JPH0854396A - Method for measuring chlamydia trachomatis antibody and preparation diagnosing chlamydia trachomatis infectious disease - Google Patents

Abstract

PURPOSE:To provide a method for measuring a Chlamydia trachomatis antibody and a preparation for diagnosing a Chlamydia trachomatis infectious disease which solve a problem of a non-specific reaction. CONSTITUTION:An elementary body refined from Chlamydia trachomatis is immobilized on a solid phase carrier, and the solid phase carrier is brought into touch with a test sample supposed to contain an antibody to Chlamydia trachomatis. Components of the test sample not reacted are removed. The generated complex of the Chlamydia trachomatis antigen and Chlamydia trachomatis antibody is brought into touch with a marker antibody to an antibody originated from the test sample, and the marker antibody not reacted is removed. The presence of the Chlamydia trachomatis antibody or amount of the antibody in the test sample is measured by measuring mass of a marker substance on the coupled marker antibody. The measuring method and diagnosing preparation featuring containing the above-mentioned steps are obtained.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a highly specific method for measuring Chlamydia trachomatis antibody for diagnosing Chlamydia trachomatis infection and a diagnostic preparation used therefor.

[0002]

[Prior Art] Chlamydi trachomatis
a trachomatis) is an obligate intracellular parasite that can only survive in the cells of the host. Its growth cycle is special and morphologically extracellular extracellular chlamydial elementary bodies (elementa).
ry body (EB) is taken up into cells by phagocytosis to form vacuole inclusion bodies, and reticulate body (R)
Change to B). RB has a proliferative ability but lacks an infectious ability, and the RB that proliferates inside the cell eventually changes into EB, breaks the inclusion body, destroys the cell membrane, and goes out of the cell. EB lacks the ability to multiply but has the ability to infect. When a person is infected with this EB,
It develops eye and genital diseases such as trachoma of the eye, virulent lymphogranuloma (LGV), non-phosphate urethritis (NGU) and cervicitis.

In recent years, sexually transmitted diseases
as one of the causative microorganisms of ed diseases)
Trachomatis is attracting attention, with 300 new cases of Chlamydia trachomatis in the US each year.
It is said to be from 10 to 10 million people (according to Etsuaki Kumamoto et al .: Chlamydia infection, Medic, 20, 1-8, 1985),
Interest in Chlamydia trachomatis infectious diseases is gradually increasing in Japan as well.

The most sensitive serodiagnosis of Chlamydia trachomatis infection is Wang and Grayston (Trachoma and related disorders c)
aused by chlamydial agents, Excerpta Medica, Amster
dam, pp.273-288, 1971), an indirect microimmunofluorescent antibody measurement method (micro-IF method). However, the micro-IF method is not adopted as a diagnostic method in clinical laboratories because the test procedure is difficult. The standard micro-IF method requires purified elementary bodies (EB) of 15 Chlamydia trachomatis serotypes. Micro-I
Method F requires the complete morphology or structure of the identified microorganism to carry out the immunofluorescence reaction. Therefore, methods that alter or destroy the EB morphology or structure cannot be used. Since this EB is infectious and toxic, it requires a special facility protected against infection in order to use it as an antigen material as it is. Therefore, an antigen treated with an immobilizing agent such as formaldehyde or acetone is usually used.

On the other hand, recently developed enzyme immunoassay (E
LISA) has the advantage that a large number of specimens can be processed simply and quickly. Although there have been reports on the measurement of Chlamydia trachomatis antibody using this ELISA, most of them are Chlamydia trachomatis L2.
Unpurified EB (which contains a large amount of host cells or fragments thereof) of the strain is used as it is or after being treated with SDS (sodium dodecyl sulfate), as an antigen material for ELISA. Therefore, it is known that there is a non-specific reaction including a cross-reaction with a Chlamydia pneumoniae antibody and a Chlamydia shita antibody by using an antigen with low purification purity. This is primarily due to the complex antigenicity of Chlamydia trachomatis. The antigenicity of Chlamydia trachomatis includes genus-specific antigens, species-specific antigens, and serotypes (bio
It is believed that there are specific antigens. As a representative genus-specific antigen, lipopolysa
ccharide: LPS) is known, which has a common antigenicity with Re mutant LPS of enterobacteria.

