JPH09229938A - Method and device for analyzing immunity of anti-syphilis treponema antibody - Google Patents

Abstract

PROBLEM TO BE SOLVED: To make it possible to simply detect anti-syphilis Treponema in a short time by providing a labeled reagent zone and a detecting zone on a matrix having a capillarity, and detecting antisyphilms Treponema antibody in a specimen coupled to the detecting zone by adding the specimen. SOLUTION: A matrix 1 is formed of a transfusible water absorbing material by capillarity, and a specimen point adhering zone 3, a labeled reagent zone 4, a detecting zone 5, and a development liquid absorbing zone 6 are provided along the development liquid transfusion direction from the development liquid zone 2 thereon. The zone 4 includes a labeled genetic recombination syphilis Treponema antigen, and the zone 5 is fixed with syphilis Treponema antigen to the matrix 1. At the time of starting the measurement, the zone 2 is dipped in a vessel filled with the development liquid, and the development liquid of the quantity arriving at the zone 6 is supplied. The zone 6 absorbs the development liquid supplied to the matrix 1 to smoothly analyze it. Thus, the anti- syphilis Treponema antibody in the specimen can be analyzed from the label coupled to the zone 5.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a labeling reagent zone consisting of a recombinant T. pallidum treponema (treponema pallidum; hereinafter referred to as TP) antigen, which is labeled on a matrix capable of being infused by capillary action, and a TP antigen is immobilized on the matrix. An immunoassay method for detecting an anti- Treponema pallidum antibody (hereinafter referred to as anti-TP antibody) in a sample bound to the detection zone by adding the sample to an immunoassay device provided with Regarding analytical equipment.

[0002]

2. Description of the Related Art Analysis of biological components and drugs contained in blood, urine, etc. is important for diagnosing pathological conditions and determining treatment progress. Then, as a method for easily analyzing the biological components, drugs, etc. from the sample by utilizing the antigen-antibody reaction, a method using a strip analyzer made of strip-shaped filter paper impregnated with a reaction reagent has been found. In this analysis method, the sample is added to the sample spotting zone provided on the filter paper of the device, the solution is developed,
This is a method of diagnosis based on the degree of color development displayed in the detection zone provided on the filter paper. These strip analyzers use a filter paper that contains the necessary reagents (enzyme-labeled antibody, substrate, coloring reagent, etc.) according to the reaction format, and perform analysis from the color development, so a simple method without a special determination device is used. It can be implemented. In addition, an analysis method using particles such as color latex and metal colloid as a label is also known. In this analysis method, an antibody or an antigen that reacts with an antigen to be detected or a reagent in which an antigen is bound to a particle is used, and the particle image bound to the detection zone is detected.

[0003] Conventionally, T cells have been used to detect blood cells in the detection of anti-TP antibody.
Method using TPHA reagent bound to P cell component, method using indirect agglutination immunoassay reagent produced by binding TP antigen to artificial particle, enzyme immunoassay reagent comprising TP antigen-bound solid phase and labeled anti-globulin antibody And the like have been known. With each of these methods, it takes more than two hours to start the measurement and obtain the result, and since the measuring equipment is used for the judgment, it is necessary to cope with the urgent inspection and the short-time measurement at the bedside. I couldn't.

Anti-TP using a conventional strip analyzer
In the antibody measurement method, a reaction occurred between a large amount of globulin present in the sample and the labeled antibody, and the measurement could not be performed. Therefore, a method using a device in which a washing tank is added to the analyzer (JP-A-5-126832), a device in which a zone for preventing a signal caused by a labeling substance in the developing solution is provided (JP-A-1-503439), and the like are available. Was found.

In these methods, the operation is complicated and it is difficult to always carry out the measurement operation under constant conditions. In addition, it was not a satisfactory method for highly sensitive measurement due to the influence of globulin contained in the sample. In order to solve this problem, when the anti-TP antibody in the sample was measured, it was attempted to detect it by the sandwich method with the TP antigen bound to the solid phase and the labeled TP antigen. It was not easy to introduce a label into the obtained mixture of TP antigens, and it was not possible to continuously obtain a labeled antigen reagent in which a certain label was substituted. Thus, heretofore, a method using a labeled TP antigen has not been known.

[0006]

The conventional methods for measuring anti-TP antibody have a problem that the operation is complicated and highly accurate analysis cannot be performed in a short time. An object of the present invention is to provide an analytical method which can obtain a result in a short time by a simple operation as compared with a conventional method and an apparatus for carrying out the analysis.

