JP2002275199A - Method for preparing antibody and method for immunodetecting acid-fast bacterium - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for preparing an antibody specifically reactive with specified species of acid-fast bacteria and a rapid and simple method for immunodetecting the acid-fast bacteria with high reliability. SOLUTION: This method for preparing the antibody specifically reactive with microbial cells of the specified species of bacteria is characterized by using a microbial cell column supporting microbial cells of another species of bacteria having cross-reactivity. The method for immunodetecting the acid- fast bacteria has a step of bringing a specimen into contact with an antibody specific for the species of the acid-fast bacteria specifically reactive with the specified species of the acid-fast bacteria and a step of detecting the antibody specific for the species of the acid-fast bacteria forming an immune complex.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a Mycobacterium tuberculosis (Mycobacterium tuberculosis).
The present invention relates to a method for producing a species-specific antibody such as an acid-fast bacterium, and an immunological detection method using an acid-fast bacterium-specific antibody and a kit for immunological detection of an acid-fast bacterium.

[0002]

2. Description of the Related Art Tuberculosis is an infection caused by the presence of Mycobacterium tuberculosis. Almost all tissues and organs of the whole body are affected sites, but they are particularly frequently found in the lungs. The local symptoms vary from patient to patient, and systemic symptoms include wasting fever, sweating, weakness, and the like, similar to sepsis. Without early diagnosis and treatment, the disease is often progressive and mortality is high. In addition, patients with pulmonary tuberculosis constantly excrete the bacteria themselves into the air by breathing, and a delay in diagnosis will promote the spread of infection.

[0003] Mycobacterium tuberculosis is a taxonomically belonging to the family Mycobacteriaceae, Mycobacterium, and Mycobacterium tuberculosis. There are several species of M. tuberculosis in addition to M. tuberculosis, which have little pathogenicity to humans. Mycobacterium mycobacteria include mycobacterium leprae, which cannot be cultivated. Mycobacterium avium complex (Mycobacter
ium avium complex, Mycobacterium kansasii, and others that are not as strong as Mycobacterium tuberculosis but have pathogenicity to humans.

[0004] Conventionally, diagnosis of mycobacterial infections including tuberculosis has been performed by smear microscopy, culture identification using a special medium, and detection of mycobacteria (tuberculosis) by molecular genetic techniques. Had been The smear staining microscopy is performed by the Thiel-Neelsen staining method (staining with a Ziel solution, decolorization in hydrochloric acid alcohol, and counterstaining with methylene blue). Mycobacteria stain red, others stain light blue. The mycobacteria stained red are confirmed under a microscope, and the number thereof is calculated to estimate the approximate amount of eradication. The culture identification method using the dedicated medium is performed by using a fixed medium (Ogawa medium) or a liquid medium having a high culturing rate for 1 to 8 weeks and performing chemical identification at the stage when the bacterial colony grows on the medium. Determine the species. The current mainstream of the molecular genetic method is PC
In this method, an acid-fast bacillus-specific gene amplified by a nucleic acid propagation method such as the R method is detected for each bacterial species by a nucleic acid hybridization method.

[0005]

As described above, smear staining microscopy, culture identification using a special medium, molecular genetic techniques, and the like are known for the detection and identification of mycobacteria in the prior art. . The Siehl-Neelsen stain of the smear microscopy method can only stain intact cells covered with hard lipid cell walls, and thus miss digested bacterial fragments in sputum. Basically, it is used as a standard for estimating the amount of bacteria, but has a problem that the detection rate is low (35% or less), the specificity is lacking, and the species cannot be distinguished. Because the culture identification method using the dedicated medium requires a long time until the result is determined,
It does not help early diagnosis of tuberculosis. Since the molecular genetic technique has high sensitivity and high specificity, false positive (caused by contamination of a negative sample with a positive sample) becomes a problem, and dedicated facilities and equipment are indispensable. Due to the specialty of sputum samples, false negatives caused by alienation of nucleic acid multiplication have been pointed out earlier. In the situation having the above-mentioned problems, it is necessary to realize an immunological test system that can easily diagnose the mycobacterial species or serotype.
There is no doubt that it will contribute significantly to the diagnosis and treatment of tuberculosis. However, the cell wall of acid-fast bacilli contains a large amount of wax-like components, making the antibody production difficult due to the strong hydrophobicity of the bacterial surface layer. At present, no specific inspection has been reported.

Accordingly, it is an object of the present invention to provide a method for producing an antibody which specifically reacts with a specific type of acid-fast bacterium, and a rapid and reliable method for immunological detection of an acid-fast bacterium. It is in.

[0007]

Means for Solving the Problems The inventors of the present invention have conducted intensive studies to achieve the above object, and have produced a cell column carrying cells having an antigen similar to the target cells for antibody production. By passing a polyclonal antibody obtained from a serum immunoglobulin fraction of an animal obtained by immunizing the target cell for antibody production through the cell column, an antigen similar to the target cell for antibody production is obtained. By absorbing an antibody that reacts with a cell having the above, an antibody that specifically reacts with the target cell for the production of the antibody was successfully produced. Furthermore, by using the acid-fast bacterium species-specific antibody prepared using the cell column, the acid-fast bacterium can be quickly detected, and the acid-fast bacterium species in the sample can be quickly detected. It has been found that it can be applied to discrimination. That is, the present inventors have completed the present invention based on these findings.

[0008] In summary, the present invention relates to a method for producing an antibody which specifically reacts with cells of a specific species of bacteria, comprising the steps of producing cells of another species having cross-reactivity. The present invention relates to an antibody production method for producing an antibody that specifically reacts with a cell of a specific bacterial species, characterized by using a cell column carrying the same. The cell is preferably an acid-fast bacterium, and further,
Preferably, the acid-fast bacterium is Mycobacterium tuberculosis, Mycobacterium kansasii or Mycobacterium avium complex.

The present invention also relates to a step of contacting a specimen with an acid-fast bacterium-specific antibody which specifically reacts with a specific acid-fast bacterium; The present invention relates to a method for detecting an acid-fast bacterium having a step of detecting an antibody. Further, in this detection method, the step of detecting the acid-fast bacterium species-specific antibody that has formed the immune complex detects the size and fluorescence of particles passing through a detection point by a flow cytometer to detect the acid-fast bacterium. A method including a step of detecting is preferable, and the specimen is preferably sputum.

[0010] The present invention also relates to a kit for immunological detection of acid-fast bacilli containing an acid-fast bacterium species-specific antibody.

The present invention also provides a step of contacting a specimen with a mixed reagent obtained by mixing two or more kinds of mycobacterial species-specific antibodies labeled with different coloring agents; The present invention relates to a method for detecting an acid-fast bacterium by a method of detecting a bacterial species-specific antibody. Further, in this detection method, the step of detecting the acid-fast bacterium species-specific antibody that has formed the immune complex includes detecting the difference in the size and fluorescence of particles passing through a detection point with a flow cytometer and detecting the acid resistance. A method for detecting the bacterial species is preferred, and the specimen is preferably sputum.

The present invention also relates to a kit for immunological detection of mycobacteria comprising a mixed reagent obtained by mixing two or more mycobacterial species-specific antibodies labeled with different coloring agents.

[0013]

BEST MODE FOR CARRYING OUT THE INVENTION The present invention provides a method for producing an antibody that specifically reacts with only a specific bacterial species by using a bacterial cell column. Generally, polyclonal antibodies obtained by immunizing cells with animals often have a cross-reactivity with cells having an antigen similar to the cells, but the present invention relates to the production of antibodies. A cell column supporting cells having an antigen similar to the target cell was prepared, and a polyclonal antibody obtained from a serum immunoglobulin fraction of an animal immunized with the target cell for antibody production was prepared using the cell column. The antibody is characterized by absorbing an antibody that reacts with a cell having an antigen similar to the target cell for preparing the antibody, thereby producing an antibody that specifically reacts with the target cell.

