placed in a plastic container, and after the liquid temperature reached 80 ° C. The reaction was carried out for 30 minutes to complete the heat treatment under alkaline conditions to obtain a hot-alkaline-treated Q heat Rickettsia microbial cell suspension.

By the same solvent extraction as in 1-4 of Example 1, a Q heat antigen having a protein concentration of 200 μg / ml was obtained.

The evaluation using the Q fever infection test reagent was carried out in Example 1.
The same operation as 1-5 was performed.

(Results) The performance evaluation of the Q fever infection test reagent was carried out using 1 sample of a healthy subject and 1 sample of a patient with Q fever infection. The results are shown in Table 1. Sensitization concentration 200 μg / m
The highest performance was shown by l.

Regarding the reagent performance, a positive image was not detected even when the sample of a healthy subject was diluted 8 times or less, whereas a positive image could be detected when the sample of a patient with Q fever infection was diluted 512 times. As in Example 1, a non-specific agglutination reaction did not occur in a healthy subject sample,
A good Q fever infection test reagent, which is highly sensitive to a Q fever infection patient sample, could be prepared.

Comparative Example 1 Production of Q heat antigen by heat treatment under acidic conditions Cultivation of Q heat rickettsia cells and recovery of Q heat rickettsia cells were performed according to 1-1 and 1-2 of Example 1. . Solvent extraction was performed without heat treatment under alkaline conditions. Solvent extraction was performed according to 1-4 of Example 1.

(Heat Treatment under Acidic Conditions) The aqueous suspension of Q heat Rickettsia cells was added with hydrochloric acid so that the final concentration was 0.1 M, and then placed in a plastic container at a liquid temperature of 100 ° C.
Then, the heat treatment under acidic conditions was completed by reacting for 30 minutes.

The aqueous suspension containing the Q heat rickettsiae cells was placed in a dialysis tube and dialyzed against PBS to remove acid to obtain a heat acid treated Q heat rickettsiae cell suspension. By the same solvent extraction as in 1-4 of Example 1, a Q heat antigen having a protein concentration of 20 μg / ml was obtained.

The evaluation using the Q fever infection test reagent was carried out in Example 1.
The same operation as 1-5 was performed.

(Results) The performance of the Q fever infection test reagent was evaluated using one sample of a healthy subject and one sample of a patient with Q fever infection. The results are shown in Table 1.

Regarding the reagent performance, no positive image was detected in both normal and Q fever infection patient specimens even at 8-fold dilution or less.

The sensitivity of the Q fever infectious disease test reagent was lower than that in Example 1, and it was revealed that the alkaline condition when the heat treatment is carried out in the production process of the Q fever antigen is essential.

Comparative Example 2 Production of Q fever antigen without heat treatment The cultivation of Q heat rickettsiae cells and the recovery of Q heat rickettsiae cells were carried out according to 1-1 and 1-2 of Example 1. Solvent extraction was performed without heat treatment. Solvent extraction is 1 of Example 1
-4.

(Alkaline treatment) Q An aqueous suspension of heat-resistant Rickettsia cells was added with sodium hydroxide to a final concentration of 0.2 M, then placed in a plastic container and the liquid temperature was 4 ° C.
Then, the alkali treatment was completed by reacting for 30 minutes to obtain alkali-treated Q heat rickettsiae cells.

The aqueous suspension containing the Q heat rickettsiae cells was placed in a dialysis tube and dialyzed against PBS to remove the alkali to obtain a hot alkali treated Q heat rickettsiae cell suspension.

By the same solvent extraction as in 1-4 of Example 1, a Q heat antigen having a protein concentration of 50 μg / ml was obtained.

The evaluation using the Q fever infection test reagent was carried out in Example 1.
The same operation as 1-5 was performed.

(Results) The performance of the Q fever infection test reagent was evaluated using one sample of a healthy subject and one sample of a patient with Q fever infection. The results are shown in Table 1.

Regarding the reagent performance, no positive image was detected in both normal and Q fever infection patient specimens even at 8-fold dilution or less.

Compared to Example 1, the sensitivity of the Q fever infectious disease test reagent is low, and the performance of the Q fever infectious disease test reagent is not sufficient if only the Q fever rickettsia cells are subjected to alkaline conditions during the production of the Q fever antigen. Sufficient, and it became clear that heat treatment was essential.

Comparative Example 3 Method for Producing Q Fever Antigen without Solvent Treatment Cultivation of Q fever rickettsiae cells, recovery of Q fever rickettsiae cells, and heat treatment under alkaline conditions were 1-1 and 1 of Example 1.
2, the same operation as 1-3 was performed.

The Q heat antigen was used without solvent extraction. The protein concentration was 200 μg / ml.

Evaluation of the Q fever antigen with the Q fever infection test reagent was performed according to Example 1.

(Results) The performance evaluation of the Q fever infection test reagent was carried out using 1 sample of a healthy subject and 1 sample of a patient with Q fever infection. The results are shown in Table 1. Sensitization concentration 200 μg / m
The highest performance was shown by l.

Regarding the reagent performance, a positive image was not detected even when the sample of a healthy subject was diluted 8 times or less, whereas a positive image could be detected when the sample of a patient with Q fever infection was diluted 256 times.

Q produced in Comparative Example 3 as compared to Example 1
It was revealed that the Q fever infection reagent prepared with a heat antigen had low sensitivity to Q fever infection patient specimens, and solvent extraction was essential.

[0074]

[Table 1]

Claims (1)

[Claims] 1. Q heat rickettsia (Coxiella b)
urnetii) After heat-treating the aqueous suspension in which the bacterial cells are dispersed under alkaline conditions, the aqueous suspension is contacted with an organic solvent to extract the organic solvent-soluble component, and the dissolved component in the remaining aqueous layer is removed. A method for producing a Q fever antigen, which comprises using an antigen.