[0007]

Means for Solving the Problems As a result of intensive studies by the present inventors, a labeling reagent zone composed of a recombinant TP antigen labeled on a matrix capable of infusion by capillary action and a TP antigen immobilized on the matrix. In the anti- Treponema pallidum antibody analyzer equipped with the detection zone, the sample is added, the immunoassay method for detecting the anti- Treponema pallidum antibody in the sample bound to the detection zone, and the infusible matrix by capillary action. New anti-T with a labeling reagent zone consisting of labeled recombinant TP antigen
The inventors have found an immunoassay device for P antibody and completed the present invention. More specifically, a developing solution zone containing a substrate in a matrix that can be infused by capillary action, a labeling reagent zone consisting of enzyme-labeled genetically modified TP antigen, a detection zone in which TP antigen is immobilized on a matrix, and a specimen spotting Using an anti-TP antibody immunoanalyzer provided with a zone and a developing solution absorption zone, the sample solution is spotted on the sample spotting zone, and then the developing solution is supplied to the developing solution zone, Immunoassay method characterized by carrying out the activity of the enzyme immobilized in the detection zone in an amount depending on the anti-TP antibody in the substrate, and a radioisotope in a matrix infusible by capillary action, Transgenic T labeled with latex, metal colloid particles, fluorescent particles or colored particles
Using an anti-TP antibody immunoassay device provided with a developing solution zone for adding a developing solution containing P antigen and a specimen, a detection zone in which TP antigen is immobilized on a matrix, and a developing solution absorption zone After spotting the sample solution on the sample spotting zone, the developing solution is supplied to the developing solution zone to detect a labeled substance immobilized on the detection zone in an amount depending on the anti-TP antibody in the sample solution. It is an immunoassay method characterized by being carried out.

Hereinafter, the immunoassay method of the present invention will be described in more detail with an apparatus. In this immunoassay device, the matrix is composed of a water-absorbing material that can be infused by capillary action. As the preferable material, for example, cellulose, cellulose such as nitrocellulose or a derivative thereof,
Examples include filter paper and porous membranes formed of glass fiber or the like. The size of this matrix is not limited, but a strip-shaped one having a width of 3 mm to 10 mm and a length of 30 mm to 100 mm is preferable because it is easy to handle. It is preferable to use a matrix having a thickness of 100 μm to 1 mm. The matrix can be used by blocking a part of it with, for example, bovine serum albumin (BSA), casein, sucrose or the like in order to prevent adsorption of the protein due to a non-specific reaction.

(Labeling reagent zone) The labeling reagent zone is a labeled recombinant TP provided on the matrix.
This is the zone containing the antigen. This zone can be provided upstream of the detection zone in the direction of infusion of the developing solution from the developing solution zone. This zone can be provided by a method of spotting the recombinant TP antigen labeled on the matrix or a method of attaching a water-absorbing pad containing the labeled recombinant TP antigen on the matrix.

[0010] The TP antigen has surface antigens of various molecular weights present on the cell surface of TP, and the major one has a molecular weight of 47 kDa.
(TP47), 42kDa (TP42), 17kDa
(TP17), 15kDa (TP15), 50kDa
Antigens such as (TPN50) and TmpA are known. Genes encoding these antigens have already been cloned, and the antigens have been produced by genetic engineering (Tokuhyo 2-500403, INFECTION AND IMMUNITY, Vol. 5).
7, No. 17, PP. 3708-3714, 1989; Molecular Microbiolo
gy, 4 (8), 1371-1379, 1990 etc.). The recombinant TP antigen of the present invention can be obtained by using the antigens of the above-mentioned molecular weights, for example, the method of Norgard et al. (INFECTION AND IMMUNIT
Y, Vol.61, PP.1202-1210, 1993), the Escherichia coli transformed into a vector by culturing is transformed according to the method described in Y. Vol. 61, PP. Further, the gene recombinant TP antigen of the present invention has a derivative of the antigenic protein of each molecular weight,
For example, it is also possible to use a genetically engineered TP antigen in which glutathione-S-transferase (GST) is fused to the N-terminal of a protein (JP-A-7-28).
7017). Further, the recombinant TP antigen is produced by fusing two or more antigens selected from the above-mentioned respective antigens, for example, TP15-17 antigen, TP15-42 antigen, TP15-
47 antigen, TP17-47 antigen, TP42-47 antigen,
TP15-17-42 antigen, TP15-42-47 antigen, TP15-17-47 antigen, TP15-42-47
It may be an antigen, a TP15-17-42-47 antigen, or a fusion TP antigen in which the order of these is reversed.