The term "cells" as used herein includes cells themselves and cell fragments. Cell fragments can also be obtained by fragmenting cells by ultrasonic treatment or the like.

The strain to be applied to the method for producing the antibody of the present invention is not particularly limited, but a mycobacterium that causes tuberculosis or a tuberculosis-like disease in animals such as humans and rabbits is preferably applied. As an example, Mycobacterium tuberculosis Mycobacterium tuberculosis Aoyama B (Mycobacterium t.
uberculosis Aoyama B: hereinafter referred to as MTAB, Mycobacterium kansasii which is a kind of atypical acid-fast bacterium (hereinafter referred to as MK), Mycobacterium avium complex (hereinafter referred to as Mycobacter)
ium avium complex: hereinafter, referred to as MAC).

As an example of a preferred embodiment, a method for producing an antibody that specifically reacts with MTAB will be described. MTAB obtained by immunizing rabbits with MTAB
Rabbit polyclonal antibody (available from Japan Bioscience Center Co., Ltd.) shows cross-reactivity with MK and MAC, and a MK cell column carrying MK cells showing this cross-reactivity and MAC A MAC cell column supporting the cells was prepared, and a rabbit polyclonal antibody against the MTAB was passed through each of the cell columns.
An antibody that specifically reacts with MTAB (MTAB-specific antibody) can be prepared by absorbing and removing antibodies having cross-reactivity with K cells and MAC cells. Similarly, a rabbit polyclonal antibody against MK (available from the Japan Biological Science Center, Inc.)
Antibodies (MK-specific antibodies) that specifically react with
An antibody that specifically reacts with MAC (MAC-specific antibody) can be prepared from a rabbit polyclonal antibody (available from Japan Bioscience Center Co., Ltd.).

Here, an antibody that specifically reacts with a cell is reactive with a specific cell, but has no cross-reactivity with other similar cells, or is extremely low. Antibodies that show only cross-reactivity are meant. For example, an MTAB-specific antibody that specifically reacts with MTAB refers to an antibody that has no cross-reactivity with MK, MAC, etc., which are cells having similar shapes, or that has only extremely low cross-reactivity. means.

The microbial cell column of the present invention is used to absorb an antibody contained in a sample by utilizing an interaction (affinity) between a substance such as an antigen and an antibody. The specific antibody production method of the present invention immunizes a target cell for antibody production by utilizing an antigen-antibody reaction, that is, the affinity between a cell as an antigen and an antibody reactive with the cell. An antibody which cross-reacts with a cell having an antigen similar to the cell to be prepared for the antibody is absorbed and removed from the polyclonal antibody obtained from the serum immunoglobulin fraction of the sensitized animal.

The cell column is used to immobilize cells and cell fragments on an insoluble carrier usually used for immunological measurement by a conventional method (physical adsorption, polymerization by cross-linking, Immobilization by sealing or non-covalent bonding)
It is prepared by packing the insoluble carrier into a column made of glass, plastic, stainless steel or the like. Specific examples of the insoluble carrier include synthetic resins such as polystyrene, polyethylene, polypropylene, polyvinyl chloride, polyester, polyacrylate, nylon, polyacetal, and fluorine resin, polysaccharides such as cellulose and agarose, glass, and metals. Is mentioned. As the shape of the insoluble carrier, various shapes such as a tray shape, a spherical shape, a fiber shape, a rod shape, and a disk shape can be used.

As the insoluble carrier used in the method of the present invention, any insoluble carrier can be used as long as it can immobilize cells or cell fragments. For example, a commercially available product, Amersham Pharmacia Biotech's Sepharose 2B, Sepharose 4B, Sepharose 6B, CN
Br-activated Sepharose 4B, AH-Sepharose 4B, CH-Sep
harose 4B, Activated CH-Sepharose 4B, Epoxy-activa
ted Sepharose 6B, Activated thiol-Sepharose 4B, Se
phadex, CM-Sephadex, ECH-Sepharose 4B, EAH-Sepharo
se 4B, NHS-activated Sepharose or Thiopropyl
Bio-Gel manufactured by Bio-Rad, such as Sepharose 6B
A, Cellex, Cellex AE, Cellex-CM, Cellex PAB, Bio-
Gel P, Hydrozide Bio-GelP, Aminoethyl Bio-Gel P, B
io-Gel CM, Affi-Gel 10, Affi-Gel 15, Affi-Prep 1
0, Affi-Gel Hz, Affi-Prep Hz, Affi-Gel 102, CM Bio
-Gel Affi-Gel heparin, Affi-Gel 501 or Affi-Ge
l 601 etc., Wako Pure Chemical Industries, Ltd. chromagel A, chromagel P, Enzafix P-HZ, Enzafix P-SH
Or AE-Cellu lose, CM-Cellulose or PAB Cellul manufactured by Serva, such as Enzafix P-AB
ose and the like, and among these, CNBr-ac
tivated Sepharose 4B is preferred.

The present invention provides a method for immunologically detecting an acid-fast bacterium in a sample, using an acid-fast bacterium-specific antibody prepared by the antibody preparation method of the present invention. The immunological detection method,
Since an antibody that specifically reacts with a specific acid-fast bacterium is used, the acid-fast bacterium can be easily and reliably identified and quantified in a specific manner.

The immunological detection method of the present invention is not particularly limited as long as it includes a step of contacting a sample with the antibody and measuring an immune complex formed, and any known method can be applied. . Based on the measurement method, there are a direct antibody method, an indirect antibody method, a competitive method, a sandwich method using a combination of two types of monoclonal antibodies or polyclonal antibodies (or monoclonal and polyclonal antibodies) having different recognition sites for antigens, Any of these can be employed. Specifically, agglutination reaction,
Agglutination inhibition reaction, immunonucleic acid method, immunoturbidimetry, competitive reaction method,
Examples of the immunodetection method using a labeling substance include known radioimmunoassays, enzyme immunoassays, fluoroimmunoassays, and chemiluminescene immunoassays, and any of these can be employed. Each of these immunoassay methods is well known in the art and can be easily performed by those skilled in the art.

In the assay method, if necessary, the antigen or antibody is held on a suitable carrier (gel particles, cellulose particles, polyacrylamide gel, physical adsorbent (glass, styrene resin), etc.), if necessary. can do.

The anti-antibody used in the immunological detection method of the present invention
The body used is labeled if necessary. This
Such labeling substances include enzymes, fluorescent substances, luminescent substances and
Radioactive materials and the like can be used. In particular,
¹⁴C,^ThreeH,³²P,¹²⁵I,^{13 1}Of radioisotopes such as I
Other, horseradish peroxidase, alkaline phospha
Enzyme, β-galactosidase, glucose oxidase
Enzymes such as acridium ester and isoluminol
Body, luminescent substances such as luciferin, fluorescein,
Olesamine, fluorescein isothiocyanate, raw
Damine, Rhodamine X isothiocyanate, sulfolo
-Damine 101, Lucifer Yellow, Acridine, A
Clidine isothiocyanate, riboflavin, AMCA
Fluorescent substance, metal colloid particles such as gold, silver, copper colloid
Child. In addition, biotin labeling and avidin
It is also possible to adopt a method of using them in combination. But these
Without limitation, immunity using antigen-antibody reaction
Other methods that can be used for biological detection
Can be used. Labeling methods are well known in the art.
It is known and can be easily implemented by those skilled in the art.

When the labeling substance is an enzyme, its activity
The substrate and, if necessary, the color former are used to measure
You. When using peroxidase for the enzyme,
And H _TwoO_TwoAnd 2,2'-azinobis (3-E
Tylbenzothiazoline-6-sulfonic acid) (ABTS)
When alkaline phosphatase is used as the enzyme
Can use PNPP or the like as a substrate. I
However, the present invention is not limited to these, and the enzyme activity is measured.
Others that can be determined can be used.

