JPH0712815A - Method for quickly diagnosing infectious disease - Google Patents

Abstract

PURPOSE:To extremely quickly and easily detect a phatogenic factor in a specimen with high sensitivity by detecting the phatogenic factor by performing enzyme immunoassay using a high polymer material on the surface of which a large amount of the antibody against the phatogenic factor is stably immobilized. CONSTITUTION:At the time of detecting a phatogenic factor by performing enzyme immunoassay after catching the photogenic factor by using a high polymer material on the surface of which the antibody against the phatogenic factor is immobilized by covalent bonding through an acid anhydride group, the phatogenic factor is detected by measuring the enzyme activity by using a water soluble compound using a marker enzyme as a substrate.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a rapid diagnostic method for infectious diseases using an antibody-immobilized polymer material, which is used in fields such as clinical tests and food tests. More specifically, it is a substrate in a reaction solution. The present invention relates to a rapid diagnostic method for an infectious disease using an antibody-immobilized polymer material capable of extremely rapidly detecting a pathogenic factor in a sample by the color development of.

[0002]

2. Description of the Related Art In order to correctly diagnose and treat a pathogenic factor, it is necessary to accurately and promptly test the causative factor causing the pathogenic factor. When the causative agent is a microorganism, the culture test method that is generally used requires skill in the test, and 10 to 16 hours of enrichment culture and 18 to 24 hours of separate culture are required, and the test results are revealed. It takes a long time to do so, and it is not always possible to meet the demands of clinicians for treatment. Therefore, in the examination of pathogenic factors, the development of a test method that is suitable for clinical use is desired.

Recently, as a means for solving these problems,
Although the DNA probe method and immunological method have been developed and researched, among them, the immunological method is superior in specificity, rapidity, and convenience because it utilizes an antigen-antibody reaction, and moreover, is direct. It is expected that specimens can be searched and diagnosis can be made extremely quickly.

[0004]

In recent years, methods for detecting pathogenic factors using these immunological techniques have been extensively studied. One of them is to allow latex particles to nonspecifically adsorb an antibody against a toxin. , There is a method of examining the latex agglutination reaction associated with the antigen-antibody reaction (latex agglutination method), but it has handling problems such as relatively low sensitivity, long inspection time, difficult determination, and difficult to obtain stable results. Was.

[0005] In addition, a method which is widely used at present is a method in which an antibody against a pathogenic factor is physically adsorbed by a hydrophobic interaction between a protein and a polymer material, of which polystyrene is representative. There is a method of reacting a sample with a microplate consisting of or using beads such as polystyrene, nylon, agarose, glass, etc., and then adding an enzyme-labeled antibody, and measuring the enzyme activity recovered on the plate or beads. At this time, the product generated by the decomposition of the substrate in the reaction solution is near ultraviolet (190-400 nm)
Alternatively, when it has an absorption in the visible region (400 to 800 nm) or produces a fluorescent substance, the detection measurement is performed using a spectroscopic instrument. However, in general, in the case of detection by such an immobilization method by physical adsorption, since the protein and the polymer material are bound by a nonspecific hydrophobic interaction, they are bound during the analytical operation. Desorption of the antibody causes problems such as low sensitivity and poor reproducibility, and conversely, when the number of washings is reduced, a nonspecific positive reaction occurs.

On the other hand, these problems can be overcome by using a method of chemically immobilizing a polymer material and an antibody, but the detection sensitivity does not increase as expected as compared with the amount of antibody bound. However, it has a problem that it must be stably bound but must be stored in a solution, and the storage period is relatively short (about 4 weeks).

[0007] As a representative of the currently known chemical immobilization methods, a method of immobilizing an antibody on a hydrazide-group-introduced polystyrene treated with glutaraldehyde (Katsuru Ishii: clinical examination, vol.34) , 759 (1990)), a method of immobilizing an antibody on glutaraldehyde-treated polystyrene having an alkylamino group introduced therein (Eiji Ishikawa, "Enzyme Immunoassay", Tokyo Kagaku Dojin, 198).
9 years), a method of immobilizing antibodies on polystyrene treated with Sanger reagent treated with glutaraldehyde (Sa
nger: Biochem. J., 39, 507 (1945)), a method of immobilizing antibodies on partially hydrolyzed nylon treated with glutaraldehyde or carbodiimide (Hendry, Herrma.
nn: J. Immunol. Methods, 35,285 (1980)), a method in which succinic anhydride is allowed to act on polystyrene having an alkylamino group introduced, and the resulting carboxyl group and the amino group of the antibody are bound using a carbodiimide ( Masaru Ishii: Clinical examination, vol.34, 759 (1990)). However, in these methods, one molecule of antibody is bound to one reactive group on the surface of the polymer material, and the immobilization reaction of the antibody is performed under relatively severe reaction conditions. Since a large amount of antibody is required, it has not always been popular.

[0008] It is an object of the present invention to provide a rapid diagnostic method for infectious diseases which detects pathogenic factors in a sample quickly, simply and with high sensitivity.

