JPH07248329A - Detecting method of tubercular infectivity inspection membrane and tubercular infectivity related antibody therewith - Google Patents

Abstract

PURPOSE:To provide a tubercular infectivity inspection membrane, which is simple in operation, requiring no special equipment, and enables the detection of such a tubercular infectivity related antibody as high being high in sensitivity and peculiarity, and an inspection method of the tubercular infectivity related antibody in use of this membrane as well as tubercular infectivity inspection kit. CONSTITUTION:A tubercular infectivity inspection membrane in this invention is featured that a mycolic acid content glycolipid is fixed on an amphipathic high polymer membrane such as PVDF(polyvinylydendeflorid) or the like. This membrane is made contact with a specimen of blood serums, etc., being doubtful to contain a tubercular infectivity related antibody, and then a labeled second antibody is connected together, whereby the tubercular infectivity related antibody is made so as to be detectable speedily and simply.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a tuberculosis infectious disease test membrane and a method for detecting an antibody associated with tuberculosis infectious disease using the membrane.

[0002]

2. Description of the Related Art The number of tuberculosis patients is decreasing with the development of powerful drugs and improvement of hygiene environment, but the number of tuberculosis patients in Japan still reaches about 50,000 per year. It is also one of the widespread and irreversible diseases in developing countries. Furthermore, recently, tuberculosis infection of HIV-infected persons has become a big problem.

As a test method for tuberculosis infection, a culture method using Ogawa's medium has been used for a long time, but this method requires a long period of 1-2 months for culture. Recently, a PCR method and a DNA probe method have been developed as a rapid test method. However, the PCR method and the DNA probe method have a large practical problem that they require skill in operation and require a special facility.

In addition, H. He et al. (FEMS Microbiol Immun
ol. 1991, 76 (4), 201-4), R.A. Maekura et al. (A
m. Rev. Respir. Dis. 1993, 148 (4), 997-1001), and International Publication No. WO90 / 08323 disclose the detection of Mycobacterium tuberculosis antibody by the microplate ELISA method. In these methods, a mycolic acid-containing glycolipid (coding factor, TDM, etc.), which is a characteristic bacterial membrane surface layer component of Mycobacterium tuberculosis and has toxicity, is adsorbed on a microplate as an antigen, and an antigen-antibody reaction is utilized. Antibodies associated with tuberculosis infection are detected. This microplate E
The LISA method is useful as one of rapid tuberculosis infection test methods. However, it has a drawback in that it requires a large amount of antigen and requires an expensive device such as a microplate reader. Therefore, it is desired to develop a simpler, cheaper, and faster inspection method.

On the other hand, a method of detecting an antigen or an antibody by immobilizing a protein on a nitrocellulose membrane and carrying out an antigen-antibody reaction on the membrane is well known in, for example, Western blotting and immunoscreening. However, since the nitrocellulose membrane used in these methods is not resistant to organic solvents, it cannot be used for immobilizing mycolic acid-containing glycolipid, which is insoluble in water. Further, although immobilization of proteins using a carrier film other than nitrocellulose has been reported, no example of immobilizing mycolic acid-containing glycolipid is known.

[0006]

From the above, the present invention eliminates the above-mentioned drawbacks, is easy to operate, does not require a special facility, and has high sensitivity and specificity for a tuberculosis test. The purpose is to provide a reagent for use.

[0007]

Means for Solving the Problems As a result of intensive studies aimed at achieving such an object, the present inventors have found that an amphipathic polymer film is extremely useful as a carrier film for immobilizing water-insoluble mycolic acid-containing glycolipids. They have found that they are excellent and have completed the present invention.

The present invention provides a membrane for inspecting tuberculosis infection characterized in that a glycolic acid containing mycolic acid is immobilized on an amphipathic polymer membrane.

As used herein, the term "amphipathic" means a property of having an affinity for water and an organic solvent, but being insoluble in both water and the organic solvent.
Here, the organic solvent is an organic solvent generally used for dissolving glycolipids in the field of biochemistry, for example, chloroform, ethanol, methanol, acetone, n-.
It means hexane and the like.