As preferred embodiments of the immunological detection method of the present invention, a fluorescent antibody method, an indirect method using an ELISA method and a sandwich method will be briefly described below. is not.

In the case of the fluorescent antibody method, the antibody obtained by the method of the present invention is fluorescently labeled, and the fluorescent-labeled antibody is reacted with a sample which may contain an acid-fast bacterium. And measuring the fluorescence emitted by the immune complex formed by the method. For example, a sample is bound to an appropriate solid phase, the fluorescence-labeled antibody solution is added thereto, and the mixture is incubated at room temperature for a predetermined time. Thereafter, the reaction solution is removed, and the well is washed well to remove unreacted antibodies. And a method of measuring the fluorescence emitted by the immune complex bound to the solid phase. Fluorescence can be measured quantitatively by a fluorometer or directly by using a fluorescence microscope.

The labeling of the fluorescent substance can be performed by a method generally used in the art. That is, the antibody of the present invention and the fluorescent substance are mixed in an appropriate buffer at an appropriate molar ratio, stirred under light shielding for several hours, and then released using a pre-equilibrated Sephadex column or the like. It can be prepared by removing the fluorescent substance.

In the case of the immunological detection method by an indirect ELISA method using an enzyme-labeled antibody, first, a sample that may contain acid-fast bacilli in a suitable carrier such as a 96-well plate or a glass plate is used. After blocking with proteins such as bovine serum albumin, casein, and gelatin, the antibody prepared in the present invention was reacted as a primary antibody, washed, and then washed as a secondary antibody.
After reacting with a labeled antibody such as an enzyme-labeled anti-rabbit IgG antibody and washing, the anti-rabbit IgG antibody is measured based on the enzymatic reaction with the labeled enzyme to detect and quantify the acid-fast bacterium in the sample. can do.

In the case of the immunological detection method by the sandwich method of the ELISA method using an enzyme-labeled antibody, first, 9
After immobilizing the antibody prepared in the present invention on a suitable carrier such as a 6-well plate or a glass plate, and blocking with a protein such as bovine serum albumin, casein, gelatin, etc. After allowing the sample to stand and react with a sample that may be contained, washing, and reacting with a labeled antibody such as an enzyme-labeled anti-rabbit IgG antibody, and washing,
By measuring the anti-rabbit IgG antibody based on the enzymatic reaction of the labeled enzyme, the acid-fast bacterium in the sample can be detected and quantified.

The specimen used in the immunological detection method of the present invention is not particularly limited as long as it is excreted from a living body that may widely contain acid-fast bacilli and the like. , Serum, plasma, tears, saliva, cerebrospinal fluid, human tissues, organ extracts, feces, urine, and the like, but are not limited thereto. Of these, sputum is particularly preferred in the present invention.

The present invention also provides a kit for immunological detection of acid-fast bacilli. The kit contains the acid-fast bacillus species-specific antibody prepared in the present invention as an essential component.
The antibody may be in the form of a solution dissolved in an appropriate buffer and freeze-dried, or may be fixed on a solid carrier such as a 96-well microplate or glass beads. When the kit includes the antibody immobilized on a fixed carrier, the fixed carrier is preferably one treated with a blocking agent. In addition, the antibody may be, for example, R
It is labeled with I, an enzyme, a fluorescent substance, or the like.

The kit may further include, as the second component, a secondary antibody labeled with RI, an enzyme, a fluorescent substance, or the like when the indirect antibody method is used. When the sandwich method is used as a measurement principle, a labeled antibody having a different recognition site, for example, an anti-rabbit IgG antibody can be included.

[0034] The standard antigen may preferably contain an acid-fast bacterium inactivated by heat treatment or the like.
When the label is an enzyme, a coloring reagent containing a substrate of the enzyme can be further included in the kit. However, a commercially available coloring reagent can be used and is not an essential component of the present invention.

Further, the kit of the present invention can contain, as optional components, a sample diluting buffer, a washing solution and the like. Further, it may contain an encapsulant for a specimen for fluorescence measurement with a fluorescence microscope.
Glycerol-containing PBS, polyvinyl alcohol-containing P
BS and the like are preferably exemplified. In addition, non-reactive components of the kit include instructions, a mixing container, a stirring means, a pipette, etc., but are not essential components, and the relative amounts of various reagents substantially optimize assay sensitivity. It can be varied widely to achieve the solution concentration of the reagents.

The present invention also provides a method for contacting a specimen with a mixed reagent obtained by mixing two or more kinds of acid-fast bacterium-specific antibodies labeled with different color-forming agents, whereby the bacteria of the acid-fast bacterium in the specimen are determined. It is intended to provide a method for immunological detection of a specific acid-fast bacterium. The two or more kinds of acid-fast bacillus species-specific antibodies specifically mean two to four kinds of acid-fast bacillus species-specific antibodies, preferably three kinds of acid-fast bacterium species-specific antibodies. Suitable examples include MTAB-specific antibodies, MK-specific antibodies, and MAC-specific antibodies, which are antibodies against bacterial strains that frequently occur in humans clinically. These are preferably produced by the antibody production method of the present invention. Is what is done. The strain-specific antibody is preferably labeled, and more preferably, is labeled with a coloring agent that differs depending on the type of acid-fast bacterium with which it reacts. Fluorescein (green label), rhodamine (red label), A
Labeling with a fluorescent label having a different color tone such as MCA (blue label) is exemplified, but the label is not limited thereto. The labeling substance and the labeling method are as described above.

The method is not particularly limited, as long as it is a method in which a specimen can be reacted with two or more of the above-mentioned species-specific antibodies to detect an immune complex obtained by an antigen-antibody reaction. In addition, any known immunological detection method can be employed, and any known analysis method such as an analysis method using a flow cytometer can be used.

As preferred embodiments of the method for immunological detection of bacterial species of the present invention, an immunohistological staining method using a fluorescent label,
An analysis method using a flow cytometer using a fluorescent label is briefly described below, but is not limited to these methods.

The immunohistochemical staining is a method in which a specific antibody labeled for visualization is bound to an antigen contained in a sample such as a separated cell or a biological excrement such as a tissue or sputum, and a fluorescent microscope is used. It is a method of observing using. Alternatively, an unlabeled antigen-specific antibody can be reacted and detected with a secondary antibody labeled with a substance necessary for visualization. For example, after immobilizing a sample using a known fixing means, dehydrating with ethyl alcohol, etc., two or more types of acid-fast bacterium species which are labeled with fluorescent labels having different color to the respective acid-fast bacterium types that react with each other. After contacting with a mixed reagent containing the antibody, washing well to remove unreacted labeled antibody, and sealing,
By observing with a fluorescence microscope, the acid-fast bacterium is directly detected for each bacterial type. By using an acid-fast bacterium species-specific antibody labeled with a fluorescent label having a different color for the type of acid-fast bacterium to be reacted, the acid-fast bacterium in the specimen can be reliably and easily detected by the cell type.

The flow cytometer applies a flow cytometry principle to a sample that has been reacted with a fluorescently labeled antibody, flows through a nozzle, and emits a fluorescent light emitted by an immune complex of the fluorescently labeled antibody and cells that passes through a detection point. It is a device for measuring the strength.

As an example of a preferred embodiment of the analysis using a flow cytometer, first, a sample obtained by solubilizing sputum or the like which may contain mycobacteria is reacted with a sufficient amount of a fluorescently labeled antibody. After washing away the unreacted excess labeled antibody, the immune complexes formed by the antigen-antibody reaction are resuspended in a buffer, for example, MB buffer, and they are resuspended under normal pressure, for example, Bright HS flow cytometry. It can be analyzed by a conventional method using a metrology system (manufactured by Bio-Rad). Upon irradiation with light, the acid-fast bacterium to which the fluorescently labeled antibody is bound emits fluorescence, and by detecting this, the presence of the acid-fast bacterium can be detected. If the antibody that specifically reacts with the acid-fast bacterium is a mixed reagent that is a mixture of two or more kinds of acid-fast bacillus-specific antibodies labeled with different fluorescent labels, the size of the particles passing through the detection point and the fluorescence By detecting, bacterial cells or cell fragments of acid-fast bacteria can be detected, and the bacterial species of acid-fast bacteria can be determined easily and quickly.