[0009]

Means for Solving the Problems As a result of intensive studies for solving the above problems, the present inventors have found that many acid anhydride groups present on the surface of the polymer material or introduced are present. After capturing the virulence factor in the sample using a polymeric material in which a large amount of the antibody against the virulence factor has been immobilized by covalent bonding in a stable and efficient manner, the captured virulence factor and the enzyme-labeled antibody The present invention was found to be capable of stably detecting a pathogenic factor present in a sample by causing the substrate solution to develop a color by binding to a substrate solution of a labeling enzyme which produces a soluble product. .

That is, the present invention uses an antibody-immobilized polymer material in which an antibody against the pathogen is immobilized on the surface of the polymer material by a covalent bond via an acid anhydride group, and after capturing the pathogen, In detecting a pathogenic factor by an enzyme immunoassay, a water-soluble compound is used as a substrate for a labeling enzyme, and a rapid diagnostic method for infectious diseases characterized by detecting a pathogenic factor by measuring an enzyme activity is summarized. To do.

The present invention will be described in detail below. The pathogenic factor in the present invention is a causative agent of a disease, and includes, for example, Mycobacterium tuberculosis, Staphylococcus, Streptococcus, Neisseria gonorrhoeae, Syphilis, Pertussis, Tetanus, Escherichia coli, Pneumococcus, Pseudomonas aeruginosa, Shigella, Diphtheria, Typhoid fever, paratyphoid fever, cereus,
Bacteria such as Yersinia, Campylobacter, Clostridium perfringens, Difficile, Salmonella, Vibrio parahaemolyticus, Cholera, fungi, pneumonia virus, hepatitis virus, AIDS virus, rotavirus, chlamydia virus, herpes virus, RS virus, influenza virus, measles virus Pathogenic microorganisms such as viruses, and botulinum toxin produced by Clostridium botulinum, enterotoxin of Staphylococcus, TSS toxin, hemolytic poison of Vibrio parahaemolyticus, cholera enterotoxin of Vibrio cholerae, Shiga toxin of Shigella,
Enterotoxin of toxigenic Escherichia coli, diphtheria toxin of diphtheria bacterium, tetanus toxin of tetanus bacterium, pertussis toxin of B. pertussis, toxin A of D. faecalis, exotoxin A of Pseudomonas aeruginosa, phospholipase C (α-toxin) of C. perfringens, Examples include toxins and colonization factors produced by microorganisms such as enterotoxin.

Among the above-mentioned virulence factors, cholera toxin (CT, molecular weight of about 3) is known as a virulence factor of Vibrio cholerae as a cholera toxin.
5,000) (Masaaki Iwanaga: Medical Bacteriology Volume 3, p.2)
43-269, 1988).

As a Vibrio parahaemolyticus toxin, a thermostable hemolytic toxin (Thermostable Direct Hemolysi) known as a virulence factor of Vibrio parahaemolyticus
n; TDH, molecular weight about 46,000) and toxin similar to TDH (TDH related hemolysin; TRH, molecular weight about 48,000)
[Toshio Miwatani, Makoto Ohashi (supervised), Yoshifumi Takeda, Yasuo Kudo, Sumio Shinoda, Takeshi Honda (edited): Vibrio parahaemolyticus III, Modern Publishing].

As a colonization factor for toxigenic Escherichia coli, among Escherichia coli having intestinal pathogenicity, bacteria colonize and grow in the small intestine,
Factors that attach to and proliferate human intestinal cells, which are the fimbriae of Enterotoxigenic Escherichia coli, that cause diarrhea due to the action of the produced toxin (Takeshi Honda: Medical Bacteriology Volume 2, p.65-95) , 1987), a toxin produced by toxin-producing Escherichia coli is a heat-labile enterotoxin (heat-labile ent) which is inactivated by heating at 60 ° C for 10 minutes.
erotoxin [LT]) and heat-stable enterotoxin (heat-stable enterot) that retains its activity even after heating at 100 ° C for 30 minutes.
oxin [ST]) (Takeshi Honda, Tetsuya Iida: Microorganisms Vol. 3, p. 248-257, 1987).

As a botulinum toxin, Clostridium botulinum (Clo
stridium botulinum) produces neurotoxins A, B, C, D, E, and F types with a molecular weight of about 300,000 (Keiji Oguma: Medical Bacteriology Vol. 1, p.211-247, 1987).

An enterotoxin produced by Staphylococcus aureus is a toxin protein having a molecular weight of about 29,000 (Shogo Masuda: Medical Bacteriology, Vol. 4, p.
119-138, 1989).

Clostridium perfringens
The enterotoxin produced by S. cerevisiae includes a toxin protein with a molecular weight of about 35,000, and phospholipase C (α-toxin) includes a protein with a molecular weight of about 43,000 (Junichi Sakurai: Medical Bacteriology 4, p.315-364, 1987). ).

As diphtheria toxin, diphtheria bacteria (Cor
ynebacterium diphtheriae) produced molecular weight of about 58,000
The toxin protein of the above is mentioned (Eisuke Mada: Medical Bacteriology, Vol. 4, p.39-57, 1989).

Bordetella is a pertussis toxin.
pertussis) produces a toxin protein with a molecular weight of about 110,000 (Yuji Sato: Medical Bacteriology Vol. 1, p.57-79, 1986).
).

As Shiga toxin of Shigella, Shigell
The cytotoxin with a molecular weight of about 29,000 produced by a dysenteriae 1 can be mentioned (Noda Kimitoshi: Medical Bacteriology Vol. 1, p.281-300,
1986).