On the other hand, as a representative species-specific antigen and serotype-specific antigen, the major outer membrane peptide of Chlamydia trachomatis (Major Outer Membrane Protein: MOMP) is known, which is the outer membrane protein of Chlamydia trachomatis. It is said to occupy about 60% of the total. However, it is known that this MOMP also has a genus-specific antigenicity (Collett et al., Annu.Meet.Am.Soc.Microbiol., Was.
hington.DC, Abstract No. D-159, 1986). MOMP
Other outer membrane antigens of Chlamydia trachomatis are mainly genus-specific antigens, but at the same time, some of them have species-specific antigenicity. The sarcosyl insoluble molecular weight approximately 59.5 dalton peptide falls into this category.

[0007] As described above, the antigenicity of Chlamydia trachomatis is extremely complicated. Chlamydia trachomatis antibodies in Chlamydia trachomatis infected individuals show diversity corresponding to the complex antigenicity of Chlamydia trachomatis, and the pattern varies depending on the infected person. Therefore, it is virtually impossible to measure the Chlamydia trachomatis antibody using one specific type of Chlamydia trachomatis protein. Therefore, in order to measure the Chlamydia trachomatis antibody possessed by a person infected with Chlamydia trachomatis with high precision and high sensitivity while suppressing nonspecific reactions including cross-reactivity with the Chlamydia pneumoniae antibody and the Chlamydia shita antibody. , It is necessary to properly select the antigen material to be used.

[0008]

DISCLOSURE OF THE INVENTION The present invention provides a Chlamydia pneumoniae antibody and a Chlamydia shita antibody in a conventional ELISA method for measuring Chlamydia trachomatis antibody using the above-mentioned unpurified EB itself or SDS-treated EB as an antigen material. The present invention provides a method for measuring Chlamydia trachomatis antibody and a preparation for diagnosing Chlamydia trachomatis infection, which solves the problem of nonspecific reaction including cross-reactivity with.

[0009]

That is, the present invention is as follows.
(A) immobilizing purified Chlamydia trachomatis elementary bodies on a solid-phase carrier, (b) contacting the solid-phase carrier with a specimen sample suspected of containing an antibody against Chlamydia trachomatis, (c) The unreacted analyte sample component is removed, (d) the antigen-antibody complex consisting of the generated Chlamydia trachomatis antigen and Chlamydia trachomatis antibody is brought into contact with a labeled antibody against the antibody derived from the analyte sample, and (e) Unreacted labeled antibody is removed, and (f) each step of measuring the presence or amount of Chlamydia trachomatis antibody in the specimen sample by measuring the amount of the labeled substance on the bound labeled antibody is included. The present invention relates to a characteristic method for measuring Chlamydia trachomatis antibody. The present invention also relates to a preparation for diagnosing Chlamydia trachomatis infectious disease, which comprises a combination of an immobilized antigen in which a purified basic substance of Chlamydia trachomatis is immobilized on a solid phase carrier and a labeled antibody.

BEST MODE FOR CARRYING OUT THE INVENTION

Hereinafter, the present invention will be described in detail. The Chlamydia trachomatis purified EB used in the present invention can be prepared by combining already known methods. For example,
By the following method. After ultrasonically disrupting an infected culture cell suspension in which a Chlamydia trachomatis strain was infected and cultured, an infection culture supernatant was obtained, and the supernatant was layered on, for example, sucrose-containing Tris-HCl buffer. Then, the obtained centrifugal precipitate is suspended by adding, for example, sodium phosphate buffer (PBS), and this suspension is suspended in urographin (diatrizoat).
HE containing e meglumine and trizatesodium) and salt
PES (N-2-hydroxyethylpiperazine-N'-2-ethanesulf
onic acid) solution and centrifuged again, the obtained precipitate is suspended again in a sodium phosphate buffer solution, layered on a urographine discontinuous density gradient, and centrifuged. The EB sedimentation zone generated at the interface of urographine having different concentrations was collected, and SPG was collected.
The purified EB can be obtained by removing residual urographine by diluting with and repeating centrifugation.

Here, the purified EB is substantially free from contaminants other than EB, such as host cells and their constituent fragments (proteins, other contents, etc.). Generally, conventionally used unpurified EB contains about 50% by volume of such impurities. The purified EB of the present invention preferably contains 90% by volume or more of EB. The volume ratio can be measured from the area ratio of cells and the like shown in an electron micrograph. In the present invention, it is important to use the purified Chlamydia trachomatis EB as an antigen as described above, and if any one polypeptide, for example, about 39.5 is used.
It is difficult to say that the use of a purified K Dalton peptide (MOMP) causes a coagulative negative reaction, which is a clinically very serious problem, and obtains sufficient species specificity.

