JP2631796B2 - Diagnostic test slide and manufacturing method thereof - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a diagnostic test slide capable of efficiently measuring a trace substance in a body fluid by utilizing an immunochemical reaction, and a simple production method thereof.

[0002]

2. Description of the Related Art A method for measuring a trace substance in a body fluid with high sensitivity by utilizing the specificity of an immune reaction is particularly useful in the field of clinical examination. This measurement method generally includes one immunoreactive substance (ie, an antigen or an antibody) supported on polystyrene latex particles and another immunoreactive substance present in a sample and corresponding to the immunoreactive substance. It utilizes an antigen-antibody reaction with one immunoreactive substance (that is, an antibody or an antigen). The aggregate of latex particles generated by the antigen-antibody reaction is observed with the naked eye, or turbidity due to the aggregate is observed. By measuring the degree with a spectrometer, a trace substance contained in the specimen is measured. Among them, a method of detecting and / or measuring a trace substance in a specimen by observing the state of aggregation of latex particles after mixing and leaving the latex reagent and the specimen on a slide-type test plate, the reagent is inexpensive. Moreover, since it can be rapidly detected and / or measured, it is widely used in immunoserum tests and the like.

However, performing the immunoreaction by dropping and mixing both the latex reagent and the sample on the slide each time the test is performed not only complicates the operation but also makes it possible to accurately measure a fixed amount of each reagent. However, there is a drawback that it is not easy to obtain an accurate inspection result because it is difficult to drop the liquid on the surface.

As means for overcoming these drawbacks, for example, Japanese Unexamined Patent Application Publication No. Sho 59-100863 describes a diagnostic test slide to which a latex reagent has been applied in advance and a method for producing the slide. For this purpose, a specially processed sheet is required, and further, freeze-drying must be performed to fix the latex reagent applied on the sheet, so that the manufacturing process is complicated and the cost is high. was there.

Japanese Patent Application Laid-Open No. 2-173568 discloses that in order to stabilize and re-dissolve a latex reagent, a water-soluble solid polyhydroxy sugar or sugar alcohol is added and dried, and a sample is prepared at the time of use. Is described. However, even in this method, since the moisture content of the dried latex is high, there is a disadvantage that the latex reagent layer is easily tacky during slide storage. Further, when stored in such a dry state, there are drawbacks such as insolubilization upon re-dissolution depending on the type of the antibody used for sensitization, and a decrease in antibody activity, whereby the original performance of the kit cannot be exhibited. In addition, special attention had to be paid to storage conditions before use in clinical trials.

[0006]

The present inventor has studied various conditions for fixing a latex reagent on a slide in order to improve these drawbacks. It has been found that if a reagent is applied on the slide surface and air-dried, a dry test latex layer can be produced without stickiness and a diagnostic test slide having excellent resolubility and stability can be produced. In addition, the present inventor has also found that by further adding a water-soluble natural compound to the water-soluble synthetic polymer compound, a diagnostic test slide with even more excellent stability can be produced. The present invention is based on such findings.

[0007]

SUMMARY OF THE INVENTION Accordingly, the present invention provides
(A) latex particles sensitized with an immunoreactive substance, (B) a water-soluble synthetic polymer compound having a molecular weight of 1,000 to 300,000, and (C) a water-soluble natural compound, The present invention relates to a diagnostic test slide characterized in that a reagent layer containing a compound (B): a water-soluble natural compound (C) at a weight ratio of 1: 1 to 1:15 is carried on a slide surface. Further, the present invention relates to (A) latex particles sensitized with an immunoreactive substance, (B) a water-soluble synthetic polymer compound having a molecular weight of 1,000 to 300,000, and (C) a weight of a water-soluble natural compound. Ratio [the water-soluble synthetic polymer compound (B): the water-soluble natural compound (C)] 1: 1
The present invention relates to a method for producing a diagnostic test slide, which is applied on a slide surface in the presence of １ ： 1: 15 and air-dried. That is, the diagnostic test slide of the present invention comprises, on the slide surface, a latex particle sensitized with an immunoreactive substance (hereinafter, sometimes referred to as component (A)), and a water-soluble synthetic polymer compound [hereinafter, referred to as Component (B)] and a water-soluble natural compound (hereinafter sometimes referred to as component (C)) in the presence and drying. Diagnostic test slides can be provided without depending on the drying method.

Hereinafter, the method of the present invention will be described in detail. The latex particles used as a raw material of the component (A) used in the method of the present invention are not different from the latex particles used in the preparation of conventional diagnostic test slides, and may be any of natural rubber, synthetic rubber and / or plastic. It may be. Further, the surface may be modified with an amino group, a carboxyl group, an amide group, a hydrazine group and / or a hydroxyl group. Many latex particles are commercially available, and it is preferable to use these commercially available latex particles in the method of the present invention. The diameter of the latex particles is preferably 0.01-5 μm, more preferably 0.1-1 μm.

[0009] The immunoreactive substance used for preparing the component (A) is an antigen or an antibody. Examples of the antigen include drugs, metabolites, microorganisms such as fungi (eg, incomplete fungi) and their derivatives, and cells (eg, tumor cells). The antibody is an antibody against these antigens, may be either a polyclonal antibody or a monoclonal antibody, and may be IgG or IgM.