The amphipathic polymer membrane used as the carrier membrane of the tuberculosis infection test membrane of the present invention has excellent immobilization properties for glycolipids, which are antigens, and, unlike the nitrocellulose membrane, is resistant to organic solvents. It is characterized by being. Therefore, the mycolic acid-containing glycolipid, which is insoluble in water, can be dissolved in the organic solvent and can be easily and stably immobilized on the membrane. Furthermore, since this membrane also has hydrophilicity, it is suitable for use in an antigen-antibody reaction carried out in an aqueous system and a subsequent enzyme reaction. As the amphipathic polymer film, for example, a PVDF (polyvinylidene difluoride) film, a polyamide film which may have a side chain functional group, or the like can be used. Preferably, a PVDF membrane or a side chain amino group,
A nylon film having a carboxyl group or ⁺ NR ₃ group is used. Most preferably a PVDF membrane is used.

The amphipathic polymer membrane used in the present invention can be produced by a method well known to those skilled in the art,
It is also commercially available. Nylon membranes are available, for example, from Pol (USA), and PVDF membranes are available, for example, from Japan Bio-Rad Laboratories.

The mycolic acid-containing glycolipid used as an antigen in the tuberculosis infection membrane of the present invention can be prepared by a well-known method. Briefly, Mycobacter
ium tuberculosis) is shake-cultured in a Sawton's medium at 37 ° C. for 4 to 8 weeks, and then the grown tubercle bacilli are precipitated by centrifugation. The obtained precipitate is washed twice with ion-exchanged water. The microbial cells are suspended in ion-exchanged water and sonicated to disrupt the microbial cells. This suspension is mixed with 20 volumes of an organic solvent (for example, chloroform / methanol 2: 2).
1 mixed solution) and separated. The organic solvent extract is dried, dissolved in a small amount of organic solvent, and separated by thin layer chromatography using a solution of chloroform / methanol / acetone / acetic acid as a developing solution. After separation
After sufficiently removing the developing solvent, the color is developed by the iodine method or the like, and the target glycolide containing mycolic acid is collected.

The tuberculosis infection test membrane of the present invention can be produced by immobilizing the mycolic acid-containing glycolipid prepared as described above on an amphipathic polymer membrane which is a carrier membrane. Specifically, the extracted antigen is diluted with an organic solvent or the like, if necessary, and the amount of 0.
After fixing 1-10 μg, preferably 0.2-2.0 μg, it is dried. Preferably, blocking treatment is performed before or after drying. That is, an amphipathic polymer membrane having a mycolic acid-containing glycolipid immobilized thereon is treated with, for example, Tween.
20 (0.01-10%, preferably 0.05-5%)
Tris buffer (0.001-1M, preferably 0.05-0.5M, pH 6-9, preferably pH 7.
5-8.5) or the like for blocking at room temperature for 1-16 hours, and then Tris buffer (0.001-1M, preferably 0.05-0.5M, pH 6-9, preferably pH).
After washing with 7.5-8.5) or the like, it is dried.

As will be shown in Examples described later, by using the tuberculosis infectious disease test membrane of the present invention, a simple and highly sensitive test for tuberculosis infectious disease becomes possible. In addition, the tuberculosis infection test membrane of the present invention has an advantage that it can be stored in a storage solution or in a dry state at room temperature for at least 3 months. When storage for a longer period is desired, it can be stored as a dried product after being treated with a protective agent such as a preservative.

The membrane for inspecting tuberculosis infection according to the present invention is, for example,
In the form of a strip with a side of 0.2 cm-30 cm,
The antigen can be immobilized on the spot having a diameter of about 0.05 cm-1 cm. As one specific example, the membrane for inspecting tuberculosis infection according to the present invention comprises about 1 cm × 1 c
m strip, diameter about 0.3c on the strip
The antigen is immobilized on the spot m. As another specific example, the tuberculosis infection test membrane of the present invention has a size of about 1 cm × 3 cm.
Of two diameters on the strip of about 0.
Mycolic acid-containing glycolipids derived from Mycobacterium tuberculosis and mycolic acid-containing glycolipids derived from eosinophils other than Mycobacterium tuberculosis were immobilized on 3 cm spots. Antibodies specific for mycolic acid-containing glycolipids of acid-fast bacteria can be detected.
As another specific example, the membrane for inspecting tuberculosis infection according to the present invention is provided with 3 to 1000 antigen-fixing spots on one membrane, and a necessary amount may be appropriately cut during use. Can be used.