The present invention can also provide a kit for diagnosing acid-fast bacilli according to the type of bacteria. The kit contains a mixed reagent obtained by mixing two or more kinds of mycobacterial species-specific antibodies as essential components. The antibody may be in the form of a solution dissolved in an appropriate buffer or a lyophilized product. Further, the antibody is labeled as necessary with, for example, RI, an enzyme, a fluorescent substance, or the like, and these are preferably provided with a coloring agent having a different color tone depending on the type of acid-fast bacterium with which each reacts. . As such a label, the above-mentioned fluorescent labeling substance can be used.

In the case of the indirect antibody method, the kit may further include, as a second element, a secondary antibody labeled with RI, an enzyme, a fluorescent substance, or the like.

The standard antigen may preferably contain an acid-fast bacterium inactivated by heat treatment or the like.
When the label is an enzyme, a coloring reagent containing a substrate of the enzyme can be further included in the kit. However, a commercially available coloring reagent can be used and is not an essential component of the present invention.

Further, the kit of the present invention may contain a buffer for diluting a sample, a washing solution, and the like as optional components. Further, it may contain an encapsulant for a specimen for fluorescence measurement by a fluorescence microscope.
Glycerol-containing PBS, polyvinyl alcohol-containing P
BS and the like are preferably exemplified. In addition, non-reactive components of the kit include instructions, a mixing container, a stirring means, a pipette, etc., but are not essential components, and the relative amounts of various reagents substantially optimize assay sensitivity. It can be varied widely to achieve the solution concentration of the reagents.

EXAMPLES The present invention will be described in detail below with reference to examples, but the present invention is not limited to these examples.

Example 1 A method for preparing an acid-fast bacterium-specific antibody using a cell column will be described below.

1. MTA having MTAB cells and cell fragments carried in a column, which is used for absorption of MTAB antibodies
B Cell Column Preparation Method A cell column supporting MTAB cells and cell fragments was prepared by the following procedure. (1) 400 mg of wet weight of MTAB heat-killed bacteria (obtained from Department of Bacteriology, Osaka City University School of Medicine) was coupled with a coupling buffer (0.1 M bicarbonate buffer (pH 8.3)).
(+0.5 M NaCl) in 4 mL, and homogenized by sonication in an ultrasonic cleaner for 15 to 60 minutes to obtain a cell treatment solution. (2) 1 g of CNBr-activated Sepharose 4B (Amersham Pharmacia Biotech) was swelled in 1 mM HCl for 15 minutes. (3) CNBr-activated Sepharose swollen in (2)
4B (3.5 mL after swelling) was packed into an empty PD-10 column (Amersham Pharmacia Biotech), and 1 mM
HCl 50 mL, the coupling buffer 50 m
Washed in the order of L. (4) The bacterial cell treatment solution obtained in (1) was added to the column of (3), and the mixture was reacted at room temperature for 16 hours with a rotator. (5) The column of (4) is coupled with a coupling buffer of 20 m.
After washing in order of L and 10 mL of a blocking buffer (0.2 M glycine buffer (pH 8.3)), the column was filled with a blocking buffer and reacted at room temperature for 2 hours. (6) Coupling buffer 30 mL, washing buffer (0.1 M acetate buffer ((pH 4.0) +0.5
M NaCl), 30 mL, elution buffer (0.1 M
Washing was performed in the order of 30 mL of a glycine buffer (pH 2.5)) and 30 mL of a binding buffer (PBS (-)). (7) Fill the column with the binding buffer, and use 4
Stored at ° C.

The MK cell column and the MAC cell column that absorb the MK antibody and the MAC antibody, respectively, were replaced with MK heat-killed bacteria (obtained from Osaka City University Medical School Bacteriology Department) instead of MTAB heat-killed bacteria. It was prepared by the same method except that dead bacteria (obtained from the Department of Bacteriology, Osaka City University School of Medicine) were used.

Example 2 Preparation of Absorbed Antibody Absorption Treatment of Anti-MTAB Rabbit IgG Absorption treatment was performed on anti-MTAB rabbit IgG, and MK and M
An antibody that does not show cross-reactivity with AC and reacts specifically with MTAB was prepared by the following procedure. (1) 10 mg of anti-MTAB rabbit IgG in PBS
Replaced with (-). (2) The MK cell column (3.5 m) prepared in Example 1
L) was equilibrated with PBS (-), and the anti-MT
The AB rabbit IgG / PBS solution was passed through the MK cell column at a flow rate of 0.6 mL / min to adsorb the MK antibody to the MK cell column. (3) The protein fraction passed through the MK cell column was recovered. (4) The MAC cell column (3.5 mL) prepared by the method described in Example 1 is equilibrated with PBS (-), and the protein fraction collected in (3) is re-equipped with the MAC cells again. After passing through the body column, the MAC antibody was absorbed in the MAC cell column. (5) The protein fraction that passed through the MAC cell column was collected. (6) The protein fraction collected in (5) is concentrated using an ultrafiltration membrane (molecular weight cut off: 50,000), and an antibody that does not show cross-reactivity with MK and MAC but specifically reacts with MTAB is obtained. Obtained.

In the above example, the MAC cell column was used after the MK cell column was used for the absorption treatment.
After using the C cell column, the MK cell column may be used.

Absorption treatment of anti-MK rabbit IgG Absorption treatment of the MTAB antibody and the MAC antibody from the anti-MK rabbit IgG gave an antibody that did not show a cross-reactivity with MTAB and MAC but specifically reacted with MK.
Instead of combining the MK cell column and the MAC cell column in the method of the anti-MTAB rabbit IgG absorption treatment, the anti-MK rabbit IgG absorption treatment is performed using MTAB.
The procedure was performed according to the above-mentioned method except that the bacterial cell column and the MAC bacterial cell column were combined.

Absorption treatment of anti-MAC rabbit IgG Absorption treatment of MTAB antibody and MK antibody from anti-MAC rabbit IgG gave an antibody that did not show cross-reactivity with MTAB and MK and reacted specifically with MAC.
The absorption treatment of anti-MAC rabbit IgG is carried out by the method of absorption treatment of anti-MTAB rabbit IgG described above, instead of combining the MK cell column and the MAC cell column with MTA.
The procedure was performed according to the above-mentioned method except that the B cell column and the MK cell column were combined.

Example 3 Preparation of Fluorescent Labeled Antibody Fluorescein Label (Green Label) (1) Absorbing antibody 10m prepared by the above-mentioned absorption treatment
g in 50 mM bicarbonate buffer (pH 8.5) 1
Dissolved in mL. As the absorption antibody, any of an MTAB-specific antibody, an MK-specific antibody, and a MAC-specific antibody may be used. (2) 0.5 mg of NHS-fluorescein (Pierce) was dissolved in 0.5 mL of DMSO (Wako Pure Chemical Industries, Ltd.). (3) Absorbed antibody / bicarbonate buffe prepared in (1)
r While stirring the solution, the NHS-fluorescein / DMSO solution prepared in (2) was added, and the mixture was allowed to stand on ice for 2 hours. (4) PBS (-) + 0.02% on PD-10 column
NaN ₃ and ultrafiltration membrane (fraction molecular weight 5000
The solution was concentrated in 0) to obtain a fluorescein-labeled absorbed antibody. These steps (1) to (4) were performed under light shielding.