The component (A) used in the method of the present invention can be prepared by sensitizing latex particles with an immunoreactive substance by a conventional method. Sensitization is generally pH 6
Performed in ~ 9 buffer solutions. Preferably, a GBS buffer solution is used. This buffer contains 0.14 in addition to glycine.
Molar sodium chloride and a minimum amount (eg, 0.005
Mol sodium hydroxide) (hereinafter referred to as glycine buffer). Sensitization is carried out by using 0.01 to 30% by weight of latex particles (preferably 0.05 to 1%).
0% by weight) and 0.01 of the immunoreactive substance.
Or approximately 30% by weight (preferably 0.05 to 10% by weight) of a buffer solution, or 0.01 to 30% by weight (preferably 0.05 to 10% by weight) of latex particles.
Wt%) of the immunoreactive substance in an aqueous suspension containing 0.01% by weight.
It can be carried out by adding and mixing in an amount corresponding to ３０30% by weight (preferably 0.05 to 10% by weight). The mixing temperature is 0-60 ° C (preferably 4-50 ° C).
° C). Mixing is performed for 0.5 to 24 hours (preferably 1 to 24 hours).
12 hours). Centrifuge the sensitized latex particles. The separated sensitized latex particles are preferably suspended again in a buffer and centrifuged. By performing the purification treatment consisting of resuspension in a buffer solution and centrifugation two to four times, it is possible to obtain sensitized latex particles of high purity.

The water-soluble synthetic polymer compound of the component (B) is
There is no particular limitation as long as it meets the purpose of the method of the present invention. As the component (B), for example, polyitaconic acid,
Mention may be made of polymethacrylic acid, polyethyleneimine, polyacrylamide, polyamide, polyacrylic acid, polypeptide and polyvinylpyrrolidone. Preferably, polyvinylpyrrolidone is used. The molecular weight of component (B), 1, 000～300,000, good Mashiku 3,
000-100,000, more preferably 5,000-
50,000. When the molecular weight is less than 1,000, the dried latex becomes sticky, and when the molecular weight exceeds 300,000, insolubilization is apt to occur at the time of re-dissolution, which is not preferable. Examples of the water-soluble natural compound as the component (C) include α, β or γ cyclodextrin, and furthermore, a derivative thereof, and a methylated derivative is preferable.

[0012] Component (A) is suspended in a buffer, and components (B) and (C) are dissolved in the buffer, and then the suspensions and solutions are separately or mixed and then combined. Can be applied on a slide. When the component (A), the component (B) and the component (C) are mixed, not only each component is suspended or dissolved in a buffer solution and then mixed, but also the components (B) and (C) are mixed. The component (A) can be added to and mixed with the above solution. Further, the component (B) and the component (C) can be added to the suspension of the component (A) and mixed.
By any of the mixing methods, the component (A) in the mixed solution,
The concentration of each of the component (B) and the component (C) is preferably 0.01 to 30% by weight, and more preferably 0.05 to 30% by weight.
10% by weight. The ratio of component (B) to component (C) is
It is 1: 1 to 1:15, preferably 1: 1 to 1:10.

The buffer containing component (A), component (B) and component (C) can be applied on a slide in an amount of 5 to 100 μl. After the application, it is naturally dried at 15 to 80 ° C, preferably 20 to 60 ° C, for 0.1 to 72 hours, preferably 0.5 to 48 hours. When the suspension of the component (A) and the solution of the component (B) and the solution of the component (C) are sequentially and separately applied, the component (A) is added (preferably 0.01 to 30% by weight, more preferably 5 to 100 μl of the suspension containing (in an amount of 0.05 to 10% by weight) are applied to the slides and subsequently the solution of component (B) and component (C) is added while the suspension does not dry. 5-100 μl of the suspension containing (preferably in an amount of 0.01 to 30% by weight, more preferably 0.05 to 10% by weight) is spread on a slide, and
It air-drys at 0.1 degreeC, Preferably it is 20-60 degreeC, for 0.1-72 hours, Preferably it is 0.5-48 hours. Alternatively, the solution containing the component (B) and the component (C) may be applied on a slide first, and then the suspension containing the component (A) may be applied on the slide.

The method of application is not particularly limited, and examples include spontaneous dropping and application using a stick. The drying method is natural drying. Here, the natural drying means ordinary drying at the above-mentioned temperature, and also includes ventilation and vacuum drying, and does not include freeze-drying or the like. Thus, a reagent layer containing the component (A), the component (B), and the component (C) is formed on the slide surface. This reagent layer does not need to be formed on the entire surface of the slide, and can be formed in a circular shape or an arbitrary shape, preferably at the center of the slide surface.

The material of the slide is not particularly limited, and examples thereof include glass, paper and plastic. It is preferable to use a slide made of paper or plastic. This is because these can be incinerated, and therefore, infection with a virus or the like in an examination room or the like can be avoided.

[0016] 10 to 100 µl of the sample liquid is dropped on the coated portion of the diagnostic test slide prepared by the method of the present invention, and after 1 to 5 minutes from the well mixing, the state of aggregation is observed.
The diagnostic test slide prepared by the method of the present invention comprises:
It can be used for detection and measurement of trace substances (eg, drugs, metabolites, microorganisms and their derivatives or cells) contained in bodily fluids, for example, serum, plasma, cerebrospinal fluid, urine, vaginal contents, and the like. .