According to another aspect of the present invention, the present invention uses a membrane for inspecting tuberculosis infection, which comprises an amphipathic polymer membrane having a mycolic acid-containing glycolipid immobilized thereon. A method for detecting an antibody against a contained glycolipid is provided. This method comprises contacting the tuberculosis infection test membrane of the present invention with a sample suspected of containing an anti-mycolic acid-containing glycolipid antibody, and detecting the presence of the antibody by an antigen-antibody reaction.

As a sample, serum of human or animal suspected of being infected with Mycobacterium tuberculosis or other mycobacteria is appropriately diluted and used. Antibody detection by antigen-antibody reaction
It can be carried out according to the usual ELISA method, for example, as follows. The tuberculosis infection test membrane of the present invention is equilibrated with 50% methanol or the like, and then Tris buffer solution (0.001-1M, preferably 0.05-0.5M, pH 6-) containing a blocking agent such as skim milk powder. 9, preferably pH
7.5-8.5) (hereinafter, referred to as a wash diluent), the sample serum was added, and the mixture was added at 37 ° C for 10 minutes-
Incubate for 16 hours, preferably 30 minutes to 2 hours. The membrane is then washed with wash diluent to remove anti-human Ig
Add a wash diluent containing G-enzyme labeled antibody at room temperature for 10
Incubate for 2 minutes to 2 hours, preferably 20 minutes to 1 hour. After washing the membrane with the wash diluent, a substrate solution is added and color development due to the enzymatic reaction is observed.

The enzyme-labeled antibody and the substrate used in the method of the present invention are well known to those skilled in the art, and for example, alkaline phosphatase-labeled antibody can be treated with 5-bromo-4-chloro-3-indolyl phosphate p-toluidine salt. (BCI
P) and nitroblue tetrazolium (NBT). Further, peroxidase and orthophenylenediamine can be used in combination. Further, it is within the skill of those skilled in the art to modify or improve the method of the present invention by using a tertiary antibody, a color-promoting agent, a non-specific antigen absorbent, a surfactant, biotin-avidin and the like.

According to still another aspect of the present invention, the present invention comprises detection of a tuberculosis infection-related antibody comprising a tuberculosis infection test membrane having a mycolic acid-containing glycolipid immobilized on an amphipathic polymer membrane. A kit for use. This kit may contain an enzyme-labeled antibody, a substrate, a washing solution, a diluting solution, a non-specific antigen absorbent, and the like, in addition to the tuberculosis infection test membrane of the present invention. Further, it may contain negative and positive standard sera for control, or color samples for judgment or quantification.

The kit of the present invention contains, for example, the following reagents: Tuberculosis infection membrane 2. Equilibration solution 3. Wash diluent 4. Alkaline phosphatase labeled goat anti-human IgG antibody 5.5-Bromo-4-chloro-3-indolyl phosphate
p-toluidine salt 6. Nitro blue tetrazolium 7. Negative control serum 8. Positive control serum 9. Judgment film.

If the membrane for inspecting tuberculosis infection according to the present invention is used,
The anti-mycolic acid-containing glycolipid antibody in the sample can be easily and approximately 3 times.
It can be detected visually by a short time, that is, within a time. Further, the detection method using the tuberculosis infectious disease test membrane of the present invention is carried out by the conventional microtiter plate ELIS.
It has a higher sensitivity than Method A. For example, in the conventional method, about 2.5 μg of antigen is required per sample, and 160-fold diluted serum can be tested using this, whereas in the present invention, about 1 μg of antigen and 128 are used.
It can be detected with 0-fold diluted serum. Therefore,
The present invention has extremely high detection sensitivity as compared with the conventional microtiter plate method, and can detect a low concentration of antibody that cannot be detected by the conventional method. Further, since the tuberculosis infection test membrane of the present invention has high specificity for antibodies, it is possible to simultaneously distinguish between antibodies against Mycobacterium tuberculosis and antibodies against other mycobacteria, which makes it possible in conventional diagnostic methods. It is expected that it is possible to distinguish between tuberculosis and mycobacteriosis, which were difficult to distinguish. Further, the membrane for inspecting tuberculosis infection according to the present invention can be produced at low cost, and is expensive when used, such as expensive equipment such as plate reader and DNA.
Since it does not require any special technique or facility for handling the, it has an advantage that the cost required for diagnosis is much lower than that of the conventional method.