A label is bound to this genetically modified TP antigen to give a reagent. Examples of the label include enzymes, radioisotopes, latex, metal colloid particles,
Examples thereof include fluorescent particles and colored particles. Examples of the enzyme include various enzymes used in enzyme immunoassay (EIA), and examples of the enzyme include alkaline phosphatase, peroxidase, and β-D-galactosidase. Examples of radioactive isotopes include isotopes such as iodine, tritium and carbon. Examples of the latex include particles of a polymer compound such as polystyrene latex. The metal colloid particles are particles made of various metal colloids, and examples thereof include metal colloids such as selenium, platinum, and gold. The particle diameter is 10 nm ~
It is preferable to use one having a thickness of 1 μm.

Examples of the fluorescent particles include polystyrene containing a fluorescent substance such as fluorescein, rhodamine and platinum cyanide, styrene-butadiene copolymer, styrene-
Examples thereof include particles of acrylic acid copolymer and glass. Colored particles are particles composed of an organic polymer compound or an inorganic compound colored with various dyes or pigments,
For example, polystyrene, polymethyl acrylate, polyacryl amide, polypropylene, polycarbonate, glass or the like is used alone or in a mixture thereof. It is preferable to use the fluorescent particles and the colored particles having a particle size of 10 nm to 1 μm.

The method for binding the labeled substance to the recombinant TP antigen can be produced by utilizing a known method for forming a covalent bond or a non-covalent bond. Examples of the binding method include glutaraldehyde method, periodate method, maleimide method,
The pyridyl disulfide method, a method using various cross-linking agents, and the like can be mentioned (see, for example, “Protein Nucleic Acid Enzyme”, Supplement No. 31, pages 37 to 45 (1985)). In the coupling method using a crosslinking agent, examples of the crosslinking agent include N-succinimidyl-4-maleimidobutyric acid (GMBS), N-succinimidyl-6-maleimidohexanoic acid, N-succinimidyl-4- (N-maleimidomethyl) cyclohexane-1. -Carboxylic acid and the like can be used. In the method using a covalent bond, a functional group existing in the recombinant TP antigen can be used, or after a functional group such as a thiol group, an amino group, a carboxyl group or a hydroxyl group is introduced into the recombinant TP antigen by a conventional method. The labeled gene recombinant TP antigen can be produced by the binding method. In addition, examples of the non-covalent bonding method include a physical adsorption method.

As a method of labeling the genetically modified TP antigen with a radioisotope, for example, Bolton Hunter reagent or the like can be used.

The amount of the labeled gene recombinant TP antigen contained in the labeling reagent zone can be appropriately changed depending on the method of incorporating the matrix into the spotting or water-absorbing pad, the test object, and the amount of the sample used for the measurement. The dry weight is usually about 0.01 μg to 5 μg. Marker gene recombinant T
P antigens are, for example, TP47, TP42, TP17, TP
It is possible to add at least one kind of antigen from labeled recombinant TP antigens such as 15, TPN50, TmpA, and the fusion TP antigen to the labeling reagent zone for use.

(Specimen spotting zone) The specimen spotting zone can be provided on the downstream side in the direction of infusion of the developing solution in the developing solution zone, and in the matrix on the upstream side of the detection zone without particularly containing reagents and the like. Further, the sample spotting zone can be provided on the downstream side of the developing solution zone in the developing solution infusion direction, on the upstream side of the labeling reagent zone, on the downstream side of the labeling reagent zone, on the upstream side of the detection zone, or on the labeling reagent zone. Further, in the apparatus in which the sample spotting zone is provided in the labeling reagent zone, it is preferable to attach a water-absorbing pad containing the labeled recombinant TP antigen as the labeling zone for efficient analysis. In the device to which the pad is added, a large amount of the sample liquid can be spotted, so that the detection sensitivity of the trace component in the sample can be enhanced and the analysis can be performed. Examples of the water absorbent pad include polyvinyl alcohol (PV
Materials composed of porous synthetic or natural polymer compounds such as A), non-woven fabric, and cellulose can be used alone or in combination. Although the size and thickness of the pad are not limited, it is preferable to use a pad having a length and width of about 3 mm to 10 mm and a thickness of about 0.5 mm to 4 mm for efficient measurement.