[0017]

EXAMPLES The present invention will be described below in more detail with reference to examples, but these examples do not limit the scope of the present invention. Reference Example 1 (1) Anti-Candida-derived mannan antigen monoclonal antibody
Preparation of body A monoclonal antibody was prepared according to the method described in JP-A-59-187794. That is, cultured Candida tropicalis (Candida tropical)
alis: ATCC750 strain) PBS containing 2 × 10 ⁷ cells / ml was mixed with Freund's complete adjuvant, and 0.2 ml of the obtained mixture was mixed with BALB / C mice (8 weeks old,
(Female) was administered intraperitoneally. After repeating the second to third immunizations, 100 days later, the spleens of the immunized mice were excised and the cells were collected. The spleen cells of this immunized mouse were mixed with rat myeloma cells Y3-Ag1.2.3, and cell fusion was performed at 37 ° C. in the presence of a 45% polyethylene glycol 4000 solution. After the fusion, the cells were washed, HAT medium was added at 37 ° C to suspend the cells well, dispensed into a cell culture plate, and culture was started at 37 ° C. Two weeks after the start of the culture, the hybridoma growth was observed and
Cloning of hybridomas in the wells in which the production of the desired antibody was observed in the culture supernatant by Method A was performed by the limiting dilution method. After the cloning, a stable clone having a high antibody secreting ability, excellent proliferative property and being selected was selected and cloned again in the same manner as above to establish an antibody-producing hybridoma. This hybridoma was confirmed by the EIA method to produce an antibody that specifically reacts with a Candida-derived mannan antigen. This hybridoma is
The suspension was suspended at 1 × 10 ⁵ cells / ml in RPMI1640 medium prepared for hybridoma culture containing 0% fetal bovine serum, and the obtained suspension was placed in a tissue culture flask at 2 × 10 ⁵ cells / ml.
The mixture was dispensed in 5 ml portions and cultured at 37 ° C. On the fourth day, the culture supernatant was collected and the antibody fraction was separated by ammonium sulfate salting out, and then purified by an antibody affinity column to obtain an anti-Candida-derived mannan antigen monoclonal antibody, ie, IgM.

(2) For detecting Candida-derived mannan antigen
Manufacture of slides A latex reagent sensitizing anti-Candida mannan antigen monoclonal antibody was prepared by the following method. That is, a glycine buffer solution (pH 8.4) containing the monoclonal antibody obtained in Reference Example 1 (1) (antibody concentration 1 mg / m2)
l) 5 ml of glycine buffer (pH 8.4) containing polystyrene latex particles (average particle size: 0.525 μm; manufactured by Sekisui Chemical Co., Ltd.) (solid content concentration: 1% by weight) was left at 25 ° C. for 3 hours. After that, centrifugation (15,000 rpm; 10 minutes) was performed under cooling at 4 ° C. Next, the separated monoclonal antibody-sensitized latex particles were suspended in a glycine buffer (pH 8.4), and
Centrifugation (15,000 rpm; 10 minutes) under cooling at 0 ° C. Subsequently, the purified monoclonal antibody-sensitized latex particles were added to polyvinylpyrrolidone (molecular weight 4).
Glycine buffer containing 2.5% by weight (pH
8.4) In 10 ml, the sensitized latex particle concentration is 0.5
The suspension was re-suspended to a weight% to obtain an anti-Candida-derived mannan antigen monoclonal antibody-sensitized latex reagent.
Transfer the obtained latex reagent to a slide test plate (made of paper)
Was applied in an amount of 20 ± 2 μl and dried naturally at 20 ° C. for 12 hours to obtain a slide for detecting a Candida-derived mannan antigen (diagnostic test). The dried slide did not stick. The finish was well adhered to the slide.

(3) for detecting Candida-derived mannan antigen
Immediately after the slide test example was prepared, Candida tropicalis (Candid tropicalis) was placed on the surface of the Candida fungus-derived mannan antigen detection slide.
a tropicalis: ATCC750 strain-derived mannan antigen at various concentrations (pH 8.
4) 20 μl was added dropwise, mixed well with the latex reagent, and observed 5 minutes later. As a result, an agglutination reaction was observed from a concentration of 10 ng / ml or more of Candida-derived mannan antigen. The resolubility in this system was good.

Reference Example 2 (1) Preparation of a slide for detecting mannan antigen derived from Candida
The monoclonal antibody (Ig) used in Reference Example 1 (2)
M) -containing glycine buffer (pH 8.4) (concentration 1 mg /
5 ml) was added to 5 ml of a glycine buffer (pH 8.4) (solid content concentration 1% by weight) containing polystyrene latex particles (average particle size 0.550 μm; manufactured by Sekisui Chemical Co., Ltd.),
After standing at 4 ° C. for 12 hours, centrifugation (15,000 rpm; 10 minutes) was performed while cooling at 4 ° C. next,
The isolated anti-Candida-derived mannan antigen-antibody-sensitized latex particles were suspended in glycine buffer (pH 8.4),
Centrifugation (15,000 rpm; 10 minutes) under cooling at 0 ° C. Subsequently, the purified monoclonal antibody-sensitized latex particles were treated with polyvinylpyrrolidone (molecular weight 10,10).
Glycine buffer containing 1.25% by weight (pH
8.4) The suspension was resuspended in 10 ml to adjust the concentration of the sensitized latex particles to 0.5% by weight to obtain a latex sensitized latex reagent for a mannan antigen monoclonal antibody derived from anti-Candida bacteria. The obtained latex reagent was accurately applied to a slide-type test plate (made of paper) in an amount of 20 ± 2 μl.
The slide was dried naturally for a period of time to detect a Candida-derived mannan antigen (diagnostic test). The finish was well adhered to the slide.

(2) For detection of Candida-derived mannan antigen
Test Examples of Slides Using a slide for detecting a Candida bacillus-derived mannan antigen immediately after obtained in Reference Example 2 (1), detection of a Candida bacillus-derived mannan antigen in vaginal contents of 20 Candida vaginitis patients was attempted. That is, the contents of the vagina are glycine buffer (pH 8.4)
To prepare a sample solution. 20 μl of these sample solutions were dropped on the surface of the mannan antigen detection slide, mixed well with the latex reagent, and observed 5 minutes later. As a result, all 20 cases showed agglutination in the sample solutions of Candida vaginitis patients. On the other hand, a control solution [glycine buffer (p
H8.4) alone], no aggregation reaction was observed. Therefore, its usefulness in clinical tests was recognized. The resolubility in this system was good.