As exotoxin A of Pseudomonas aeruginosa, Pseudomonas aeruginosa
A cytotoxic protein having a molecular weight of about 66,000 produced by Pseudomonas aeruginosa can be mentioned (Seki Matsumoto: Medical Bacteriology Vol. 1, p.367-436, 1986).

As toxin A of Difficile bacterium, the molecular weight of Clostridium difficile produced by Clostridium difficile is about
There are 440,000 to 600,000 proteins (Shinichi Nakamura: Medical Bacteriology Vol. 5, p.55-97, 1990).

As a tetanus toxin of tetanus, tetanus C
A neurotoxin protein with a molecular weight of about 150,000 produced by lostridium tetani is mentioned (Morihiro Matsuda: Medical Bacteriology Vol. 3, p.
307-358, 1988).

Next, in the present invention, the polymer material for immobilizing the antibody against the pathogenic factor is, for example, ethylene-
Vinyl acetate copolymer, polyvinyl chloride, polyurethane,
Examples thereof include synthetic polymers such as polyethylene, polystyrene, nylon, polyester and polycarbonate, natural polymers such as starch, gluten, chitin, cellulose and natural rubber, and derivatives thereof. Further, agarose derivatives having a hydrophobic group, nitrocellulose, and their derivatives are also included. In addition, inorganic polymer compounds such as porous glass, silica gel and hydroxyapatite can also be used.

These polymeric materials have shapes such as microplates, beads, films, sheets, tubes, fibers and sticks depending on the method of use.

In the present invention, in order to immobilize the antibody on the surface of the polymer material, it may be immobilized by a covalent bond via an acid anhydride. As a method for immobilizing an antibody by covalent bonding via an acid anhydride, the antibody may be immobilized via an acid anhydride group present on the surface of the polymer material, or a carboxyl group present on the surface of the polymer material, After introducing an acid anhydride group into another reactive functional group such as a formyl group, an amino group, an azido group, an isocyanate group, a chloroformyl group and an epoxy group, the antibody may be immobilized via this. If neither the acid anhydride group nor the reactive functional group is present on the surface of the polymer material, the acid anhydride group may be directly introduced to the surface of the material to immobilize the antibody, or the reactive functional group may be added. After the introduction, an acid anhydride group may be introduced to immobilize the antibody.

As a method of introducing a reactive functional group onto the surface of a polymer material, for example, when introducing a carboxyl group into an ethylene-vinyl acetate copolymer, after saponifying the ethylene vinyl acetate copolymer, carboxymethyl is introduced. It is introduced by converting. Further, the carboxyl group can be converted to an azide group via a hydrazyl group. Further, the carboxyl group can be converted to a chloroformyl group by chlorination with thionyl chloride, acetyl chloride or the like.

To introduce an amino group into the ethylene-vinyl acetate copolymer, the saponified ethylene-vinyl acetate copolymer may be aminoacetalized. The epoxy group can be introduced by reacting with epichlorohydrin, diethylene glycol diglycidyl ether or the like. The isocyanate group can be introduced by reacting with hexamethylene diisocyanate, tolylene diisocyanate or the like. The formyl group can be introduced by reacting with glutaraldehyde, dialdehyde starch and the like.

As a method of introducing a reactive functional group such as a carboxyl group or an amino group onto the surface of a polymer material having no reactive functional group such as polystyrene, there is a method by plasma treatment. Plasma treatment includes glow discharge treatment, corona discharge treatment, and the like, and the gas used includes reactive gas such as oxygen, nitrogen, and ammonia, nonreactive gas such as helium and argon, or mixed gas such as air. Oxygen gas is preferably used when introducing a carboxyl group to the surface of the polymer material, and ammonia gas is preferably used when introducing an amino group. The discharge output is preferably 10 to 1000 W, and the treatment time is preferably 1 second to 1 hour.

The acid anhydride group can be introduced by reacting it with a styrene maleic anhydride copolymer, an ethylene-maleic anhydride copolymer, a methyl vinyl ether-maleic anhydride copolymer or the like. For example, maleic anhydride can be added to polyurethane. It is also possible to introduce an acid anhydride group by graft-polymerizing with an γ-ray or an electron beam.

When carrying out the immobilization treatment, for example, an antibody solution can be used. The antibody solution used at this time is
The antibody is preferably in water or saline, preferably
A solution diluted to a concentration of 10 to 1000 times can be used. The antibody solution may optionally contain an antibacterial agent, a stabilizer and the like, and the temperature and time conditions for the treatment with the antibody solution are preferably room temperature or lower, preferably 1 More than time.

As the antibody against the pathogenic factor of infectious disease in the present invention, for example, in the case of a monoclonal antibody, a clone of a fused cell of spleen cells and myeloma cells of a mouse immunized with a purified pathogenic factor, that is, a toxin or a bacterial cell is used. The mouse ascites is used as a guide. The polyclonal antibody used is purified from serum obtained by immunizing rabbits, goats, rats, sheep, chickens, pigs, donkeys, guinea pigs, dogs, cattle, etc. using antigens such as toxins and bacterial cells.