In the preparation of the present invention in which such purified Chlamydia trachomatis EB is used as an antigen raw material for measuring Chlamydia trachomatis antibody, the antigen is used as a solid phase carrier such as a plastic material such as polystyrene or vinyl chloride, nitrocellulose. It can be fixed on fiber materials such as nylon and nylon, and inorganic materials such as glass and silica gel, and its shape is titer plate, beads,
All shapes such as magnetic beads, paper disks, threads are possible. Of these, polystyrene beads or polystyrene microtiter plates are particularly preferred. Further, the preparation of the present invention is not limited to the method of immobilizing on the above-mentioned solid phase carrier by a method based on physical adsorption,
In principle, the antigen material may be immobilized on a carrier by a covalent bond method using BrCN ₂ or the like.

In the method for measuring human Chlamydia trachomatis antibody using the preparation of the present invention, human serum is mainly used as a specimen sample. However, the method of the present invention can be applied to a specimen sample having a low Chlamydia trachomatis antibody titer due to its low non-specific reactivity. Therefore, human tears, human cervical endometrial secretions, and prostatic fluid human semen extruded from the urethra can also be used as sample samples.

The above-mentioned antigen-immobilized solid-phase carrier is brought into contact with a specimen sample suspected of containing an antibody against Chlamydia trachomatis, which has been appropriately diluted, incubated for a certain period of time, reacted, and then left unreacted. The analyte sample component of the reaction is removed to obtain a complex of Chlamydia trachomatis outer membrane-constituting polypeptide antigen and Chlamydia trachomatis antibody on the carrier. Next, this antigen / antibody complex is brought into contact with a labeled antibody against an antibody derived from a sample, preferably an enzyme-labeled anti-human IgG, IgA or IgM antibody, and reacted. Then, the unreacted labeled antibody is removed, and the amount of the labeled substance bound to the bound labeled antibody is measured to measure the IgG, IgA, or IgM Chlamydia trachomatis antibody present in the specimen sample.

The labeling substance may be a radioisotope, a fluorescent dye or the like, and is not limited to an enzyme. As a labeling enzyme, malate dehydrogenase (enzyme number 1.1.
1.37), glucose-6-phosphate dehydrogenase (the same 1.1.1.
49), glucose oxidase (same as 1.1.3.4), horseradish peroxidase (same as 1.11.1.7), acetylcholinesterase (same as 3.1.1.7), alkaline phosphatase (same as 3.1.3.1), glucoamylase (same as 3.2.1.3), Lysozyme (3.2.1.17), β-galactosidase (3.
2.1.23) and the like can be used, but preferably alkaline phosphatase or horseradish peroxidase is used. The quantification of the labeling substance can be performed according to a conventional method selected according to the type of the labeling substance.

It has been revealed that the Chlamydia trachomatis antibody titer in the specimen sample thus measured well reflects the clinical picture of Chlamydia trachomatis infection. That is, it is considered that the IgM antibody appears 1 to 2 weeks after the infection due to the infection of the body surface with Chlamydia trachomatis, and the IgG antibody is recognized relatively early and decreases with time, but lasts for a long time. On the other hand, secretory IgA antibody is considered to be useful for protection against re-infection, as in cell-mediated immunity. Regarding human genital infection of Chlamydia trachomatis, IgG, IgA, in serum and cervical endometrial secretions, especially in women,
It is known that IgM antibody is detected, and the amount of IgA antibody is inversely proportional to the decrease of the pathogen Chlamydia trachomatis, and regulates the excretion and diffusion of the pathogen (Brunha
m., Infect. Immun., 39, 1491-1494, 1983).

[0017]

[Examples] The following examples show the chlamydia of the present invention.
The method for measuring the Trachomatis antibody will be described in more detail, but the present invention is not limited thereto.

A) Purification of Chlamydia trachomatis elementary bodies (EB) Chlamydia trachomatis strain L2 / 434 / Bu
bo was used. 10% fetal bovine serum (FBS)
McCoy cells grown in supplemented Eagle MEM medium were inoculated. For inoculation of Chlamydia trachomatis, cells of 3 days in culture were used, and 150 × g, 5
The cells were collected by centrifugation for 1 minute, and washed with Hank's BSS three times. For adsorption, 10 Chlamydia trachomatis are reacted per host cell, and the host cell concentration is 10 ⁷ / ml.
At 37 ° C. for 2 hours in warm water with shaking. After adsorption, the cells were resuspended in FBS-added medium so that the cell concentration became 5 to 7 × 10 ⁵ / ml, and cultured at 37 ° C. for 2 days. After culturing, a part thereof was collected, the infected cells were collected by centrifugation to prepare a smear, and it was confirmed by Giemsa staining that EB was sufficiently grown.