Reference Example 3 (1) Sampling of Immunosuppressive Substance from Ascites of Colorectal Cancer Patient An immunosuppressive substance (IS) was collected according to the method described in JP-A-56-145297. That is, 25 ml of ascites collected from a colorectal cancer patient was subjected to 10,000 centrifugation by a cooling centrifuge.
The supernatant was collected by centrifugation at G for 30 minutes. This supernatant was mixed with an amphoteric carrier solution (using an ampholine manufactured by LKB) having a pH of 2.5 to 4.0 and 4.0 to 6.0, and then mixed with an amphoteric carrier solution having a pH of 2.5 to 6.0. Using a Sephadex G-75 gel (manufactured by Pharmacia) as a support, electrophoresis was performed by applying electricity at 8 watts for 40 hours using a flat plate isoelectric focusing apparatus manufactured by LKB. Final voltage 120
Electrophoresis was terminated at 0V. After electrophoresis, pH 2.6 ~
The fraction of 3.6 was taken out together with the Sephadex gel support, the fraction was washed out of the Sephadex gel with pure water, and then dialyzed with running water to remove ampholine and the like coexisting in the above fraction. The obtained fraction was freeze-dried to obtain an IS substance.

(2) Preparation of a slide for detecting an immunosuppressive substance The goat was immunized with the IS substance obtained in Reference Example 3 (1), an antiserum was collected, and fractionated and purified to obtain an anti-IS polyclonal antibody. That is, IgG was obtained. A glycine buffer solution (pH 8.4) in which this polyclonal antibody was dissolved (at a concentration of 1 m
g / ml) was added to 5 ml of a glycine buffer solution (pH 8.4) (solid content concentration: 1% by weight) containing polystyrene latex particles (average particle size: 0.485 μm; manufactured by Sekisui Chemical Co., Ltd.) and left at 37 ° C. for 3 hours. Then, centrifuge at 4 ° C (1
(5,000 rpm; 10 minutes). Next, the separated polyclonal-sensitized latex particles are suspended in a glycine buffer (pH 8.4) and centrifuged at 4 ° C. (15,
000 rpm; 10 minutes). The purified polyclonal antibody-sensitized latex particles were added to 10 ml of a glycine buffer (pH 8.4) containing 1.25% by weight of polyvinylpyrrolidone (molecular weight 10,000) so that the concentration of the sensitized latex particles was 0.5% by weight. Re-suspend in anti-IS
A polyclonal antibody-sensitized latex reagent was obtained. The obtained latex reagent is placed on a slide-type test plate (made of paper) for 30 minutes.
The mixture was applied accurately in an amount of ± 3 μl, and naturally dried at 20 ° C. for 12 hours to obtain a slide for IS detection (diagnostic reagent). No stickiness was observed on the slides. The finish was good and the adhesion to the slide was good.

(3) Test of IS Detection Slide The surface of the IS detection slide immediately after prepared in Reference Example 3 (2) contains the IS prepared in Reference Example 3 (1) at various concentrations. Glycine buffer (pH 8.4) 30 ±
3 μl was added dropwise and mixed well with the latex reagent. The redispersibility was good. When observed 5 minutes later, IS
An agglutination reaction was observed at a concentration of 25 ng / ml or more of the antigen.

Reference Example 4 (1) Anti-Aspergillus-derived galactomannan antigen
Preparation of Noclonal Antibody Monoclonal antibody was prepared according to the method described in JP-A-59-187795. Aspergillus
Fumigatus (Aspergillus fumiga)
(tus: IAM3006 strain) was filtered through a membrane filter (0.45 μm), and the filtrate was dialyzed at 4 ° C. for 4 days and lyophilized to obtain a crude antigen powder. A mixture of a solution prepared by dissolving this antigen in PBS at a concentration of 100 μg / ml and complete Freund's adjuvant 0.2 m
The l CDF ₁ mice; administered subcutaneously (8 weeks old female) further repeated twice immunized with 14 day intervals, the tiles organs of immunized mice were removed after 73 days, the cells were harvested. The spleen cells of the immunized mouse were mixed with mouse myeloma cells P3-X63-Ag8 cultured in vitro in advance, and cell fusion was performed at 37 ° C. in the presence of a 45% polyethylene glycol 4000 solution. . After the fusion, wash the cells,
Dulbecco containing 10% fetal calf serum
o's) The cells were suspended in a MEM medium, dispensed into 24-well tissue culture plates, and culture was started. Thereafter, the medium was replaced with a HAT medium for hybridoma selection, and hybridomas having a growth ability were selected. Two weeks after the start of the culture, the growth of the hybridoma was observed, and the cloning of the hybridoma in the well in which the production of the desired antibody was observed in the culture supernatant by the EIA method was performed by the limiting dilution method. After the cloning, a stable clone having a high antibody secreting ability, excellent proliferative property and being selected was selected and cloned again in the same manner as above to establish an antibody-producing hybridoma. It was confirmed by EIA that this hybridoma produced an antibody that specifically reacted with galactomannan derived from Aspergillus. This hybridoma was added to an RPMI1640 medium prepared for hybridoma culture containing 20% fetal bovine serum at 1 × 10 6
^The cells were suspended at a concentration of ⁵ cells / ml, and the suspension was dispensed into tissue culture flasks at 25 ml each and cultured at 37 ° C. On the fourth day, the culture supernatant was collected, the antibody fraction was separated by ammonium sulfate salting out, and then purified by an antibody affinity column to obtain an anti-Aspergillus-derived galactomannan antigen monoclonal antibody, that is, IgM.