In the present invention, an antigen, that is, a pathogenic factor is directly captured by using a polymer material on which an antibody against the pathogenic factor is immobilized and detected by an enzyme immunoassay method. For example, a sandwich method is used as the enzyme immunoassay method. be able to. This is because after capturing the pathogenic factor with the antibody immobilized on the polymer material, for example, the antibody bound with the enzyme is allowed to act on the captured pathogenic factor to qualitatively and quantitatively determine the activity of the labeled enzyme bound. This is a method of detecting a pathogenic factor in a sample by measuring. In addition, the captured pathogenic factor was reacted with an antibody prepared in a different animal from the antibody immobilized on the polymer material before the antibody conjugated with the enzyme was allowed to react, and then the enzyme was conjugated. Antibodies may act.

Examples of the labeling enzyme used for detection include peroxidase, β-galactosidase, alkaline phosphatase, urease, glucose oxidase, glucoamylase, carbonic anhydrase, acetylcholinesterase and the like. On the other hand, other than the enzyme, examples of the label used for detection include gold colloid particles and silver colloid particles. Further, a method using avidin-biotin, or a method of introducing a labeled substance via protein A, protein G or lectin having antibody binding property can also be used.

Regarding the substrate of the enzyme which produces the water-soluble product used in the present invention, for example, when peroxidase is used as the labeling enzyme, hydrogen peroxide solution and 3- (4-hydroxyphenyl) are used for measurement by the fluorescence method. To measure with a colorimetric method using propionic acid, etc., use hydrogen peroxide solution and 4-
Luminol (Amersham) can be used for luminescence measurement using hydroxyphenylacetic acid, 3,3 ', 5,5'-tetramethylbenzidine, 1,2-phenylenediamine, etc.
Company) is used. When alkaline phosphatase is used, 4-methylumbelliferyl phosphate or the like is used for measurement by the fluorescence method, and 4-nitrophenyl phosphate, NADP or the like is used for measurement by the colorimetric method. β-D-
When galactosidase is used, 4-methylumbelliferyl-β-D-galactoside or the like is used for the fluorescence method, and 2-nitrophenyl-β-D-galactoside or the like is used for the colorimetric method. To use.

In order to detect a pathogenic factor using a polymeric material on which an antibody against the pathogenic factor is immobilized, first, before reacting with the pathogenic factor which is an antigen, a portion of the surface of the material that nonspecifically binds to an antibody or the like. It is necessary to perform an operation (blocking) to block the protein with antiserum or a non-interfering protein. Examples of proteins used for blocking include bovine serum albumin (BSA), ovalbumin, hemoglobin, gelatin, etc., and these proteins include 10 mM Tris-HCl buffer (pH 7.2) or phosphate buffer containing 0.9% sodium chloride. Dissolve in (pH7.2), 37 ℃, 1.5 hours or 4
Allow the material to react overnight at ℃. After reaction, 0.05
% Polyethylene sorbitan monolaurate (Tween 20),
Wash 2 to 6 times with 10 mM Tris-HCl buffer or phosphate buffer containing 0.9% sodium chloride.

The samples used in the present invention include diarrhea stool, sputum,
Human patient materials such as body fluids may be used directly, or culture supernatants of bacterial cells separated from the patient materials may be used. When a patient material is directly used as a sample, the sample is diluted 2 to 50 times with 10 mM Tris-HCl buffer containing 0.05% Tween 20, 0.9% sodium chloride and used. On the other hand, when using the culture supernatant of the bacterial cells separated from the patient material, as an example, Vibrio parahaemolyticus
Parahaemolyticus is exemplified by strains isolated from patient material in heptone-saline medium (1% polyheptone (manufactured by Difco), 3% sodium chloride, 0.5% disodium hydrogen phosphate) or SPP medium (1% polypeptone (Difco).
Company), 0.5% sodium chloride, 0.2% glucose, 0.5
The supernatant is centrifuged (5,000 rpm, 15 minutes) after culturing for 3 hours to overnight with (% disodium hydrogen phosphate). In the case of Vibrio cholerae, the strain isolated from the patient material is 2% Casamino acid (manufactured by Difco),
0.6% yeast extract (manufactured by Difco), 0.25% sodium chloride, 0.871% dipotassium hydrogen phosphate, 0.25% glucose,
Centrifuge (5,000 rpm, 15 minutes) using 5% magnesium chloride, 0.5% manganese chloride, 0.5% ferric chloride and 0.001% sulfuric acid, and incubate for 3 hours to overnight. Use the supernatant as the sample.

The macromolecular material which has captured the pathogenic factor is then made to act on the polyclonal antibody against the pathogenic factor in 10 mM Tris-HCl buffer or phosphate buffer containing 0.9% sodium chloride diluted 500 to 2000 times. The reaction conditions at this time are preferably room temperature to 37 ° C. for 10 to 90 minutes. Alternatively, an antibody against a pathogenic factor to which a labeling enzyme is directly bound may be used. After the reaction, the material is washed 2 to 6 times with 10 mM Tris-hydrochloric acid buffer solution or phosphate buffer solution containing 0.05% polyethylene sorbitan monolaurate and 0.9% sodium chloride.