[0019] The above-mentioned infected cell suspension was sonicated at 20 KHz by an ultrasonic disrupter (Nippon Seiki Seisakusho US-300) at 6 kHz.
After sonication for 0 second, centrifugation was performed at 20 ° C. for 20 minutes to obtain an infected culture supernatant. 5% of this supernatant was added to 30% sucrose (w /
v), and overlaid with 1 volume of 0.033 M Tris-HCl buffer (pH 7.2) containing 10,000 xg at 4 ° C.
It was centrifuged for 60 minutes. 0.01 M sodium phosphate buffer (PBS) containing 0.14 M sodium chloride in this centrifugal sediment (pH
7.2) was added and suspended, and this suspension was mixed with 35% urographin (diatrizoate meglumine and trizatesodium).
And 0.01M HEPES (N- containing 0.15M sodium chloride)
2-hydroxyethylpiperazine-N'-2-ethanesulfonic aci
d) Layered on the solution and centrifuged at 43,000 xg, 4 ° C for 60 minutes (Hitachi Koki Co., Ltd. separation ultracentrifuge 70P-72,
Swing rotor SPR28SA). The precipitate was suspended in 0.01 M sodium phosphate buffer (SPG, pH 7.2) containing 0.25 M sucrose and 5 mM glutamic acid, and overlaid with a urographine discontinuous density gradient (40% / 44% / 52%). , 43,000 × g, and 4 ° C. for 60 minutes.
The EB sedimentation zone that occurs at the interface of 44% and 52% urographine was collected, diluted with SPG, and washed with 30,000 × g, 4
Remaining urographin was removed by repeating centrifugation at 30 ° C. for 30 minutes to obtain purified EB (containing 90% by volume or more of whole cells and fragments thereof).

B) Method for measuring Chlamydia trachomatis antibody Using the purified EB obtained by the above method as an antigen, a carrier fixing buffer (NaHCO ₃ and Na ₂ CO _{2 in} 1 liter)
Each containing 2.93 g and 1.59 g) was adjusted to 0.5 μg / ml, 100 μl was dispensed into each well of a 96-well polystyrene microtiter plate, and the mixture was reacted at 4 ° C. overnight.
PBS containing 0.05% Tween 20 (pH
7.2) (0.05% Tween 20-PBS) 300
It was washed 3 times with μl to remove unadsorbed antigen. Next 5% BS
A blocking buffer containing A (manufactured by KPL) (blocking / specimen dilution buffer) (250 μl) was added to each well and reacted at 37 ° C. for 1 hour to give 0.05% Tween 20-P.
An antigen-immobilized plate was prepared by washing twice with 300 μl of BS.

100 μl of diluted specimen serum diluted with blocking / specimen dilution buffer was added to the above-mentioned antigen-immobilized plate and reacted at 37 ° C. for 1 hour, followed by 0.05% Tween 20.
-Washed 3 times with 300 μl of PBS. 0.05% anti-human IgG antibody labeled with horseradish peroxidase
Dilute to 1 μl / ml with Tween 20-PBS, dispense 100 μl into each well, and incubate at 37 ° C for 1 hour.
% Tween 20-PBS (300 μl) was washed 3 times. Next, the peroxidase substrate ABTS reagent (KP
L) 100 μl was added to each well and reacted at room temperature for 5 minutes, and then 25 μl of 2% oxalic acid was added to stop the reaction.
P-22 type) absorbance (λ ₁ : 415 nm, λ ₂ : 492n
m) was measured.

C) Sensitivity and specificity to human patient serum (measurement of Chlamydia trachomatis IgG antibody), 20 samples of Chlamydia trachomatis-infected patient serum (antigen positive) confirmed to be isolated from Chlamydia trachomatis, Chlamydia・ B) using 9 sera of trachomatis antibody-positive sera (antigen-negative) and 23 normal human sera
The sensitivity and specificity tests were carried out according to the measuring method (ELISA method) described in 1. The micro-IF method was performed as a control test method. Human serum was diluted 80 times with a sample dilution buffer containing 1% BSA to obtain a sample solution for measuring Chlamydia trachomatis antibody.