Rhodamine label (red label) (1) Absorbed antibody 10m produced by the above-mentioned absorption treatment
g was dissolved in 1 mL of PBS (-). As the absorption antibody, any of an MTAB-specific antibody, an MK-specific antibody, and a MAC-specific antibody may be used. (2) 0.5 mg of NHS-rhodamine (Pierce) in DM
Dissolved in 0.5 mL of SO. (3) NHS-rhodamine / D prepared in (2) while stirring the absorbed antibody / PBS (-) solution prepared in (1)
The MSO solution was added and left on ice for 2 hours. (4) PBS (-) + 0.02% on PD-10 column
NaN ₃ and ultrafiltration membrane (fraction molecular weight 5000
The solution was concentrated in 0) to obtain a rhodamine-labeled absorbed antibody.
These steps (1) to (4) were performed under light shielding.

AMCA label (blue label) (1) Absorbing antibody 10m produced by the above-mentioned absorption treatment
g was dissolved in 1 mL of 50 mM boratebuffer (pH 8.5). The absorbed antibody is an MTAB-specific antibody, MK
Either a specific antibody or a MAC-specific antibody may be used. (2) 0.5 mg of AMCA-NHS (Pierce) in DMS
O was dissolved in 0.2 mL. (3) AMCA-NHS / D prepared in (2) while stirring the absorbed antibody / borate buffer solution prepared in (1)
The MSO solution was added and left on ice for 2 hours. (4) PBS (-) + 0.02% on PD-10 column
NaN ₃ and ultrafiltration membrane (fraction molecular weight 5000
The resultant was concentrated in 0) to obtain an AMCA-labeled absorbed antibody. These steps (1) to (4) were performed under light shielding.

Example 4 Comparison of Absorbed Anti-MTAB Rabbit IgG Absorbed and Unabsorbed Absorbed Antibody Performance The cross-reactivity of the anti-MTAB rabbit IgG-absorbed antibody with the MK and MAC of the unabsorbed antibody was examined to determine the MTA of the anti-MTAB rabbit IgG-absorbed antibody.
The specificity for B was examined.

The anti-MTAB rabbit IgG unabsorbed antibody had an antibody titer of 25600 (Abs.
0.659). The anti-MTAB rabbit IgG absorption-treated antibody used was the MTAB-specific antibody obtained in Example 2 above.

The examination was performed by an indirect ELISA method. The experimental procedure is shown below. (1) 100 mg / mL MTAB suspended in PBS,
After preparing each of the heat-killed bacteria of MK and MAC at 100 μg / mL (pH 9.6) with a coating buffer, 60 μL of each of the killed cells was dispensed into each well of a 96-well ELISA microplate. Incubated for 5 hours and adsorbed to the wells. The plate was discarded immediately before use. After washing twice with a plate washer, 160 μl of a blocking solution was further dispensed into each well, and the plate was washed at 37 ° C.
For 1 hour. (2) The blocking solution in each well was discarded and washed three times with a plate washer. (3) As the primary antibody, the anti-MTAB rabbit IgG unabsorbed antibody and the anti-MTAB rabbit IgG absorbed antibody were used. These antibodies were used in a sample diluent at 100 μg /
2 -fold serial dilution from mL to 2 x 10 ^-7 ,
80 μL of each serial dilution of the B-rabbit IgG-unabsorbed antibody and the serial dilution of the anti-MTAB rabbit IgG-absorbed antibody were dispensed into each well at 80 μL.
Incubated at 7 ° C for 1 hour. (4) After incubation, the reaction solution was discarded and washed three times with a plate washer. (5) Biotin-labeled anti-rabbit Ig as a secondary antibody
Dispense 80 μL of the G antibody solution into each well and incubate at 37 ° C. for 1 hour. (6) After the plate on which the reaction of the secondary antibody has been completed is washed with a plate washer, the enzyme substrate is added to each well at 80 μl.
L was dispensed, and 30 minutes after the start of the reaction, the absorbance at a wavelength of 405 nm was measured with a plate reader. The performance of the unabsorbed antibody and the absorbed antibody was compared at the highest dilution ratio at which the absorbance difference between the target sample and the control cleared 0.1 or more. Table 1 shows the composition of each reagent.

[0060]

TABLE 1 20x coating buffer _{Na 2 CO 3 1.272 g NaHCO 3} 2.344 g NaN 3 0.160 g milli-Q water at constant volume 25xPBS in 40 ml (-) KCl 5g KH 2 PO 4 5g Na 2 HPO 4 72.5g NaCl 200g millimeter Washing solution 25 × PBS (-) 400 ml Tween 20 10 ml NaN ₃ 10 g Make up to 10 L with Milli-Q water Blocking solution Geratin 10 g 25 × PBS (-) 40 ml NaN ₃ 1 g Milli-Q water 1000 mL Dissolve Geratin by heating in constant volume hot water. Sample diluent Dilute the blocking solution 10-fold with the washing solution. Enzyme substrate (for AP): p-Nitrophenylphosphate disodium salt 100 mg 0.5 mM MgCl ₂ solution 100 ml Diethanolamine 100 μl

Table 2 shows the measurement results.

[0062]

[Table 2]

The anti-MTAB rabbit IgG-unabsorbed antibody was used to compare the titer of each killed cell with the MT cell.
For the reactivity with AB dead cells, the MK dead cells and 25
% (2560/10240), 50% of the MAC dead cells and
% (5120/10240) cross-reactivity. On the other hand, when comparing the titers of the killed cells with the anti-MTAB rabbit IgG-absorbed antibody, 0.4% (20%) of the killed MK cells with respect to the reactivity with the killed MTAB cells
/ 5120), and 0.2% (10/5
120). From this, it was confirmed that the MTAB-specific antibody prepared in Example 2 showed little cross-reactivity with MK and MAC, and was an antibody that specifically reacted with MTAB.

2. By measuring the detection limit concentrations of the anti-MTAB rabbit IgG-absorbed antibody and the unabsorbed antibody against MTAB, MK, and MAC, the anti-MTAB rabbit IgG was detected.
The performance of the rabbit IgG absorbed and unabsorbed antibodies was compared. The anti-MTAB rabbit IgG-absorbed antibody and the unabsorbed antibody were obtained in Example 41 above. The same antibodies were used as described in.

The examination was performed by the sandwich method of the ELISA method. The experimental procedure is shown below. (1) The anti-MT adjusted to 10 μg / mL with PBS
The AB rabbit IgG non-absorbed antibody and the anti-MTAB rabbit IgG-absorbed antibody were dispensed at 100 μL into each well of a 96-well ELISA microplate and allowed to stand at 37 ° C. for 2 hours to adsorb to the wells. The plate was discarded immediately before use, washed twice with a plate washer, and 160 μl of a blocking solution was further dispensed into each well.
Incubated for 1 hour at ° C. (2) 100 mg / mL MTAB suspended in PBS,
Heat-killed bacteria of MK and MAC are diluted to 2 × 10 ^-7 with PBS.
The serial dilution of the MTAB killed cells, the respective serial dilutions of the killed MK cells, and the respective serial dilutions of the killed MAC cells were dispensed into each well by 80 μL. Incubated at 37 ° C. for 1 hour. (4) After incubation, the reaction solution was discarded and washed three times with a plate washer. (5) As a primary antibody, a biotin-labeled rabbit IgG (1
0 μg / mL) was dispensed into each well in an amount of 80 μL.
Incubated for 1 hour at ° C. (6) After incubation, the reaction solution was discarded and washed three times with a plate washer. (7) As a secondary label, avidin-AP (Dako) diluted 2000-fold was dispensed into each well by 80 μL,
Incubated at 37 ° C for 1 hour. (8) After washing the plate after the reaction three times with a plate washer, 80 μL of the enzyme substrate was dispensed into each well, and one hour after the start of the reaction, the absorbance at a wavelength of 405 nm was measured with a plate reader. The performance of the unabsorbed antibody and the absorbed antibody was compared at the highest dilution ratio at which the absorbance difference between the target sample and the control cleared 0.1 or more.