(Detection Zone) The detection zone is upstream of the developing solution absorption zone and downstream of the sample spotting zone in the direction of infusion of the developing solution, and can be provided with the TP antigen fixed. This zone can be provided by immobilizing it on the matrix by a chemical bond such as a covalent bond or a physical adsorption method. Further, the TP antigen may be bound to an insoluble carrier and contained in the matrix. Examples of the insoluble carrier include particles obtained by insolubilizing a mixture of gelatin, gum arabic and sodium hexametaphosphate (Japanese Patent Publication No. 63-29223), polystyrene latex, various animal red blood cells, glass fibers and the like. The insoluble carrier and the TP antigen can be bound by the chemical bond or physical adsorption. The detection zone is
Although it may have any shape, it is preferable to form it linearly so as to be orthogonal to the infusion direction of the developing solution and immobilize the TP antigen thereon in order to improve the detection sensitivity.

In the detection zone, the above-mentioned TP47, TP4
2, a recombinant TP antigen such as TP17, TP15, and the above fusion TP antigen can be immobilized individually on a matrix to form a detection zone. Further, these antigens can be fixed at different positions on the matrix to provide two or more detection zones. An apparatus provided with two or more detection zones can separately detect and detect antibodies against each antigen. Furthermore, two or more of these recombinant TP antigens can be mixed to form one detection zone. Further, in this detection zone, in addition to the gene recombinant TP antigen, TP cultured in vivo according to a method well known to those skilled in the art, such as a rabbit testis.
A so-called cultured TP antigen (see Japanese Patent Laid-Open No. 58-71457) obtained by decomposing bacterial cells of (Nichols strain) and purifying them by combining methods such as extraction and centrifugation can be used.

(Developing liquid zone) The developing liquid zone is a zone provided at one end of the matrix and supplied with the developing liquid. To start the measurement, this zone can be immersed in a container containing at least the amount of the developing solution that reaches the developing solution absorption zone. Further, for supplying the developing solution, it is possible to start the measurement by adding a liquid tank containing the developing solution to the developing solution zone and breaking the liquid tank to bring the developing solution into contact with the matrix. The developing solution can appropriately contain a surfactant, a buffering agent and the like. As a buffer solution containing a buffer,
For example, acetic acid buffer, borate buffer, Tris-hydrochloric acid buffer, diethanolamine buffer and the like can be mentioned. Further, a water-absorbent filter paper or the like can be attached to the developing solution zone depending on the form of supplying the developing solution.

(Development liquid absorption zone) The development liquid absorption zone is provided at the other end of the development liquid zone provided at one end of the matrix. This zone is provided in order to absorb the developing solution supplied to the matrix and smoothly perform the analysis. The developing solution absorption zone may be formed by forming a long matrix to secure this zone. Further, it is possible to attach a water absorbing material to the matrix to promote the development. In the case of adding this water-absorbing material, a filter paper having a high water retention property made of a natural polymer compound, a synthetic polymer compound, or the like,
A sponge or the like can be used. By stacking the absorbent material on the matrix, a miniaturized immunoassay device can be manufactured.

The device of the present invention can take various forms depending on the type of the labeling substance. When an enzyme is used as the labeling substance, a developing solution zone containing a substrate in a matrix that can be infused by capillary action is used. , A labeling reagent zone comprising an enzyme-labeled recombinant TP antigen, and TP
The anti-TP antibody immunoassay device is provided with a detection zone in which an antigen is fixed to a matrix, a sample spotting zone, and a developing solution absorption zone.

In a device using the above-mentioned enzyme as a label, various substrates can be used to measure the enzyme activity. As the substrate, various color-developing substrates, fluorescent substrates, luminescent substrates, etc. shown below corresponding to the enzyme can be used.

(A) Chromogenic substrate for peroxidase: 2,2'-in combination with hydrogen peroxide
Azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), 3,3 ', 5,5'-tetramethylbenzidine (TMB), diaminobenzidine (DA
B) For alkaline phosphatase: 5-bromo-4-chloro-
3-indolyl phosphate (BCIP)

(B) Fluorescent substrate For alkaline phosphatase: 4-methylumbellyphenyl-phosphate (4MUP) For β-D-galactosidase: 4-methylumbellyphenyl-β-D-galactoside (4MUG)

(C) Luminescent substrate For alkaline phosphatase: 3- (2'-spiroadamantane) -4-methoxy-4- (3 "-phosphoryloxy) phenyl-1,2-dioxetane disodium salt ( AMPPD) For β-D-galactosidase: 3- (2′-spiroadamantane) -4-methoxy-4- (3 ″ -β-D-galactopyranosyl) phenyl-1,2-dioxetane (AMGPD) per For oxidase: Luminol and isoluminol combined with hydrogen peroxide

These substrates are usually used by being added to the developing solution, but the substrates can be provided as a substrate zone on the matrix. The matrix zone can be formed in the matrix by usually dissolving the matrix in a solution, adding the matrix to the matrix, and then drying the matrix.