Then, the polymer material was treated with an enzyme-labeled antibody prepared by binding a labeling enzyme with an antibody against immunoglobulin of the same animal species from which a polyclonal antibody against a pathogenic factor was obtained.
It is made to react with 10 mM Tris-hydrochloric acid buffer solution or phosphate buffer solution containing 0.05% polyethylene sorbitan monolaurate, 0.9% sodium chloride diluted ˜2000 times. The reaction conditions at this time are preferably room temperature to 37 ° C. for 10 to 90 minutes. After the reaction, the material is washed 2 to 6 times with 10 mM Tris-hydrochloric acid buffer solution or phosphate buffer solution containing 0.05% polyethylene sorbitan monolaurate and 0.9% sodium chloride.

The polymer material obtained by the above reaction is reacted with an enzyme substrate solution which produces a soluble product after the enzymatic reaction. When a substrate having low solubility in an aqueous solution is used, dimethyl sulfoxide (DMSO) is used. ), Dimethylformamide (DMF), methanol, ethanol, or the like, which is previously dissolved in an organic solvent and then mixed with an aqueous solution.

[0041]

EXAMPLES The present invention will be described in more detail below with reference to examples. Example 1 Cholera enterotoxin (CT) purchased from the Institute for Chemistry and Serum Therapy was detected as follows.

A polyclonal antibody against CT was obtained by the following method. That is, purified CT was dissolved in 10 mM phosphate buffer solution (pH 7.0: abbreviated as PBS hereinafter) containing 0.9% sodium chloride so that the concentration of Freund was 25 μg / ml.
Rabbits weighing approximately 2 kg were immunized with complete adjuvant (Difco), and 25 days later, CT was immunized again with an equal amount of Freund incomplete adjuvant (Difco) to immunize the animals. Serum was obtained. 50 to this
% Ammonium sulfate is added to cause precipitation, and this is repeated twice, and the resulting precipitate is mixed with 10 mM phosphate buffer (pH 7.
The immunoglobulin obtained after being dissolved in 0) and dialyzed against the same buffer was used as a polyclonal antibody.

The monoclonal antibody against CT was obtained by the following method. That is, 6-8 week-old BALB / c mice were immunized with 0.5 ml of CT dissolved in PBS at 25 μg / ml together with an equal amount of Freund complete adjuvant, and 21 days later, CT was further treated with an equal amount of Freund incomplete adjuvant. Immunization was performed using the added CT, and mouse splenocytes were obtained on day 32. The obtained spleen cells were fused with SP2 / o myeloma cells by polyethylene glycol (PEG1500, Boehringer Mannheim) at a concentration of about 1 × 10 ⁸ cells each, 2.5% bovine serum, 1 × 10 ⁴ M hypoxanthine, 4 ×. Ten
Celg containing ^-7 M aminopterin and 1.6 × 10 ^-5 M thymidine
Hybridomas were selected in rosser-H medium (Sumitomo Pharmaceutical Co., Ltd.). The obtained hybridomas were injected into the abdominal cavity of BALB / c mice at 2 × 10 ⁶ times, and ascites was recovered at 7 to 14 days, and the ascites was collected and centrifuged at 2,500 rpm for 10 minutes to obtain a supernatant. Was used as a monoclonal antibody.

1 cm in length and 1 in width made of ethylene vinyl acetate copolymer (EVA manufactured by Mitsui / Dupont Polychemical Co., Ltd.)
cm, thickness 0.5mm sheet 15% sodium hydroxide 80%
It was immersed in a methanol solution at 50 ° C. for 2 hours, washed with distilled water, and then immersed in a 0.5% hydrochloric acid solution of 2% aminoacetal at 58 ° C. for 5 hours. After washing and drying, soak in a dehydrated acetone solution of 1% (w / v) maleic anhydride-methyl vinyl ether copolymer (Gantrez AN-169) at room temperature for 2.5 hours, wash with acetone and vacuum-dry the sheet. Used for conversion. Immobilization was performed by immersing this sheet in a buffer solution containing a monoclonal antibody obtained from mouse ascites for CT.

Antibody-immobilized EVA thus obtained
Sheet contains 0.9% 1% bovine serum albumin (BSA)
After blocking by immersing in 10 mM phosphate buffer solution (pH 7.2, abbreviated as PBS hereinafter) containing 10% sodium chloride, it was immersed in PBS containing 500 ng / ml CT for 1.5 hours. For detection of toxin, after washing with PBS containing 0.05% polyethylene sorbitan monolaurate, rabbit polyclonal antibody against CT diluted with PBS 500 times is reacted at room temperature for 1.5 hours, and washed again with horseradish peroxidase-labeled anti-rabbit. IgG (manufactured by Cappel) was diluted 500 times and reacted with PBS containing 0.05% polyethylene sorbitan monolaurate at room temperature for 1.5 hours, and after washing 0.1
It was carried out by reacting in 20 mM Tris-HCl buffer (pH 7.2) containing 3 mg / ml 3,3 ′, 5,5′-tetramethyltylbenzidine and 0.5 μl / ml hydrogen peroxide solution at 37 ° C. for 30 minutes. It was

The reaction solution containing the antibody-immobilized EVA sheet reacted in this manner developed a yellow color. On the other hand, no color development was observed in the reaction liquid of the antibody-immobilized EVA sheet which was subjected to the same operation as the previous operation except that it was immersed in a solution containing no CT.