Hereinafter, the present invention will be described in more detail with reference to examples.

[0023]

【Example】

Example 1 Production of Membrane for Inspecting Tuberculosis Infection Mycolic acid-containing glycolipid used as an antigen was prepared as follows. That is, three strains of bacteria (Mycobacterium tuberculos
is AOYAMA B, Mycobacterium tuberculosis H37Rv, or Mycobacterium smegmatis) was shake-cultured in Sawton's medium at 37 ° C. for 4-8 weeks, and the grown cells were precipitated by centrifugation. The obtained precipitate was washed twice with ion-exchanged water, the cells were suspended in ion-exchanged water, and the cells were disrupted by ultrasonic treatment. This suspension was mixed with 20 volumes of chloroform / methanol 2: 1.
Extract with a mixed solution, dissolve in a small amount of organic solvent after drying,
Separation was performed by thin layer chromatography using a solution of chloroform / methanol / acetone / acetic acid as a developing solution. After the separation, the developing solvent was sufficiently removed, and then the color was developed by an iodine method or the like to collect the target glycolic acid containing mycolic acid.

As the carrier film, a PVDF film (trade name P
A VDF protein sequencing membrane, manufactured by Nippon Bio-Rad Laboratories, Inc.) and a nylon membrane (trade name: Biodyne, manufactured by Pol Co., Ltd.) were used.

The mycolic acid-containing glycolipid prepared as described above was added to a 2: 1 mixture of chloroform / methanol to a concentration of 0.
It was dissolved at 25 mg / ml, fixed at 1 μg per sample on a PVDF membrane or nylon membrane, and then dried in a constant temperature bath (37 ° C.). The antigen-immobilized membrane was shaken in 50% methanol at room temperature for 10 minutes for equilibration, and then the solution was removed. This membrane is 100 mM Tr
1 at room temperature in is-HCl buffer (pH 8.3)
Incubated for 0 minutes (twice). Next, this film is
100 mM Tris-HCl containing 1% Tween 20
Blocking was performed by incubating in 2 ml of buffer solution (pH 8.3) at room temperature for 1 hour,
100 mM Tris- containing 0.1% sodium azide
Incubate with HCl buffer (pH 8.3) for 10 minutes at room temperature to wash (twice). After washing, the excess washing liquid was removed with a paper towel, dried in a thermostat (37 ° C.) for 30 minutes, and stored in a desiccator. Example 2 Detection of Anti-Mycolic Acid-Containing Lipid Antibody Using Antigen-Immobilized Membrane According to Example 1, Mycobacteria
antigen-immobilized PVDF membrane (1 cm) on which 1 μg of mycolic acid-containing glycolipid derived from ium tuberculosis AOYAMA B) was immobilized
X 1 cm) and an antigen-immobilized nylon membrane were prepared. As a control, a PVDF membrane and a nylon membrane on which an antigen was not immobilized were used. The membranes were incubated in a 50% methanol solution for 10 minutes at room temperature to equilibrate and remove the solution. This membrane is washed with a diluent (20 mM Tris-HCl buffer containing 0.5% skim milk powder, pH)
8.3) Incubate in 2 ml at room temperature for 10 minutes to equilibrate (twice), then remove by aspiration and add 2 ml wash diluent. Here, 10 μl of serum of a patient whose tuberculosis infection-related antibody has been found to be positive in advance
(Final dilution 1: 200) was added and incubated at 37 ° C. for 1 hour. Next, it was washed twice with a wash diluent (2 ml), and alkaline phosphatase-labeled anti-human IgG was added.
After adding a wash diluent containing a goat antibody and incubating at room temperature for 30 minutes, the solution was removed and washed twice with 2 ml of the wash diluent. Remove the solution and add a new substrate solution (BCIP
3.8 mg / 100 ml, NBT 7.6 mg / 100
2 ml) was added and incubated at room temperature for 10 minutes to develop color.