The method of preparing the biotin-labeled rabbit IgG (biotinylation of rabbit IgG antibody) was carried out as follows. A rabbit IgG antibody dissolved in PBS (-) and adjusted to 2 mg / mL was added to a biotin solution (75 μL / mL) (EZ-Link sulfo-NHS-LC).
-Biotin adjusted to 1 mg / mL with PBS (-))
Was added and mixed, followed by reaction in ice for 2 hours. After the reaction, unreacted biotin was removed by passing it through a PD-10 column, followed by final protein quantification to prepare biotin-labeled rabbit IgG.

The measurement results are shown in FIGS. 1 (a) to 1 (c).
FIG. 1A is a graph showing the results of detecting the dead MTAB cells using the anti-MTAB rabbit IgG-absorbed antibody and the anti-MTAB rabbit IgG-unabsorbed antibody. Both showed similar reactivity. FIG. 1 (b)
Using the anti-MTAB rabbit IgG-absorbed antibody and the anti-MTAB rabbit IgG-unabsorbed antibody,
It is the graph which showed the result of having detected K dead bacterial body. The anti-MTAB rabbit IgG-absorbed antibody was prepared using the undiluted M
It showed little reactivity against K-killed cells. Figure 1
(C) is a graph showing the results of detecting the dead MAC cells using the anti-MTAB rabbit IgG-absorbed antibody and the anti-MTAB rabbit IgG-unabsorbed antibody. The anti-MTAB rabbit IgG-absorbed antibody hardly showed reactivity with the undiluted dead MAC cells.

The absorbance at which the anti-MTAB rabbit IgG-absorbed antibody and the anti-MTAB rabbit IgG-unabsorbed antibody showed a limit for detecting the killed MTAB, the killed MK, and the killed MAC. Table 3 shows the concentrations of the respective bacterial cells in Table 1.

[0069]

[Table 3]

As described above, in the anti-MTAB rabbit IgG-unabsorbed antibody, the killed MK cells and the MA
C. Cross-reactivity with dead cells was observed. In the dead MK cells, the detection limit cell concentration was about one-eighth of the detection limit cell concentration of the MAC dead cells.
It was found that the antibody showed higher cross-reactivity with the anti-MTAB rabbit IgG unabsorbed antibody than the killed C cells. On the other hand, the anti-MTAB rabbit IgG-absorbed antibody has almost no cross-reactivity to the killed MK cells and the killed MAC cells (the absorbances of undiluted cells are 0.055 and 0.024, respectively). Therefore, it was confirmed that the antibody specifically binds to MTAB.

Example 5 Performance Comparison of Absorbed and Unabsorbed Anti-MK Rabbit IgG Antibodies The specificity of the absorption-treated anti-MK rabbit IgG antibody to MK was examined by examining the cross-reactivity between the antibody-absorbed antibody of anti-MK rabbit IgG and MTAB and MAC of the unabsorbed antibody.

The anti-MK rabbit IgG unabsorbed antibody had an antibody titer at the time of blood collection of 25,600 (Abs. 1.1).
19). Anti-MK rabbit IgG absorption treated antibody
The MK-specific antibody obtained in Example 2 above was used.

The experimental procedure was such that the dead bacteria were adsorbed to the wells at 4 ° C. for 16 hours, and the anti-M antibody was used as a primary antibody.
K rabbit IgG unabsorbed antibody and the anti-MK rabbit Ig
Example 4 above, except that G was used. Was carried out according to the method described in (1).

Table 4 shows the measurement results.

[0075]

[Table 4]

The anti-MK rabbit IgG unabsorbed antibody was tested for reactivity with the killed MK cells by comparing the titer of each killed cell with the MTAB killed cells.
Both AC killed cells had a cross-reactivity of 12.5% (5120/40960). On the other hand, the anti-MK rabbit Ig
When comparing the titers of the killed cells with the G-absorbed antibody, the MTAB-killed cells were 0.8% (80/10240) and the MAC-killed cells were reactive with respect to the reactivity with the MK-killed cells. The body was only 0.1% (10/10240). Thus, the MK-specific antibody prepared in Example 2 was MTAB
And showed little cross-reactivity to MAC, MK
It was confirmed that the antibody specifically reacted with.

2. By measuring the detection limit concentrations of the anti-MK rabbit IgG-absorbed antibody and the unabsorbed antibody against MTAB, MK, and MAC, the anti-MK rabbit I
The performance of the gG absorption-treated antibody and the non-absorbed antibody was compared. Anti-MK rabbit IgG-absorbed antibody and unabsorbed antibody were obtained in Example 5 above. The same antibodies were used as described in.

The experimental procedure was as follows. The antibodies to be adsorbed on each microplate were the anti-MK rabbit IgG non-absorbed antibody and the anti-MK rabbit IgG-absorbed antibody.
Example 4 except that the sample was allowed to stand for a period of time to adsorb to the well. Was carried out according to the method described in (1).

The measurement results are shown in FIGS. 2 (a) to 2 (c).
FIG. 2A is a graph showing the results of detecting the dead MTAB cells using the anti-MK rabbit IgG-absorbed antibody and the anti-MK rabbit IgG-unabsorbed antibody.
FIG. 2 (b) is a graph showing the results of detecting the dead MK cells using the anti-MK rabbit IgG-absorbed antibody and the anti-MK rabbit IgG-unabsorbed antibody. FIG.
(C) is a graph showing the results of detecting the MAC killed cells using the anti-MK rabbit IgG-absorbed antibody and the anti-MK rabbit IgG-unabsorbed antibody.
The anti-MK rabbit IgG-absorbed antibody showed almost no reactivity with the undiluted dead MAC cells.

The absorbance at which the anti-MTAB rabbit IgG absorption-treated antibody and the anti-MTAB rabbit IgG non-absorbed-treated antibody showed a limit in detecting the dead MTAB, the dead MK, and the dead MAC cells. Are shown in Table 5.

[0081]

[Table 5]

As described above, in the anti-MK rabbit IgG unabsorbed antibody, the killed MTAB cells and the MA
Cross-reactivity with C-killed cells was observed. In the MAC-killed cells, the detection limit cell concentration was about half that of the MTAB-killed cells, and therefore, the antibacterial activity was higher than that of the MTAB-killed cells. It was found to show high cross-reactivity with MK rabbit IgG unabsorbed antibody. On the other hand, the anti-MK rabbit I
The sensitivity of the antibody treated with gG to the MTAB-killed cells was 1% of the sensitivity of the MK-killed cells from the detection limit cell concentration.
It is about 1/0, and further, cross-reactivity with the MAC dead cells is hardly recognized (absorbance of undiluted cells is 0.071). Therefore, it was confirmed that the anti-MK rabbit IgG-absorbed antibody was an antibody that specifically binds to MK.

Example 6 Performance Comparison between Absorbed and Unabsorbed Anti-MAC Rabbit IgG Antibodies By examining the cross-reactivity between the anti-MAC rabbit IgG-absorbed antibody and the unabsorbed antibody MTAB and MK, the specificity of the anti-MAC rabbit IgG-absorbed antibody to MAC was examined.

The antibody titer of the unabsorbed anti-MAC rabbit IgG was 25600 (Abs.
0.776). As the anti-MAC rabbit IgG absorption-treated antibody, the MAC-specific antibody obtained in Example 2 above was used.

The experimental procedure was such that the dead bacteria were adsorbed to the wells at 4 ° C. for 16 hours, and the anti-M
Example 4 1. Except that the unabsorbed AC rabbit IgG-treated antibody and the anti-MAC rabbit IgG-absorbed antibody were used. Was carried out according to the method described in (1).

Table 6 shows the measurement results.