Example 2 A sheet made of polyurethane (manufactured by THER MEDICS) having a length of 1 cm, a width of 1 cm and a thickness of 0.2 cm was placed in distilled water to give 80
After dipping at 6 ° C for 6 hours, it was washed with distilled water. After drying 2
% (W / v) maleic anhydride-methyl vinyl ether copolymer was immersed in a dehydrated acetone solution at room temperature for 1 hour, washed with acetone, and then vacuum-dried for immobilization. Immobilization was carried out by immersing this sheet in a buffer containing a monoclonal antibody obtained from mouse ascites to CT as in Example 1.

The thus-obtained antibody-immobilized polyurethane sheet is a 10 mM phosphate buffer solution (pH 7.) containing 0.9% sodium chloride containing 1% bovine serum albumin (BSA).
2) After blocking by immersion in 50), 50
It was immersed in PBS containing 0 ng / ml CT for 1.5 hours. For detection of toxins, after washing with PBS containing 0.05% polyethylene sorbitan monolaurate, a rabbit polyclonal antibody against CT diluted 500 times with PBS was used at room temperature for 1.
React for 5 hours, wash again, and dilute 500-fold with alkaline phosphatase-labeled anti-rabbit IgG (Cappel) 0.05%
React with PBS containing polyethylene sorbitan monolaurate for 1.5 hours at room temperature, and after washing, 0.1 mg Tris-hydrochloric acid buffer solution (pH 9.6) containing 1 mg / ml ρ-nitrophenyl phosphate
It was carried out by immersing it in 37 ° C for 15 minutes.

The reaction solution containing the antibody-immobilized polyurethane sheet reacted in this way developed a yellow color. On the other hand, CT
No color development was observed in the reaction solution containing the antibody-immobilized polyurethane sheet, which was subjected to the same operation as the previous operation except that the reaction solution was immersed in a solution not containing.

Example 3 Nylon 6 (manufactured by Unitika Ltd.) 1 cm long, 1 cm wide,
A 0.2 cm-thick sheet was immersed in 3N hydrochloric acid at 30 ° C. for 30 minutes and then washed with distilled water. After drying, soak in a 1: 5 mixture of 10% (w / v) polyethyleneimine aqueous solution and methanol at room temperature for 30 minutes, then add 2 times the amount of 5% dicyclohexylcarbodiimide in methanol and continue soaking at room temperature for 2 hours. did. After washing with methanol and drying 2
% (W / v) maleic anhydride-methyl vinyl ether copolymer was immersed in a dehydrated acetone solution at room temperature for 1 hour, washed with acetone, and vacuum-dried to be used for immobilization. Immobilization was performed by immersing this sheet in a buffer solution containing a monoclonal antibody obtained from mouse ascites for CT.

The antibody-immobilized nylon sheet thus obtained contains 1% bovine serum albumin (BSA).
10 mM phosphate buffer (pH 7.2) containing 9% sodium chloride
After blocking by dipping in 500 ng /
It was immersed in PBS containing mlCT for 1.5 hours. For detection of toxin, after washing with PBS containing 0.05% polyethylene sorbitan monolaurate, rabbit polyclonal antibody against CT diluted with PBS 500-fold is reacted at room temperature for 1.5 hours, and after washing again, alkaline phosphatase-labeled anti-rabbit IgG. (Cappel) was reacted with PBS containing 0.05% polyethylene sorbitan monolaurate diluted 500 times at room temperature for 1.5 hours, washed, and washed with 0.1M tris-hydrochloric acid buffer containing 1 mg / ml ρ-nitrophenyl phosphate ( pH 9.6)
It was performed by immersing at 37 ° C for 15 minutes.

The reaction solution containing the antibody-immobilized nylon sheet reacted in this manner developed a yellow color. On the other hand, no color development was observed in the reaction liquid containing the antibody-immobilized nylon sheet which was subjected to the same operation as the above except that it was immersed in a solution containing no CT.

Example 4 Polystyrene beads (1/4 inch diameter; manufactured by Pierce Chemical Co.) were used as a plasma reactor (produced by Yamato Scientific Co., Ltd .; PR-5).
01A type), the output was 300 W, and plasma treatment was performed for 10 minutes in the presence of ammonia gas. Next, the beads were immersed in a dehydrated acetone solution of a 2% (w / v) maleic anhydride-methyl vinyl ether copolymer at room temperature for 1 hour, washed with acetone, and vacuum dried beads were used for immobilization. The immobilization was carried out by immersing the beads in a buffer solution containing a monoclonal antibody obtained from mouse ascites to CT as in Example 1.

The thus-obtained antibody-immobilized polystyrene beads were 10 mM phosphate buffer solution (pH 7.) containing 0.9% sodium chloride containing 1% bovine serum albumin (BSA).
2) After blocking by immersion in 50), 50
It was immersed in PBS containing 0 ng / ml CT for 1.5 hours. For detection of toxins, after washing with PBS containing 0.05% polyethylene sorbitan monolaurate, a rabbit polyclonal antibody against CT diluted 500 times with PBS was used at room temperature for 1.
React for 5 hours, wash again, and dilute 500-fold with alkaline phosphatase-labeled anti-rabbit IgG (Cappel) 0.05%
React with PBS containing polyethylene sorbitan monolaurate at room temperature for 1.5 hours, wash and then use 1M tris-hydrochloric acid buffer solution (pH 9.6) containing 1 mg / ml ρ-nitrophenyl phosphate.
It was performed by immersing at 37 ° C for 15 minutes.