Purple color development of spots was observed on both the antigen-immobilized PVDF membrane and the antigen-immobilized nylon membrane. PVDF is the background (coloring of the part other than the spot)
The membrane was lower. On the other hand, no spot color was observed on the PVDF membrane on which the antigen was not immobilized and the nylon membrane.

That is, the tuberculosis infectious disease test membrane of the present invention develops a color by specifically reacting with a tuberculosis infectious disease-related antibody, and is useful for a simple and rapid test for tuberculosis infectious disease-related antibody. Was shown. Example 3 Assay of Human Serum Sample Using Antigen-immobilized Membrane According to Example 1, PVDF membrane (1 cm × 2 c)
m) 3 spots on 3 spots (Mycobacterium tube
rculosis AOYAMA B, Mycobacterium tuberculosis H37R
Antigen-immobilized membranes were prepared by immobilizing 1 μg each of mycolic acid-containing glycolipids derived from v and Mycobacterium smegmatis). As samples, normal human serum, typical tuberculosis patient serum and atypical tuberculosis patient serum, which were distributed by Osaka City University School of Medicine, were used.

Assay of anti-mycolic acid-containing glycolipid antibody was carried out in the same manner as in Example 2. 50% of antigen-immobilized membrane
Equilibration with methanol solution followed by wash diluent. Add 10 μl of sample serum (final dilution 1: 200), 37 ℃
Incubated for 1 hour. Next, after washing twice with a wash diluent (2 ml), a wash diluent containing an alkaline phosphatase-labeled anti-human IgG goat antibody was added and incubated at room temperature for 30 minutes, then the solution was removed and 2 ml of wash diluent was used. Washed twice. The solution was removed, 2 ml of the substrate solution was newly added, and the mixture was incubated at room temperature for 10 minutes for color development. A photograph of the colored antigen-immobilized membrane is shown in FIG.

The results were visually evaluated. Those with no color difference between the antigen-immobilized membrane and control (antigen-immobilized membrane) are negative (-), those with a color difference are positive (+), and those with a large color difference are strong. Positive (+
+), False positives (±) if it is difficult to judge either
And These results were measured by the plate ELISA method, conventional culture (Ogawa medium), and sputum staining (Guf).
fky determination method) and clinical diagnostic results. The plate ELISA was performed as follows. 2.5 μg of mycolic acid-containing glycolipid was adsorbed to each well of a 96-well microtiter plate, 50 μl of patient serum (dilution ratio 1: 160) was added, and the mixture was incubated at room temperature for 1 hour. Wells were washed and peroxidase labeled anti-human Ig
G goat antibody (1: 500) was added and incubated. Ortho-phenylenediamine was added as a substrate, and the absorbance was measured with a microplate reader. In addition, culture with Ogawa medium and sputum staining were performed according to known methods.

The results are shown in Table 1.

[0031]

[Table 1] As shown in Table 1, by using the tuberculosis infection test membrane of the present invention, it is possible to detect a tuberculosis infection-related antibody with a sensitivity equal to or higher than that of the conventional method by an extremely simple operation. It was

[Brief description of drawings]

FIG. 1 is a photograph showing detection of an antibody associated with tuberculosis infection using the tuberculosis infection test membrane of the present invention. The three spots on each strip are from the left, Mycobacteriumt
uberculosis AOYAMA B, Mycobacterium tuberculosis H
Mycolic acid-containing glycolipids derived from 37Rv and Mycobacterium smegmatis are immobilized. The numbers in each figure correspond to the patient numbers in Table 1.

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Ken Suehiro 4-4-216 Higashi-Shisui, Shisui-machi, Inba-gun, Chiba Prefecture

Claims (3)

[Claims] 1. A membrane for inspecting tuberculosis infection, wherein a glycolic acid containing mycolic acid is immobilized on an amphipathic polymer membrane. 2. A method for detecting an antibody against a mycolic acid-containing glycolipid, which comprises using the membrane for inspecting tuberculosis infection according to claim 1. 3. A kit for detecting an antibody against a mycolic acid-containing glycolipid, which comprises the membrane for inspecting tuberculosis infection according to claim 1.