[0087]

[Table 6]

The anti-MAC rabbit IgG unabsorbed antibody was used to compare the titer of each killed cell with the MAC.
For the reactivity with dead cells, the MTAB dead cells and 1
It had a cross-reactivity of 2.5% (2560/20480) and 50% (10240/20480) with the killed MK cells. On the other hand, when comparing the titers of the killed cells with the anti-MAC rabbit IgG-absorbed antibody, the MTAB killed cells showed 0.8% (2%) relative to the reactivity with the MAC killed cells.
0/2560), and 0.4% (10/2
560). From this, it was confirmed that the MAC-specific antibody prepared in Example 2 showed almost no cross-reactivity to MTAB and MK, and was an antibody that specifically reacted with MAC.

2. The performance of the anti-MAC rabbit IgG-absorbed and unabsorbed antibodies was compared by measuring the detection limit concentration of the anti-MAC rabbit IgG-absorbed antibody and the unabsorbed antibody against MTAB, MK, and MAC. Anti-MAC rabbit IgG absorbed and unabsorbed antibodies were prepared in Example 6 above. The same antibodies were used as described in.

In the experimental procedure, the antibodies to be adsorbed to each microplate were the anti-MAC rabbit IgG non-absorbed antibody and the anti-MAC rabbit IgG-absorbed antibody, and allowed to stand at 4 ° C. for 16 hours to adsorb to the wells. Example 4 except for the above. Was carried out according to the method described in (1).

The measurement results are shown in FIGS. 3 (a) to 3 (c).
FIG. 3A is a graph showing the results of detecting the dead MTAB cells using the anti-MAC rabbit IgG-absorbed antibody and the anti-MAC rabbit IgG-unabsorbed antibody. FIG. 3 (b) is a graph showing the results of detecting the dead MK cells using the anti-MAC rabbit IgG-absorbed antibody and the anti-MAC rabbit IgG-unabsorbed antibody. The anti-MAC rabbit IgG-absorbed antibody showed almost no reactivity against the undiluted killed MK cells. FIG. 3 (c) is a graph showing the results of detecting the dead MAC cells using the anti-MAC rabbit IgG-absorbed antibody and the anti-MAC rabbit IgG-unabsorbed antibody.

The anti-MAC rabbit IgG-absorbed antibody and the anti-MAC rabbit IgG non-absorbed antibody were combined with the MT antibody.
Table 7 shows the concentration of each bacterial cell at an absorbance showing a limit for detecting AB dead cells, the MK dead cells, and the MAC dead cells.

[0093]

[Table 7]

As described above, in the anti-MAC rabbit IgG unabsorbed antibody, the dead MTAB cells and the M
Cross-reactivity with K-killed cells was observed, and in the MK-killed cells, it was found that the MK-killed cells showed high cross-reactivity with the MK-killed cells because the detection limit cell concentration was lower than the MAC-killed cells. did. On the other hand, the sensitivity of the anti-MAC rabbit IgG absorption-treated antibody to the MTAB-killed cells is about one-eighth of the sensitivity of the MAC-killed cells, based on the detection limit cell concentration. Almost no cross-reactivity (absorbance in undiluted cells is 0.071). For this reason, the anti-MAC rabbit IgG
It was confirmed that the absorption-treated antibody was an antibody that specifically bound to MAC.

Example 7 Detection of Mycobacteria in Smears The MTAB-specific antibody, MK-specific antibody, and MAC-specific antibody prepared according to the present invention correspond to MTAB, M
We examined whether it is effective to detect K and MAC respectively.

MTAB-specific antibody, MK-specific antibody, M
As the AC-specific antibodies, those prepared in Example 2 were used. As a sample, a slide glass smeared with three kinds of acid-fast bacteria, MTAB, MK, and MAC, was used.

The immunohistological staining was performed as follows. (1) Heat-killed cells of three types of acid-fast bacterium, MTAB, MK, and MAC, were smeared on a slide glass, fixed with a flame, and washed with 0.05 M Tris-HCl (pH 7.6).
Washed at room temperature for minutes. This was repeated three times. (2) Rabbit immunoglobulin fraction not infected with mycobacteria 0.005 mg / mL (DAKO)
For 30 minutes at room temperature. (3) The plate was washed with 0.05 M Tris-HCl (pH 7.6) for 5 minutes at room temperature. This was repeated three times. (4) The fluorescein-labeled MTAB-specific antibody (0.005 mg / mL) prepared in Example 3 above was added, and reacted at room temperature for 1 hour. (5) The plate was washed with 0.05 M Tris-HCl (pH 7.6) for 5 minutes at room temperature. This was repeated three times. (6) After washing, the cells were sealed by gel mounting. (7) Fluorescence microscope (Ex. 490 nm, Em. 520n)
m, Eclipse, Nikon), and the confirmed image was photographed.

FIG. 4 (a) shows the result of immobilizing MTAB on a slide glass and reacting it with a fluorescein-labeled MTAB-specific antibody. FIG. 4 (a) shows the result of reacting MAC with a fluorescein-labeled MTAB-specific antibody immobilized on a slide glass. FIG. 4 (b) shows the result of immobilizing MK on a slide glass and reacting it with a fluorescein-labeled MTAB-specific antibody. The green fluorescent dye of fluorescein was detected only in the sample in which MTAB was immobilized on a slide glass (the green fluorescent dye can be confirmed in the original image). From this, the MTAB-specific antibody prepared according to the present invention was: It was confirmed that the antibody could specifically detect MTAB.

In the above procedure (4), the fluorescein-labeled MK-specific antibody is used instead of the fluorescein-labeled MTAB-specific antibody, whereby the MK-specific antibody specifically detects MK from the specimen. We examined whether it was effective. FIG. 5 (a) shows the results obtained by fixing MK to a slide glass and reacting with a fluorescein-labeled MK-specific antibody. FIG. 5 () shows the results of fixing MTAB to slide glass and reacting with a fluorescein-labeled MK-specific antibody. FIG. 5 (c) shows the results obtained by immobilizing the MAC on a slide glass and reacting it with a fluorescein-labeled MK-specific antibody. The fluorescein green fluorescent dye was detected only in the sample in which MK was immobilized on a slide glass (the green fluorescent dye can be confirmed in the original image). From this, the MK-specific antibody prepared according to the present invention was: It was found that the antibody could specifically detect MK.

Further, in the above procedure (4), by using a fluorescein-labeled MAC-specific antibody instead of a fluorescein-labeled MTAB-specific antibody, the MAC-specific antibody specifically detects MAC from the specimen. We examined whether it was effective. FIG. 6 (a) shows the result of reacting the MAC with a fluorescein-labeled MAC specific antibody immobilized on a slide glass, and FIG. 6 (a) shows the result of reacting MTAB with a fluorescein-labeled MAC specific antibody immobilized on a slide glass. FIG. 6 (b) shows the results obtained by fixing MK to a slide glass and reacting it with a fluorescein-labeled MAC-specific antibody. The green fluorescent dye of fluorescein was detected only in the sample on which the MAC was fixed on the slide glass (the green fluorescent dye can be confirmed in the original image). From this, the MAC-specific antibody prepared according to the present invention was: It was found that the antibody could specifically detect MAC.

As described above, the MTA manufactured according to the present invention is
B-specific antibody, MK-specific antibody, MAC-specific antibody
This is effective for specifically detecting MTAB, MK, and MAC from a sample, respectively, and provides a quick and highly reliable immunological detection method for acid-fast bacilli by the simple method described above. Is what you can do.

Example 8 Identification of Mycobacterium Species Using a Mixed Reagent (Cocktail Antibody Reagent) Mixed with Labeled Bacterial Species Antibodies Fluorescently Labeled with Different Dyes Rhodamine Label Prepared in Example 3 A mixed reagent (cocktail antibody reagent) obtained by mixing a (red-labeled) MTAB-specific antibody, an AMCA-labeled (blue-labeled) MK-specific antibody, and a fluorescein-labeled (green-labeled MAC-specific antibody) is contained in a sample. It was examined whether it is effective for visually distinguishing existing mycobacterial species.