Further, as an embodiment of the device of the present invention, a radioisotope is added to a matrix infusible by a capillary action,
A developing solution zone for adding a developing solution containing a labeled gene recombinant syphilis treponema antigen labeled with latex, metal colloid particles, fluorescent particles or colored particles, and a detection zone in which a TP antigen is immobilized on a matrix,
An example is an anti-TP antibody immunoassay device provided with a developing solution absorption zone.

This analyzer is a zone in which the labeled gene recombinant TP antigen labeled with a radioisotope, latex, metal colloid particles, fluorescent particles or colored particles and a sample are added to the developing solution zone. Different from the device. A method of adding a solution containing the labeled recombinant TP antigen and a sample to the developing solution zone,
It can be carried out by a method of adding a mixed solution of the labeled gene recombinant TP antigen and the sample. If desired, a developing solution containing the above-mentioned surfactant, buffer, etc. can be added to this developing solution. The labeled gene recombinant TP antigen to be added to this developing solution zone is a labeled gene recombinant TP antigen labeled with a radioisotope, latex, colloidal particles or colored particles, and the labeled gene recombinant TP used in the above analyzer is used. The antigen can be selected and used. Further, the detection zone and the developing solution absorption zone of this analyzer can be the same zone as the above-mentioned analyzer.

(Method of Use) The analysis apparatus of the present invention can be used to analyze anti-TP antibodies in various samples. In order to perform analysis with this device, first, a sample is supplied to the matrix of the device and then developed with a developing solution. The developing solution reaches the developing solution absorbing zone by the capillary action, and the components in the sample not bound to the detection zone and the labeled gene recombinant TP antigen are absorbed and the developing is completed. After completion of the development, the anti-TP antibody can be analyzed by directly or indirectly detecting the amount of the label immobilized in the detection zone in the sample liquid, which depends on the anti- Treponema pallidum antibody. This detection can be carried out by visual inspection or by using a corresponding measuring device such as a scintillation counter, a colorimeter, a fluorometer, a photon counter, a photosensitive film, etc., depending on the labeling substance. In the analysis, for example, a method of using an enzyme as a label and qualitatively visually observing the presence or absence of a dye produced by a chromogenic substrate is convenient. Further, in this method, a semi-quantitative analysis can be performed by using a color chart (color chart) corresponding to the concentration of the anti-TP antibody.

The matrix can be used by laminating it on a supporting member such as plastic, metal or paper depending on the type of the labeling substance and fixing it. Further, the matrix can be fixed to a case made of plastic or the like, a liquid tank containing a developing solution can be attached to the developing solution zone, and a cover having a hole in each zone can be used to construct an apparatus that can be easily handled. The immunoassay device of the present invention is not particularly limited as a sample, and can be applied to the detection of anti-TP antibody in various body fluids such as serum, plasma, whole blood and urine.

[0031]

The present invention will be described in more detail with reference to the following examples.

Example 1 Preparation of recombinant TP17 antigen labeled with alkaline phosphatase Produced according to the method described in JP-A-7-287017,
Recombinant GTP-bound recombinant TP17 antigen 0.12 m
200 nmol of 2-iminothiolane was added to g, and the temperature was 30 ° C.
It was left for 60 minutes to obtain a thiolated TP antigen. Next, 300 nmol of N-succinimidyl-4-maleimidobutyric acid (GMBS) was added to 3 mg of alkaline phosphatase, and the mixture was allowed to stand at 30 ° C. for 60 minutes to obtain maleimidized alkaline phosphatase. Then the thiolated TP antigen 10
0 μg and maleimidated alkaline phosphatase 2.5 m
After mixing with g and reacting overnight at 4 ° C., unreacted antigen and enzyme were removed by a gel filtration column to obtain a recombinant TP17 antigen labeled with alkaline phosphatase.