(2) Galactomann from Aspergillus
Manufacture of a slide for detecting a Nan antigen 5 ml of a glycine buffer solution (pH 8.4) (concentration 2 mg / ml) of the monoclonal antibody obtained in Reference Example 4 (1).
With polystyrene latex particles (average particle size 0.525μ)
m) containing glycine buffer (pH 8.4) (solids concentration 1)
% By weight), left at 37 ° C. for 3 hours,
Centrifugation (15,000 rpm; 10 ° C.) under cooling at ４4 ° C.
Min). Next, the separated monoclonal antibody-sensitized latex particles are suspended in a glycine buffer (pH 8.4), and centrifuged (15,000 rpm) under cooling at 2 to 4 ° C.
m; 10 minutes). The purified monoclonal antibody-sensitized latex particles were mixed with a glycine buffer (p.p.) containing 2.5% by weight of polyvinylpyrrolidone (molecular weight: 40,000).
H8.4) The sensitized latex particle concentration was 0.1 ml in 10 ml.
The suspension was re-suspended to 5% by weight to obtain an anti-Aspergillus-derived galactomannan antigen monoclonal antibody-sensitized latex reagent. The obtained latex reagent was accurately applied to a slide-type test plate (paper) in an amount of 20 ± 2 μl, and dried at 25 ° C. for 2 hours in a blast dryer to obtain a slide for detecting galactomannan antigen derived from Aspergillus. Was. No stickiness of the slide was observed. The appearance was good and the adhesion to the slide was good.

(3) Galactomann from Aspergillus
Test Example of Slide for Detecting Nan Antigen Aspergillus fumigatus (Aspergillus fumigatus) was placed on the surface of the slide for detecting galactomannan antigen derived from Aspergillus immediately after the slide obtained in Reference Example 4 (2).
umigatus: IAM3006 strain) containing various concentrations of galactomannan antigen (pH 8.
4) When 40 μl was dropped, mixed well with the latex reagent, and observed 5 minutes later, an agglutination reaction was observed from a concentration of 30 ng / ml or more of the galactomannan antigen derived from Aspergillus. The resolubility in this system was good.

Reference Example 5 (1) Preparation of a slide for detecting an anti-Candida fungus antibody Candida albicans
ants: ATCC752 strain) glycine buffer (pH 8.4) containing the antigen obtained by disrupting the cells of the cells.
Glycine buffer (pH 8.4) containing 5 ml of polystyrene latex particles (average particle diameter 0.525 μm; manufactured by Sekisui Chemical Co., Ltd.) (concentration: 1 mg / ml) (solid concentration: 1% by weight)
5 ml, left at 25 ° C. for 3 hours, and then centrifuged (15,000 rpm; 10 minutes) under cooling at 4 ° C. Next, the separated Candida antigen-sensitized latex particles were suspended in a glycine buffer solution (pH 8.4) and centrifuged (15,000 rpm; 10 minutes) under cooling at 4 ° C. 1. Purifying the antigen-sensitized latex particles derived from Candida fungi with polyvinylpyrrolidone (molecular weight 40,000)
10 ml of a glycine buffer (pH 8.4) containing 5% by weight
Was re-suspended so that the concentration of the sensitized latex particles became 0.5% by weight to obtain a Candida fungus antigen-sensitized latex reagent. The obtained latex reagent was accurately applied to a slide-type test plate (made of paper) in an amount of 20 ± 2 μl.
The slide was allowed to dry naturally for a period of time to obtain a slide for detecting an anti-Candida antibody (diagnostic test). The dried slide did not stick. The appearance was good and the adhesion was good.

(2) Anti-Candida Antibody Detection Slide
Test Example A dilution series of the serum of a candidosis patient or a healthy person was dropped onto the surface of the slide for detecting an anti-candida fungus antibody immediately after the slide obtained in Reference Example 5 (1), and mixed well with a latex reagent for 5 minutes. Observation of aggregation later, compared to healthy people,
Significantly higher titers of 1: 8 or more were shown. The solubility in this system was also good.

Example 1 An anti-Candida-derived mannan antigen monoclonal antibody-sensitized latex reagent was prepared by the following method. That is, a glycine buffer solution (pH 8.4) containing the monoclonal antibody (IgM) obtained in Reference Example 1 (1) (antibody concentration 1)
glycine buffer (pH 8.4) containing polystyrene latex particles (average particle size 0.525 μm; manufactured by Sekisui Chemical Co., Ltd.) (solid concentration 1% by weight).
5 ml, left at 25 ° C. for 3 hours, and then centrifuged (15,000 rpm; 10 minutes) under cooling at 4 ° C. Next, the separated monoclonal antibody-sensitized latex particles are suspended in a glycine buffer (pH 8.4), and the suspension is centrifuged (15,000 rpm; 4 ° C.) under cooling.
10 minutes). Subsequently, the purified monoclonal antibody-sensitized latex particles were treated with a glycine buffer (pH 8.4) containing 2.5% by weight of polyvinylpyrrolidone (molecular weight 40,000) and 5.0% by weight of α-cyclodextrin.
In 10 ml, the sensitized latex particles were resuspended to a concentration of 0.5% by weight to obtain a latex sensitized latex reagent for a mannan antigen monoclonal antibody derived from anti-Candida bacteria. The obtained latex reagent was added to a slide-type test plate (made of paper) for 10 ±.
Accurately apply in an amount of 1 μl and air dry at 20 ° C. for 12 hours to detect Candida-derived mannan antigen (diagnostic test)
Got slides. The dried slide did not stick. The appearance was good and the adhesion was good. On the surface of the slide for detecting Candida fungus-derived mannan antigen, Candida tropicalis: A
20 μl of a buffer solution (pH 8.4) containing various concentrations of mannan antigen derived from TCC750 strain) was added dropwise, mixed well with a latex reagent, and observed 5 minutes later. Aggregation was observed from the concentration. The resolubility in this system was good.