[0103] Immunohistochemical staining was performed as follows. (1) Heat-killed cells of three types of acid-fast bacterium, MTAB, MK, and MAC, were smeared on a slide glass and fixed with a flame, and then 0.0
Washed with 5M Tris-HCl (pH 7.6) for 5 minutes at room temperature. This was repeated three times. (2) Rabbit immunoglobulin fraction not infected with mycobacteria 0.001 mg / mL (DAKO)
For 30 minutes at room temperature. (3) The plate was washed with 0.05 M Tris-HCl (pH 7.6) for 5 minutes at room temperature. This was repeated three times. (4) Rhodamine-labeled MTAB-specific antibody (0.0
5 mg / mL), AMCA-labeled MK-specific antibody (0.05 mg / mL), fluorescein-labeled MA
Mix equal amounts of C-specific antibody (0.05 mg / mL)
The cocktail antibody reagent to which% BSA was added was added to the washed sample of (3), and reacted at room temperature for 1 hour. (5) The plate was washed with 0.05 M Tris-HCl (pH 7.6) for 5 minutes at room temperature. This was repeated three times. (6) After washing, the gel was mounted and sealed. (7) Fluorescence microscope (rhodamine: Ex. 510-560)
nm, Em. 590 nm, fluorescein: Ex. 45
0-490 nm, Em. 520 nm, AMCA: Ex.
365 nm, Em. 400nm, Eclipse, Ni
kon) and the confirmed image was photographed.

The resulting stained image is shown in FIG.
Red, blue, and green fluorescence were visually confirmed from the sample (each fluorescent dye was clearly confirmed in the original image). The confirmation of these fluorescent dyes means that an immunocomplex in which rhodamine-labeled MTAB-specific antibody and MTAB specifically bind, an immune complex in which AMCA-labeled MK-specific antibody and MK specifically binds, and fluorescein, respectively. That is, there is an immune complex in which the labeled MAC-specific antibody and the MAC are specifically bound. By using the cocktail antibody reagent, the mycobacterial species present in the sample are visually identified. It turns out that it is possible. Thus, a method for diagnosing bacterial species that can quickly and easily identify the infected mycobacterial species present in the sample can be provided.

[0105]

According to the present invention, it is possible to provide a method for producing an antibody that absorbs an antibody that causes a cross-reaction by using a cell column and specifically reacts with only a specific type of acid-fast bacterium. In addition, using the acid-fast bacterium species-specific antibody prepared by the antibody production method, an acid-fast bacterium can be immunologically detected and identified, a rapid, simple, and highly reliable method for immunological detection. Kits can be provided. Furthermore, by labeling each antibody of the bacterial type of the two or more mycobacterial species-specific antibodies produced by the antibody production method with different dyes, and using a mixed reagent obtained by mixing these labeled antibodies, Thus, it is possible to provide a method for immunologically detecting the type of bacteria, which can quickly and easily identify the species of acid-fast bacillus or the like infected with A. As a result, it is possible to quickly determine whether the acid-fast bacterium is Mycobacterium tuberculosis or an atypical acid-fast bacterium, thereby contributing to the selection of a clinically appropriate therapeutic method.

[Brief description of the drawings]

FIG. 1. Anti-MTAB rabbit IgG-absorbed antibody and anti-M
The graph which showed the result of having detected each dead bacterial body using TAB rabbit IgG unabsorbed antibody. (A) Detect dead MTAB cells (b) Detect MK dead cells (c) Detect MAC dead cells

FIG. 2 is a graph showing the results of detecting each of the dead cells using an anti-MK rabbit IgG-absorbed antibody and an anti-MK rabbit IgG-unabsorbed antibody. (A) Detect dead MTAB cells (b) Detect MK dead cells (c) Detect MAC dead cells

FIG. 3: Anti-MAC rabbit IgG-absorbed antibody and anti-MA
The graph which showed the result of having detected each dead bacterial body using C rabbit IgG unabsorbed antibody. (A) Detect dead MTAB cells (b) Detect MK dead cells (c) Detect MAC dead cells

FIG. 4 is a diagram showing the results of fixing each dead cell on a slide glass and reacting with a fluorescein-labeled MTAB-specific antibody. (A) Fix dead MTAB cells on a slide glass. (B) Fix dead MAC cells on a slide glass. (C) Fix dead MK cells on a slide glass.

FIG. 5 is a diagram showing the results of fixing each dead cell on a slide glass and reacting with a fluorescein-labeled MK-specific antibody. (A) Fix MK dead cells on a slide glass. (B) Fix MTAB dead cells on a slide glass. (C) Fix MAC dead cells on a slide glass.

FIG. 6 is a view showing the results of fixing each dead cell on a slide glass and reacting with a fluorescein-labeled MAC-specific antibody. (A) MAC dead cells fixed on a slide glass (b) MTAB dead cells fixed on a slide glass (c) MK dead cells fixed on a slide glass

FIG. 7 is a diagram in which the species of acid-fast bacterium present in the sample are identified by the cocktail antibody reagent.

──────────────────────────────────────────────────の Continued on the front page (51) Int.Cl. ⁷ Identification code FI Theme coat ゛ (Reference) C12N 11/14 C12N 11/14 (C12N 11/10 (C12N 11/10 C12R 1:32) C12R 1:32 ) (C12N 11/10 (C12N 11/10 C12R 1: 325) C12R 1: 325) (72) Inventor Tomohiro Samori, Kumiyama-cho, Kuse-gun, Kyoto, Japan In-house (72) Inventor Riharu Nakajima 461-1 Isshiki, Kazu-cho, Kaizu-cho, Kaizu-gun, Gifu Japan Biological Science Center, Inc.Biological Business Division F-term (reference) BA70 CA11 DA75 EA52 FA81

Claims (11)

[Claims] 1. An antibody producing method for producing an antibody which specifically reacts with a cell of a specific species, comprising using a cell column carrying cells of another species having cross-reactivity. An antibody production method for producing an antibody that specifically reacts with a cell of a specific strain, which is characterized by the following. 2. The method for producing an antibody according to claim 1, wherein the cells are acid-fast bacteria. 3. The method according to claim 1, wherein the acid-fast bacterium is Mycobacterium tuberculosis, Mycobacterium kansasii, or Mycobacterium avium complex.
3. The method for producing an antibody according to claim 2, wherein the antibody is a bacterium avium complex. 4. A step of contacting a specimen with an acid-fast bacterium-specific antibody that specifically reacts with a specific acid-fast bacillus species, and detecting the acid-fast bacterium-specific antibody that has formed an immune complex. An immunological detection method for an acid-fast bacterium having a step. 5. The step of detecting the acid-fast bacterium species-specific antibody that has formed the immune complex comprises detecting the size of the particles passing through the detection point and fluorescence with a flow cytometer to detect the acid-fast bacilli. The method for immunologically detecting an acid-fast bacterium according to claim 4, comprising a step. 6. The method for immunologically detecting an acid-fast bacterium according to claim 4, wherein the specimen is sputum. 7. An immunological detection kit for an acid-fast bacterium containing an acid-fast bacterium species-specific antibody. 8. A step of contacting a sample with a mixed reagent obtained by mixing two or more kinds of acid-fast bacterium species-specific antibodies labeled with different color-forming agents; A method for detecting an acid-fast bacterium by an immunological method, the method comprising detecting 9. The step of detecting the acid-fast bacterium species-specific antibody that has formed the immune complex includes detecting the difference between the size and fluorescence of particles passing through a detection point with a flow cytometer and detecting the acid-fast bacterium. 9. The method for immunologically detecting the mycobacteria according to claim 8, comprising a step of detecting the bacterial species. 10. The specimen according to claim 8, wherein the specimen is sputum.
The method for immunologically detecting the acid-fast bacterium according to any one of the above items. 11. An immunological detection kit for acid-fast bacilli containing a mixed reagent obtained by mixing two or more kinds of acid-fast bacterium species-specific antibodies labeled with different coloring agents.