Example 2 Device for Analyzing Anti-TP17 Antibody As shown in FIG. 2, a matrix 1 of cellulose membrane (Millipore) having a width of 5 mm and a length of 50 mm was placed 15 m from the upper end.
The same recombinant TP17 used in Example 1 at position m.
The detection zone 5 was created by spotting the antigen in a line with an applicator and then drying it. 20 μl of the alkaline phosphatase-labeled recombinant TP17 antigen solution prepared in Example 1
Was put on a polyvinyl alcohol (PVA) sheet cut into a width of 5 mm and a length of 5 mm, and a dried pad was placed at a position 25 mm from the upper end of the matrix to form a labeling reagent zone 4 and a specimen spotting zone 3. Also, the bottom 10 of the matrix
A filter paper having a width of 5 mm and a length of 20 mm (manufactured by Millipore) was attached at a position of mm to form a developing solution zone 2. Further, a filter paper having a width of 10 mm, a length of 20 mm and a thickness of about 1 mm was attached as a developing solution absorption zone 6 at a position of 10 mm at the upper end of the matrix to obtain an apparatus for analyzing anti-TP17 antibody.

Example 3 Measurement of anti-TP17 antibody The anti-TP antibody was analyzed using the analyzer prepared in Example 2. First, after adding 15 μl of the sample to the spotting zone 3,
200 μl of a solution containing 0.3% 5-bromo-4-chloro-3-indolylphosphoric acid (BCIP) was dropped into the developing solution zone for absorption and development. After 15 minutes, the color of the detection zone 5 was visually judged. Measurements were performed on 89 human serum samples, and the results are shown in Table 1. Those with coloration were judged to be positive, and those without coloration were judged to be negative. In addition, for the same sample, a dilution series was prepared and a conventional indirect agglutination immunoassay reagent for anti-TP antibody (Serodia-TP / PA
(Registered trademark); manufactured by Fujirebio Inc.)
Shown in

Example 4 Measurement of anti-TP47 antibody The method of Norgard et al. (INFECTION AN
D IMMUNITY, Vol.61, PP.1202-1210, 1993) was used to prepare a recombinant TP47 antigen labeled with alkaline phosphatase according to the method of Examples 1 and 2, and further purified. A device for TP47 antibody analysis was obtained. Using this device, the samples shown in Table 1 were measured. The results of Examples 3 and 4 are shown in Table 1.
When the samples showing positive results in at least one of the devices were combined with Examples 3 and 4 and the positive judgment was made, the same results as in the indirect agglutination method were obtained for all the samples.

[0036]

[Table 1]

Example 5 Anti-17TP antibody and anti-47TP
Antibody analysis device As shown in FIG. 4, 12 m from the upper end of the matrix 1 of a cellulose membrane (manufactured by Millipore) having a width of 5 mm and a length of 50 mm.
Recombinant TP17 antigen was placed at the position of m, and recombinant TP47 antigen was placed at the position of 17 mm in a line with an applicator and dried to form detection zones 5 and 8. 20 μl of a mixed solution of the alkaline phosphatase-labeled recombinant TP17 antigen prepared in Example 1 and the alkaline phosphatase-labeled recombinant TP47 antigen prepared in Example 3 was used to give a width of 5 m.
25m from the upper end of the matrix with the dried conjugate pad spotted on a PVA sheet cut to a length of 5 mm.
It was placed at the position of m to make a labeling reagent zone 4. Example 2
Similarly, the filter papers of the developing solution zone 2 and the developing solution absorbing zone 6 were attached to prepare an apparatus for analysis of anti-TP17 antibody and anti-47TP antibody.

Example 6 Anti-17TP antibody and anti-47TP
Measurement of antibody Using the apparatus prepared in Example 5, the anti-TP antibody was measured for the sample that was positive by the indirect agglutination immunoassay. First, 15 μl of the sample was added to the spotting zone 3 provided in the labeling reagent zone 4, and then 200 μl of a solution containing 0.3% BCIP was dropped into the developing solution zone for absorption and development. 1
After 5 minutes, the color development of the detection zones 5 and 8 was visually judged. Those with coloration were judged to be positive, and those without coloration were judged to be negative.

The samples determined to be positive or negative by the indirect agglutination immunoassay reagent were measured by the analyzer of Example 5. Table 2 shows the results. In addition, the known initial sample of syphilis infection, second-stage infection sample, and late-stage infection sample were measured by the indirect agglutination immunoassay and the analyzer of Example 4, respectively. Table 3 shows the results.

[0040]

[Table 2]

[0041]

[Table 3]

Example 7 Alkaline phosphatase labeled T
Preparation of P15-17 Antigen A recombinant TP15-17 antigen in which the TP15 antigen and the TP17 antigen were fused was produced, and alkaline phosphatase-labeled TP15-17 antigen was obtained according to the same method as in Example 1.