The reaction liquid containing the antibody-immobilized polystyrene beads reacted in this manner developed a yellow color. On the other hand, CT
No color development was observed in the reaction solution containing antibody-immobilized polystyrene beads, which was subjected to the same operation as the previous operation except that the reaction solution was immersed in a solution not containing.

Example 5 96-well polystyrene microplate (CORNIN
Plasma reactor manufactured by G company (Yamato Scientific Co., Ltd .; PR)
-501A type), the output was 300 W, and plasma treatment was performed for 10 minutes in the presence of ammonia gas. Next, a dehydrated acetone solution of 2% (w / v) maleic anhydride-methyl vinyl ether copolymer was added to the plate, and the plate was immersed at room temperature for 1 hour, washed with acetone, and vacuum dried to be used for immobilization. Immobilization was performed by adding a buffer containing a monoclonal antibody obtained from mouse ascites to CT to this plate.

After blocking by adding 10 mM phosphate buffer (pH 7.2) containing 0.9% sodium chloride containing 1% bovine serum albumin (BSA) to the antibody-immobilized plate thus obtained, PBS containing 500 ng / ml CT was added and reacted for 1.5 hours. To detect toxins, wash with PBS containing 0.05% polyethylene sorbitan monolaurate, then dilute 500 times with PBS C
The rabbit polyclonal antibody against T was reacted at room temperature for 1.5 hours, washed again, and then reacted at room temperature for 1.5 hours with PBS containing 0.05% polyethylene sorbitan monolaurate diluted with alkaline phosphatase-labeled anti-rabbit IgG (Cappel) 500 times. After washing, wash with 0.1 M Tris-HCl buffer (pH 9.6) containing 1 mg / ml ρ-nitrophenyl phosphate.
The reaction was carried out at 15 ° C for 15 minutes.

The reaction solution of the plate reacted in this way developed a yellow color. On the other hand, no color development was observed in the reaction solution which was completely the same as the previous operation except that a solution containing no CT was added.

Example 6 Vibrio parahaemolyticus heat-resistant hemolysin (T
DH) was detected as follows.

A polyclonal antibody against TDH was obtained by the following method. That is, a rabbit of about 2 kg in weight was immunized with purified 100 μg / ml TDH together with 0.5 ml of Freund complete adjuvant (manufactured by Difco), and 25 days later, purified TDH was re-purified with an equal amount of Freund imcomplete adjuvant (Difco). Manufactured) was added to immunize to obtain antiserum. To this, 50% ammonium sulfate is added to cause precipitation, but this operation is repeated twice, and the resulting precipitate is added to 10 mM.
An immunoglobulin dissolved in a phosphate buffer (pH 7.2) and dialyzed with the same buffer was used as a polyclonal antibody.

Monoclonal antibody against TDH was prepared by dissolving 15 μg / 0.25 ml TDH in purified PBS in an equal amount of Freund.
BALB / c 6-8 weeks old with complete adjuvant
Four weeks after immunizing the mouse, an additional 20 μg / 0.25 ml of TDH
Were immunized with an equal volume of Freund incomplete adjuvant. The splenocytes of the immunized mouse were removed and the cells were
Polyethylene glycol (PEG1500, manufactured by Boehringer Mannheim) containing 10 ⁸ cells and an equal amount of X6-Ag8.653 myeloma cells
In a Celgrosser-H medium (Sumitomo Pharmaceutical Co., Ltd.) containing 2.5% bovine serum, 1 × 10 ^-4 M hypoxanthine, 4 × 10 ^-7 M aminopterin, and 1.6 × 10 ^-5 M thymidine. Hybridomas were selected. The obtained hybridoma is BA
Ascites was collected at 7 to 14 days after the injection of 2 × 10 ⁶ cells into the peritoneal cavity of LB / c mice, and the supernatant obtained by centrifugation at 2,500 rpm for 10 minutes was used as a monoclonal antibody. .

In the same manner as in Example 2, 500 ng / ml of TDH was detected using the EVA sheet on which the monoclonal antibody obtained from mouse ascites for TDH was immobilized. The detection procedure was in accordance with Example 2.

The reaction solution containing the antibody-immobilized EVA sheet reacted in this manner developed a yellow color. On the other hand, no color development was observed in the reaction solution containing the antibody-immobilized EVA sheet, which was subjected to the same operation as the previous operation except that it was immersed in a solution containing no TDH.

Example 7 Detection of 500 ng / ml of LT was performed using an EVA sheet on which a monoclonal antibody against a heat-labile enterotoxin (LT) of toxigenic Escherichia coli was immobilized by the same method as in Example 1. The toxin used was purchased from SIGMA, and the monoclonal antibody and rabbit polyclonal antibody were obtained by the same method as CT in Example 1. The method for detecting the toxin was in accordance with Example 2.

Antibody-immobilized EV with LT detected by this method
The reaction solution containing the sheet A developed a yellow color. On the other hand, LT
No color development was observed in the reaction solution containing the antibody-immobilized EVA sheet, which was subjected to the same operation as the previous operation except that the reaction solution was immersed in a solution containing no.