Example 2 In place of α-dextrin in Example 1, di-o-
A slide for antigen detection was prepared under exactly the same conditions as in Example 1 except that methyl or tri-o-methyl β-cyclodextrin was used. The dried slide did not stick. The obtained latex reagent was accurately applied to a slide-type test plate (made of paper) in an amount of 10 ± 1 μl,
For 12 hours to obtain a Candida-derived mannan antigen detection (diagnosis test) slide. The dried slide did not stick. Both appearance and adhesion were good. On the surface of the slide for detecting a Candida fungus-derived mannan antigen, Candida tropicalis (Candida tropical) is placed.
alis: ATCC750 strain) containing 20 μl of a buffer solution (pH 8.4) containing various concentrations of mannan antigen, mixed well with a latex reagent, and observed 5 minutes later. As a result, 10 ng / ml of Candida-derived mannan antigen was observed. From the above concentrations, an agglutination reaction was observed. The resolubility in this system was good.

Example 3 A latex reagent for sensitizing a monoclonal antibody to a mannan antigen derived from an anti-candida fungus was prepared by the following method. That is, a glycine buffer solution (pH 8.4) containing the monoclonal antibody (IgM) obtained in Reference Example 1 (1) (antibody concentration 1)
glycine buffer (pH 8.4) containing polystyrene latex particles (average particle size 0.525 μm; manufactured by Sekisui Chemical Co., Ltd.) (solid concentration 1% by weight).
5 ml, left at 25 ° C. for 3 hours, and then centrifuged (15,000 rpm; 10 minutes) under cooling at 4 ° C. Next, the separated monoclonal antibody-sensitized latex particles are suspended in a glycine buffer (pH 8.4), and the suspension is centrifuged (15,000 rpm; 4 ° C.) under cooling.
10 minutes). Subsequently, the purified monoclonal antibody-sensitized latex particles were added to a glycine buffer (pH 8.4) containing 2.0% by weight of polyvinylpyrrolidone (molecular weight: 40,000) and 5.0% by weight of α-cyclodextrin.
In 10 ml, the sensitized latex particles were resuspended to a concentration of 0.5% by weight to obtain a latex sensitized latex reagent for a mannan antigen monoclonal antibody derived from anti-Candida bacteria. The obtained latex reagent was added to a slide-type test plate (made of paper) for 10 ±.
After applying in an amount of 1 μl and drying by blowing at 30 ° C. for 1 hour,
Further, the slide was dried under vacuum at 25 ° C. for 1 hour to detect a Candida-derived mannan antigen. The dried slide did not stick. Both appearance and adhesion were good. On the surface of the slide for antigen detection, Candida tropicalis (Candi)
da tropicalis: ATCC750 strain) containing various concentrations of mannan antigen at various concentrations (pH
8.4) 20 μl was added dropwise, mixed well with the latex reagent, and observed 5 minutes later. As a result, an agglutination reaction was observed at a concentration of 10 ng / ml or more of the Candida-derived mannan antigen. The resolubility in this system was good.

Example 4 Goats were immunized with the IS substance obtained in Reference Example 3 (1) to collect antisera, which were further fractionated and purified to obtain anti-IS polyclonal antibodies. Glycine buffer solution (pH 8.4) in which this polyclonal antibody was dissolved (concentration 1 mg / ml) 5
ml of polystyrene latex particles (average particle size 0.48
Glycine buffer (pH 8; 5 μm; manufactured by Sekisui Chemical Co., Ltd.)
4) (5% of solid content concentration 1% by weight)
After standing for 4 hours, centrifugation (15,000 rpm)
m; 10 minutes). Next, the separated polyclonal-sensitized latex particles were added to a glycine buffer solution (pH 8.
4) and centrifuged at 4 ° C. (15,000 rpm)
m; 10 minutes). A purified polyclonal antibody-sensitized latex particle was treated with a glycine buffer (pH 8.4) containing 2.0% by weight of polyvinylpyrrolidone (molecular weight 10,000) and 5.0% by weight of α-cyclodextrin.
In 10 ml, the sensitized latex particles were re-suspended to a concentration of 0.5% by weight to obtain an anti-IS polyclonal antibody-sensitized latex reagent. The obtained latex reagent was accurately applied to a slide-type test plate (made of paper) in an amount of 15 ± 1.5 μl, air-dried at 30 ° C. for 1 hour, and further dried at 25 ° C. for 1 hour.
After vacuum drying for a period of time, a slide for IS detection (diagnostic reagent) was obtained. No stickiness was observed on the slides. Both appearance and adhesion were good. I prepared in Reference Example 3 (1)
Glycine buffer solution (pH 8.
4) 30 μl was dropped and mixed well with the latex reagent. The redispersibility was good. When observed after 5 minutes, an agglutination reaction was observed at a concentration of 20 ng / ml or more of the IS antigen. The solubility in this system was good.