Example 8 Device for Analyzing Anti-TP15-17 Antibody According to the same method as in Example 2, the alkaline phosphatase-labeled TP15-1 prepared in Example 7 was prepared on the matrix 1.
The 7-antigen and the recombinant TP15-17 antigen were spotted to obtain an anti-TP15-17 antibody analysis device.

Example 9 Device for Analyzing Anti-TP15-17 Antibody Using the analyzer prepared in Example 8, analysis of anti-TP antibody was carried out on 5 human serum samples according to the method of Example 3. The results are shown in Table 4. For the same sample,
The results of the indirect agglutination immunoassay reagent and the anti-TP47 antibody analyzer are shown together in Table 4.

[0045]

[Table 4]

[0046]

INDUSTRIAL APPLICABILITY The present invention provides a simple and highly sensitive method for analyzing anti-TP antibody in a sample. In this method, for example, the anti-TP antibody in serum can be analyzed in about 15 minutes and the result can be obtained. Moreover, the prozone phenomenon observed when measuring a sample having a high antibody titer of anti-TP antibody was not found in the analysis method of the present invention. Furthermore, by using the method of the present invention, a plurality of anti-TP antibodies in a sample can be separated and analyzed,
The time of infection can be estimated and it is also useful for the treatment of syphilis.

[Brief description of drawings]

FIG. 1 is a plan view schematically showing an example of an aspect of an immunoassay device of the present invention.

FIG. 2 is a sectional view of the immunoassay device of the present invention when a labeling reagent zone consisting of a pad containing a labeled gene recombinant TP antigen is added.

FIG. 3 is a sectional view of an apparatus when a matrix zone 1 is provided in a matrix 1 in the immunoassay apparatus of the present invention.

FIG. 4 is a sectional view of the immunoassay device of the present invention when two detection zones 5 and 8 are provided in a matrix.

FIG. 5: Measured by supplying a recombinant TP antigen labeled with a radioisotope, latex, metal colloid particles, fluorescent particles or colored particles and a sample mixed solution 9 to a developing solution zone 2 of the immunoassay device of the present invention. It is a top view at the time of doing.

[Explanation of symbols]

 1 matrix 2 developing solution zone 3 specimen spotting zone 4 labeling reagent zone 5 detection zone 6 developing solution absorption zone 7 substrate zone 8 detection zone 9 mixed solution of labeled gene recombinant TP antigen and specimen

Claims (19)