Example 8 Using an EVA sheet on which a monoclonal antibody against Clostridium boturlinum botulinum toxin type C was immobilized by the same method as in Example 1, 500 ng / ml of botulinum toxin type C was detected. The toxin is SIGMA
Monoclonal antibody and rabbit polyclonal antibody were obtained by the same method as CT of Example 1 using those purchased from the company. The method for detecting the toxin was in accordance with Example 2.

The reaction solution containing the antibody-immobilized EVA sheet in which botulinum toxin type C was detected by this method developed a yellow color. On the other hand, no color development was observed in the reaction solution containing the antibody-immobilized EVA sheet which was subjected to the same operation as the previous operation except that it was immersed in a solution containing no botulinum toxin type C.

Example 9 Using the EVA sheet on which a monoclonal antibody against Staphylococcus aureus enterotoxin was immobilized in the same manner as in Example 1, 500 ng / ml of enterotoxin was detected. Enterotoxin is
Enterotoxin B purchased from SIGMA was used, and the monoclonal antibody and rabbit polyclonal antibody were prepared as in Example 1.
It was obtained by the same method as CT. The toxin was detected according to Example 2.

The reaction liquid containing the antibody-immobilized EVA sheet in which the staphylococcal enterotoxin was detected by this method developed a yellow color. On the other hand, no color was observed in the reaction solution containing the antibody-immobilized EVA sheet, which was subjected to the same operation as the previous operation except that it was immersed in a solution containing no enterotoxin.

Example 10 Using an EVA sheet on which a monoclonal antibody against diphtheria toxin of Corynebacterium diphtheriae was immobilized by the same method as in Example 1, 500 ng / ml of diphtheria toxin was detected. The diphtheria toxin used was purchased from SIGMA, and the monoclonal antibody and rabbit polyclonal antibody were obtained by the same method as CT in Example 1. The toxin was detected according to Example 2.

The reaction solution containing the antibody-immobilized EVA sheet in which diphtheria toxin was detected by this method developed a yellow color.
On the other hand, the antibody-immobilized EV which was completely the same as the previous operation except that it was immersed in a solution containing no diphtheria toxin.
No color was observed in the reaction solution containing the A sheet.

Example 11 Using a nylon sheet on which a monoclonal antibody against Bordetella pertussis pertussis toxin was immobilized in the same manner as in Example 1, 500 ng / ml of pertussis toxin was detected. The pertussis toxin was purchased from SIGMA, and the monoclonal antibody and rabbit polyclonal antibody were obtained by the same method as CT in Example 1. The toxin was detected according to Example 2.

The reaction solution containing the antibody-immobilized EVA sheet in which pertussis toxin was detected by this method developed a yellow color. On the other hand, no color development was observed in the reaction solution containing the antibody-immobilized EVA sheet, which was subjected to the same operation as the previous operation except that it was immersed in a solution containing no pertussis toxin.

Example 12 Exotoxin A of Pseudomonas aeruginosa was detected as follows.

As Exotoxin A of Pseudomonas aeruginosa, Pseudomonas aeruginosa purchased from LIST BIOLOGICAL LABORATO-RIES, INC. Was used.

Monoclonal antibodies against exotoxin A and rabbit polyclonal antibodies were obtained by the same method as in CT of Example 1.

As in the case of CT, the purified exotoxin A was detected according to Example 2 using the nylon sheet on which the monoclonal antibody prepared above was immobilized.

The reaction solution containing the antibody-immobilized EVA sheet in which exotoxin A was detected by this method developed a yellow color. On the other hand, no color development was observed in the reaction solution containing the antibody-immobilized EVA sheet which was subjected to the same operation as the previous operation except that it was immersed in a solution not containing Exotoxin A.

Example 13 The detection of tetanus toxin of Clostridium tetani of Clostridium tetani was carried out as follows.

The tetanus toxin of Clostridium tetani is LIST BIOLOGICAL
The C-fragment purchased from LABORATORIES, INC. Was used.

The monoclonal antibody and rabbit polyclonal antibody against the tetanus toxin C-fragment were prepared in Example 1
It was obtained by the same method as CT.

Similar to CT, the purified tetanus toxin C-fragment was detected according to Example 2 using the EVA sheet on which the monoclonal antibody prepared above was immobilized.

The reaction solution containing the antibody-immobilized EVA sheet in which the tetanus toxin C-fragment was detected by this method developed a yellow color. On the other hand, in the reaction solution containing the antibody-immobilized EVA sheet, which was subjected to the same procedure as the above except that it was immersed in a solution containing no tetanus toxin C-fragment,
No color development was observed.

[0084]

INDUSTRIAL APPLICABILITY According to the present invention, a polymeric material on which a large amount of antibody against a pathogenic factor is immobilized by covalent bond through an acid anhydride group and which is stably immobilized, is used for the pathogenic factor by an enzyme immunoassay. Therefore, it is possible to detect the pathogenic factor in the sample extremely quickly, highly sensitively, and easily.

Claims (1)

[Claims] 1. An antibody-immobilized polymer material in which an antibody against the pathogen is immobilized on the surface of the polymer material by a covalent bond via an acid anhydride group, and the pathogen is captured after capturing the pathogen. When detecting by enzyme immunoassay,
A rapid diagnostic method for an infectious disease, which comprises detecting a pathogenic factor by measuring an enzyme activity using a water-soluble compound as a substrate for a labeling enzyme.