Example 5 The monoclonal antibody (Ig) obtained in Reference Example 4 (1) was used.
M) in glycine buffer solution (pH 8.4) (concentration 2 mg /
5 ml) was added to 5 ml of a glycine buffer (pH 8.4) (solid content concentration 1% by weight) containing polystyrene latex particles (average particle size 0.525 μm; manufactured by Sekisui Chemical Co., Ltd.),
After leaving at 37 ° C. for 3 hours, centrifugation (15,000 rpm; 10 minutes) was performed under cooling at 2 to 4 ° C. next,
The separated monoclonal antibody-sensitized latex particles were suspended in a glycine buffer (pH 8.4) and centrifuged (15,000 rpm; 10 minutes) under cooling at 2 to 4 ° C. 1. Purify the purified monoclonal antibody-sensitized latex particles with polyvinylpyrrolidone (molecular weight 40,000)
Resuspended in 10 ml of a glycine buffer (pH 8.4) containing 0% by weight and 5.0% by weight of α-cyclodextrin so that the concentration of the sensitized latex particles becomes 0.5% by weight;
An anti-Aspergillus-derived galactomannan antigen monoclonal antibody-sensitized latex reagent was obtained. The obtained latex reagent is placed on a slide-type test plate (made of plastic).
The mixture was applied precisely in an amount of ± 1 μl, and dried at 25 ° C. for 2 hours in a blow dryer to obtain a slide for detecting galactomannan antigen derived from Aspergillus. No stickiness of the slide was observed. Both appearance and adhesion were good. Reference Example 4
Aspergillus fumigatus (Aspergillus fumigatus) was placed on the surface of the slide for detecting galactomannan antigen derived from Aspergillus bacterium immediately obtained in (2).
us: IAM3006 strain) containing 20 μl of a buffer solution (pH 8.4) containing galactomannan antigen at various concentrations.
Was added dropwise, mixed well with the latex reagent, and observed 5 minutes later. As a result, an agglutination reaction was observed from a concentration of 10 ng / ml or more of the Aspergillus-derived galactomannan antigen. The resolubility in this system was good.

Reference Example 6 An anti-Candida mannan antigen monoclonal antibody-sensitized latex reagent was prepared by the following method. That is, a glycine buffer solution (pH 8.4) containing the monoclonal antibody (IgM) obtained in Reference Example 1 (1) (antibody concentration 1)
glycine buffer (pH 8.4) containing polystyrene latex particles (average particle size 0.525 μm; manufactured by Sekisui Chemical Co., Ltd.) (solid concentration 1% by weight).
5 ml, left at 25 ° C. for 3 hours, and then centrifuged (15,000 rpm; 10 minutes) under cooling at 4 ° C. Next, the separated monoclonal antibody-sensitized latex particles are suspended in a glycine buffer (pH 8.4), and the suspension is centrifuged (15,000 rpm; 4 ° C.) under cooling.
10 minutes). Subsequently, the purified monoclonal antibody-sensitized latex particles were added to 10 ml of a glycine buffer (pH 8.4) containing 2.5% by weight of polyvinylpyrrolidone (molecular weight: 360,000) so that the concentration of the sensitized latex particles was 0.5% by weight. % To obtain a latex reagent sensitized to a mannan antigen monoclonal antibody derived from anti-Candida. The obtained latex reagent is accurately applied to a slide-type test plate (made of paper) in an amount of 20 ± 2 μl, air-dried at 20 ° C. for 12 hours, and a slide for detecting Candida-derived mannan antigen (diagnosis test) is prepared. Obtained. The dried slide was good without stickiness. The appearance was also good. On the surface of the antigen detection slide, Candida tropicalis (Candida)
buffer containing various concentrations of mannan antigen derived from ida tropicalis (ATCC750 strain).
8.4) 20 μl was dropped, mixed well with the latex reagent, and observed 5 minutes later. As a result, an agglutination reaction was observed at a concentration of 20 ng / ml or more of the Candida-derived mannan antigen. Resolubility in this system was somewhat poor. Blocking was observed.

Reference Example 7 A slide for detecting a Candida-derived mannan antigen was prepared under the same conditions as in Reference Example 6, except that polyvinylpyrrolidone with a molecular weight of 800 was used instead of polyvinylpyrrolidone with a molecular weight of 360,000 in Reference Example 6. The slide was sticky but had good adhesion. The appearance was somewhat bad. The surface condition was somewhat bad. Immediately after preparation, the surface of the slide for antigen detection is labeled with Candida tropicalis.
buffer (pH 8.4) 2 containing various concentrations of mannan antigen derived from R. tropicalis (ATCC750 strain).
0 μl was added dropwise, mixed well with the latex reagent, and observed 5 minutes later.
An agglutination reaction was observed from a concentration of g / ml or more. The resolubility in this system was good.

Reference Example 8 An anti-Candida mannan antigen monoclonal antibody-sensitized latex reagent was prepared by the following method. That is, a glycine buffer solution (pH 8.4) containing the monoclonal antibody obtained in Reference Example 1 (1) (antibody concentration 1 mg / m2)
l) 5 ml of glycine buffer (pH 8.4) containing polystyrene latex particles (average particle size: 0.525 μm; manufactured by Sekisui Chemical Co., Ltd.) (solid content concentration: 1% by weight) was left at 25 ° C. for 3 hours. After that, centrifugation (15,000 rpm; 10 minutes) was performed under cooling at 4 ° C. Next, the separated monoclonal antibody-sensitized latex particles were suspended in a glycine buffer (pH 8.4), and
Centrifugation (15,000 rpm; 10 minutes) under cooling at 0 ° C. Subsequently, the purified monoclonal antibody-sensitized latex particles were added to polyvinylpyrrolidone (molecular weight 4).
(000) 1.4% by weight and α-cyclodextrin 3
Glycine buffer (pH 8.4) containing 0% by weight
The sensitized latex particles were re-suspended so as to have a concentration of 0.75% by weight to obtain a latex reagent for sensitizing a mannan antigen monoclonal antibody derived from an anti-Candida bacterium. The obtained latex reagent was placed on a slide-type test plate (made of paper) at 10 ± 1.
The solution was applied precisely in an amount of 0 μl, dried by blowing at 30 ° C. for 1 hour, further dried by blowing at 25 ° C. for 1 hour, and further dried at 25 ° C.
After drying under vacuum for a period of time, a slide for detecting a Candida-derived mannan antigen (diagnostic test) was obtained. The dried slide was not sticky, but cracked entirely and peeled off partly from the substrate.