In order to further clarify the effects of the present invention, purified Chlamydia trachomatis major outer membrane peptide (purified MOMP) was used as a control antigen for measuring Chlamydia trachomatis antibody, and used in the present invention. The sensitivity and specificity of the purified Chlamydia trachomatis L2 strain EB were compared. The results are shown in Table 1. In addition, purified MO
MP is a partially purified MOMP fraction prepared according to the method of Caldwell H, D et al. (Japanese Patent Laid-Open No. 57-158725) and further subjected to preparative SDS polyacrylamide gel electrophoresis to give an immunologically uniform state. The prepared standard was used. The cut-off value of each test was a value obtained by adding the standard deviation twice the average value of the absorbance of 23 normal human serum samples.

The results in Table 1 show that the method of the present invention using the purified EB of Chlamydia trachomatis has a very good agreement rate with the micro-IF method as compared with the purified MOMP tested as a control antigen. Shows. That is, there are no false positives (judgment based on the data of negative concordance (specificity)), and in particular, clinically problematic falsification (fals
The e negative) is very low compared to the purified MOMP, indicating that the clinical utility is high.

[0025]

[Table 1]

[0026]

INDUSTRIAL APPLICABILITY According to the method for measuring Chlamydia trachomatis antibody of the present invention, compared to the method using unpurified EB or purified MOMP, there is almost no clinically problematic agglutination and no agglutination. Further, it is possible to perform highly species-specific measurement with low nonspecific reaction with the Chlamydia pneumoniae antibody and the Chlamydia shita antibody, which is highly clinically useful. Further, the Chlamydia trachomatis infection diagnostic preparation of the present invention has almost no clinically problematic coagulative negative, no coagulative positive, and a low nonspecific reaction with a Chlamydia pneumoniae antibody and a Chlamydia shita antibody, Chlamydia trachomatis antibody with high species-specificity can be measured and has high clinical utility.

Claims (11)

[Claims] 1. A method comprising: (a) immobilizing purified Chlamydia trachomatis elementary bodies on a solid phase carrier; and (b) contacting the solid phase carrier with a specimen sample suspected of containing an antibody against Chlamydia trachomatis. And (c) removing unreacted components of the specimen sample, and (d) contacting the generated antigen-antibody complex of Chlamydia trachomatis antigen and Chlamydia trachomatis antibody with a labeled antibody against an antibody derived from the specimen sample. And (e) removing the unreacted labeled antibody, and (f) measuring the amount of the labeled substance on the bound labeled antibody to determine the presence or amount of the Chlamydia trachomatis antibody in the specimen sample. A method for measuring Chlamydia trachomatis antibody, which comprises a step. 2. The test sample is any one of human tears, human cervical endometrial secretions, human semen and human serum.
The method for measuring Chlamydia trachomatis antibody described. 3. The labeled antibody is anti-human IgG, IgA or I
The method for measuring Chlamydia trachomatis antibody according to claim 1 or 2, which is a gM antibody. 4. The method for measuring Chlamydia trachomatis antibody according to claim 1, 2 or 3, wherein the labeled antibody is an enzyme-labeled antibody. 5. The method for measuring Chlamydia trachomatis antibody according to claim 4, wherein the enzyme-labeled antibody is an alkaline phosphatase- or horseradish peroxidase-labeled antibody. 6. A preparation for diagnosing Chlamydia trachomatis infectious disease, which comprises a combination of an immobilized antigen in which purified purified basic bodies of Chlamydia trachomatis are immobilized on a solid phase carrier and a labeled antibody. 7. The preparation for diagnosing Chlamydia trachomatis infection according to claim 6, wherein the solid phase carrier is selected from plastic materials, fiber materials and inorganic materials. 8. The preparation for diagnosing Chlamydia trachomatis infection according to claim 6 or 7, wherein the solid phase carrier is polystyrene beads or polystyrene microtiter plate. 9. The preparation for diagnosing Chlamydia trachomatis infection according to claim 6, 7 or 8, wherein the labeled antibody is an anti-human IgG, IgA or IgM antibody. 10. The preparation for diagnosing Chlamydia trachomatis infection according to claim 6, 7, 8 or 9, wherein the labeled antibody is an enzyme-labeled antibody. 11. The preparation for diagnosing Chlamydia trachomatis infection according to claim 10, wherein the enzyme-labeled antibody is an antibody labeled with alkaline phosphatase or horseradish peroxidase.