[Claims] 1. Immunization with an anti- Treponema pallidum antibody comprising a labeling reagent zone consisting of a genetically engineered Treponema pallidum antigen labeled on a matrix capable of being infused by capillary action and a detection zone wherein the Treponema pallidum antigen is immobilized on the matrix. An immunoassay method comprising adding an analyte to an analyzer and detecting anti- Treponema pallidum antibody in the analyte bound to the detection zone. 2. A developing solution zone containing a substrate in a matrix capable of being infused by capillary action, a labeling reagent zone comprising an enzyme-labeled genetically engineered treponema pallidum treponema antigen, and a detection zone wherein treponema pallidum antigen is immobilized on the matrix. Using the anti-syphilis treponemal antibody immunoanalyzer provided with a sample spotting zone and a developing solution absorption zone, after depositing the sample solution on the sample spotting zone, the developing solution is supplied to the developing solution zone. Then, the activity of the enzyme immobilized in the detection zone in an amount depending on the anti- Treponema pallidum antibody in the sample liquid is detected by the substrate, and the immunoassay method is performed. 3. An immunoassay device is applied to a matrix and a developing solution zone, a substrate zone, a sample spotting zone, a labeling reagent zone comprising an enzyme-labeled gene-modified syphilis treponemal antigen, and a syphilis treponemal antigen are immobilized on the matrix. The method according to claim 2, wherein the analyzer is provided with the detection zone and the developing solution absorption zone. 4. The method according to claim 2 or 3, wherein an analyzer provided with a sample spotting zone on the labeling reagent zone is used. 5. The method according to claim 4, wherein the labeling reagent zone comprises a water-absorbing pad containing an enzyme-labeled recombinant treponema pallidum antigen labeled with enzyme. 6. A developing solution zone in a matrix that can be infused by capillary action, and a labeling reagent zone comprising a genetically engineered Treponema pallidum antigen labeled with a radioisotope, latex, metal colloid particles, fluorescent particles or colored particles. Using an anti-syphilis treponemal antibody immunoanalyzer equipped with a detection zone in which the T. syphilis treponemal antigen is immobilized on the matrix, a sample spotting zone, and a developing solution absorption zone, the sample liquid is spotted onto the sample spotting zone. After that, the developing solution is supplied to the developing solution zone, and the amount of the marker immobilized in the detection zone in an amount depending on the anti- Treponema pallidum antibody in the sample solution is detected to perform the immunoassay method. . 7. A radioisotope, a latex, a metal colloid particle, which is contained in a matrix infusible by a capillary action.
A developing solution zone for adding a developing solution containing genetically modified treponema pallidum antigen and a sample labeled with fluorescent particles or colored particles, a detection zone in which the syphilis treponemal antigen is immobilized on a matrix, and a developing solution absorption zone. Using the provided anti-syphilis treponemal antibody immunoassay device, the labeled genetically engineered syphilis treponemal antigen and the sample solution are added to the sample developing solution zone, depending on the anti-syphilis treponemal antibody in the sample solution. An immunoassay method, which comprises detecting an amount of a label immobilized on the detection zone. 8. The immunoassay method according to claim 1, wherein the Treponema pallidum antigen in the detection zone is a recombinant Treponema pallidum antigen. 9. A recombinant syphilis treponemal antigen is TP15 having a molecular weight of 15 kDa of the syphilis treponemal surface antigen.
Antigen, TP17 antigen with a molecular weight of 17 kDa, molecular weight of 42 k
9. The immunoassay method according to claim 1, which is at least one antigen selected from Da TP42 antigen, TP47 antigen having a molecular weight of 47 kDa, and a fusion TP antigen in which two or more of these TP antigens are fused. 10. An immunoassay apparatus for an anti- Treponema pallidum antibody, comprising a labeling reagent zone comprising a labeled recombinant Treponemal syphilis antigen in a matrix capable of being infused by capillary action. 11. A developing solution zone containing a substrate in a matrix, a labeling reagent zone comprising an enzyme-labeled genetically engineered recombinant syphilis treponemal antigen, a detection zone in which the syphilis treponemal antigen is immobilized on the matrix, and a sample spotting zone. The analyzer according to claim 10, further comprising a developing solution absorption zone. 12. A matrix containing a developing solution zone, a substrate zone, a sample spotting zone, and a labeling reagent zone comprising an enzyme-labeled genetically engineered Treponema pallidum antigen.
11. The analyzer according to claim 10, which comprises a detection zone in which the Treponema pallidum antigen is immobilized on a matrix and a developing solution absorption zone. 13. The analyzer according to claim 11, wherein a sample spotting zone is provided on the labeling reagent zone of the matrix. 14. The analyzer according to claim 13, wherein the labeling reagent zone comprises a water-absorbing pad containing an enzyme-labeled recombinant Treponema syphilis antigen labeled on the matrix. 15. A developing solution zone on a matrix, a labeling reagent zone comprising a genetically engineered syphilis treponemal antigen labeled with a radioisotope, latex, metal colloid particles, fluorescent particles or colored particles, and a syphilis treponemal antigen on a matrix. 11. The analyzer according to claim 10, comprising a fixed detection zone, a sample spotting zone, and a developing solution absorption zone. 16. The reagent zone contains genetically engineered Treponema pallidum antigen labeled with radioisotope, latex, metal colloid particles, fluorescent particles or colored particles,
The analyzer according to claim 15, wherein a reagent zone composed of a water absorbent pad to which a sample is added is additionally provided on the matrix, and a sample spotting zone is provided on this reagent zone. 17. A developing solution zone for adding a developing solution containing a genetically engineered Treponema pallidum antigen labeled with radioactive isotope, latex, metal colloid particles, fluorescent particles or colored particles to a matrix and a syphilis. 11. The analyzer according to claim 10, which comprises a detection zone in which the treponemal antigen is immobilized on a matrix and a developing solution absorption zone. 18. The analyzer according to claim 10, wherein the Treponema pallidum antigen in the detection zone is a recombinant Treponema pallidum antigen. 19. A recombinant syphilis treponemal antigen is TP1 having a molecular weight of 15 kDa of the syphilis treponemal surface antigen.
5 antigens, TP17 antigen with a molecular weight of 17 kDa, molecular weight 42
19. The analyzer according to claim 10, which is at least one antigen selected from a TP42 antigen having a kDa, a TP47 antigen having a molecular weight of 47 kDa, and a fused TP antigen in which two or more of these TP antigens are fused.