Reference Example 9 An anti-Candida-derived mannan antigen monoclonal antibody-sensitized latex reagent was prepared by the following method. That is, a glycine buffer solution (pH 8.4) containing the monoclonal antibody (IgM) obtained in Reference Example 1 (1) (antibody concentration 1)
glycine buffer (pH 8.4) containing polystyrene latex particles (average particle size 0.525 μm; manufactured by Sekisui Chemical Co., Ltd.) (solid concentration 1% by weight).
5 ml, left at 25 ° C. for 3 hours, and then centrifuged (15,000 rpm; 10 minutes) under cooling at 4 ° C. Next, the separated monoclonal antibody-sensitized latex particles are suspended in a glycine buffer (pH 8.4), and the suspension is centrifuged (15,000 rpm; 4 ° C.) under cooling.
10 minutes). Subsequently, the purified monoclonal antibody-sensitized latex particles were added to 10 ml of a glycine buffer (pH 8.4) containing 5% by weight of α-cyclodextrin,
The sensitized latex particles were resuspended to a concentration of 0.5% by weight to obtain an anti-candida-derived mannan antigen monoclonal antibody sensitized latex reagent. The obtained latex reagent is accurately applied to a slide-type test plate (made of paper) in an amount of 20 ± 2 μl, air-dried at 20 ° C. for 12 hours, and a slide for detecting Candida-derived mannan antigen (diagnosis test) is prepared. Obtained. Dry slides have slightly poor adhesion to the test plate,
Cracks were observed. On the surface of the antigen detection slide,
Candida tropical
20 μl of a buffer solution (pH 8.4) containing various concentrations of mannan antigen derived from C. calis: ATCC750 strain), mixed well with a latex reagent, and observed 5 minutes later.
From the above concentrations, an agglutination reaction was observed. Blocking was observed in this system.

Evaluation of Physical Properties The storage stability of each of the diagnostic test slides obtained in Examples 1 to 5 and Reference Examples 1 and 6 to 9 was examined. Each diagnostic test slide was packaged with a multilayer coating film and stored at 40 ° C. for one month. The resolubility of each slide was examined. Furthermore, each concentration of the antigen used in each Example was 10
ng / ml) The aggregation at 20 ng / ml or 50 ng / ml was examined, and the results in Tables 1 to 4 were obtained. The properties (appearance, surface condition, and adhesiveness to the slide substrate) are shown immediately after the preparation of each slide. Here, “appearance” is the quality of the finished appearance, and “surface state” is the dry state of the slide surface. Further, the sensitivity (aggregation ability) in Tables 1 to 4 was evaluated on the basis of the following four steps from observation of the latex aggregation image. FIG. 1 shows a schematic image of a typical example of each of these four stages.

[0040]

[Table 1]

[0041]

[Table 2]

[0042]

[Table 3]

[0043]

[Table 4]

[0044]

According to the method of the present invention, a diagnostic test slide can be produced by a simple operation in which an immunoreactive substance-sensitized latex reagent is applied in the presence of a water-soluble synthetic high-molecular compound and air-dried. The diagnostic test slide obtained in this way has a non-sticky coating surface and is excellent in resolubility and stability. The addition of water-soluble natural compounds further enhances their physical properties.

[Brief description of the drawings]

FIG. 1 is an explanatory view schematically showing the degree of aggregation as a reference for evaluation when measuring the sensitivity of a slide.

Claims (14)

(57) [Claims] 1. A latex particle sensitized with (A) an immunoreactive substance and (B) a molecular weight of 1,000 to 300,000.
Of the water-soluble synthetic polymer compound (C) and the water-soluble natural compound (C) in a weight ratio of the water-soluble synthetic polymer compound (B) to the water-soluble natural compound (C) of 1: 1 to 1:15. A diagnostic test slide, wherein a reagent layer is carried on a slide surface. 2. A latex particle sensitized with (A) an immunoreactive substance is mixed with (B) a molecular weight of 1,000 to 300,000.
0 in the presence of the water-soluble synthetic polymer compound (C) and the water-soluble natural compound (C) [the water-soluble synthetic polymer compound (B): the water-soluble natural compound (C)] in the presence of 1: 1 to 1:15. A method for producing a diagnostic test slide, wherein the method is applied on a slide surface and air-dried. 3. The method according to claim 2, wherein the water-soluble synthetic polymer is polyvinylpyrrolidone. 4. The method according to claim 2, wherein the water-soluble natural compound is cyclodextrin. 5. The method according to claim 2, wherein the immunoreactive substance is an antibody. 6. The method according to claim 5, wherein the antibody is IgG. 7. The method according to claim 6, wherein the IgG is a tumor antibody. 8. The method according to claim 5, wherein the antibody is IgM. 9. The method according to claim 8, wherein the IgM is an antibody against Candida cells. 10. The method according to claim 8, wherein the IgM is an antibody against Aspergillus cells. 11. The method according to claim 2, wherein the immunoreactive substance is an antigen.
The described method. 12. The method according to claim 11, wherein the antigen is an antigen derived from Candida cells. 13. The method according to claim 11, wherein the antigen is an antigen derived from Aspergillus cells. 14. The method according to claim 11, wherein the antigen is derived from a tumor.