JPH08129014A - Inspection plate and inspection method - Google Patents

Abstract

(57) [Summary] [Structure] A slide plate for observing with a microscope and an upper member detachable on the slide plate are provided, and a plurality of through holes are provided in the upper member to slide the upper member. An inspection plate in which a plurality of through holes provided in the upper member form independent sections when mounted on the plate. [Effect] When used in a fluorescent antibody method or an enzyme antibody method using a microscope, an efficient test can be performed.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention has a test plate for use in a clinical test or research using a microscope, a test plate having a slide plate on which an antigen is fixed, and a slide plate on which an antigen is fixed. The present invention relates to a method for manufacturing an inspection plate and an inspection method using the inspection plate.

[0002]

2. Description of the Related Art A fluorescent antibody method, an enzyme antibody method, or pathological histochemistry using a microscope has become an extremely important test method for testing autoimmune diseases, infectious diseases, pathologies and the like. That is, for autoimmune diseases, tests for antinuclear antibody, anti-DNA antibody (Clysidia method), anti-mitochondrial antibody, etc., for infectious disease, syphilis test (FTA-ABS), for pathology, histological diagnosis of cancer, etc.
Many tests have been performed.

This inspection method is classified into an indirect method and a direct method. The indirect method is usually a method in which an antigen such as a cell or a tissue is immobilized on a slide plate and an antibody against this antigen in a test sample is detected. For example, for antinuclear antibodies, He
The p2 cells and the like are fixed on a slide plate and used as an antigen. A test sample such as serum or plasma is reacted with this, and the specific antibody in the test sample is reacted with the antigen substance in the fixed cells. After allowing the bound antibody to react with anti-immunoglobulin (labeled antibody) labeled with fluorescence or enzyme, the stained image is observed with a microscope. According to this method, antibodies against a plurality of antigens can be detected, and the antigens to which the antibodies correspond can be estimated by the staining pattern. The direct method is an inspection method in which a test sample such as tissue or cells is fixed on a slide plate, reacted with an antibody labeled with fluorescence, enzyme, or biotin, and the immunologically stained image is observed under a microscope. It is used for detecting cells.

[0004]

DISCLOSURE OF INVENTION Problems to be Solved by the Invention In the conventional fluorescent antibody method or enzyme antibody method inspection using a microscope, a test sample or reagent is dispensed onto a slide plate and the slide plate is washed in a beaker or the like. 2 of washing with
Automation of the inspection process was the most difficult field because various types of operations were repeated alternately. Further, in the method of immobilizing the antigen on the slide plate, there are many steps similar to those of the inspection, and automation is difficult, so that the method must be done manually.

A detailed explanation will be given by taking an indirect fluorescent antibody method of an antinuclear antibody as an example. The procedure for manufacturing an inspection plate by immobilizing an antigen on a general slide plate, which has been conventionally performed, is as follows.

Cancer cells Hep2 on slide plate
Mount the suspension and allow the cells to attach to the glass. The slide plate is washed with physiological saline or the like.

The cells are fixed with acetone. Air dry and use as a test plate. Next, a general inspection procedure using the inspection plate will be shown.

Serum diluted with physiological saline or the like,
It is placed on a slide plate, and the specific antibody (antinuclear antibody) in serum is reacted with the antigen on the slide plate. Immerse the slide plate in saline in a beaker and wash.

Fluorescently labeled anti-immunoglobulin is placed on a slide plate and reacted with a specific antibody bound to the antigen on the slide plate. Immerse the slide plate in saline in a beaker and wash.

An aqueous glycerin solution is placed on a slide plate, covered with a cover glass, and observed with a fluorescence microscope. In this way, in the process of immobilizing the antigen on the slide plate and the inspection process using the slide plate on which the antigen is immobilized, the dispensing on the slide plate and the washing of the slide plate in the beaker are repeated, which is automated. However, most of the steps have been done manually. as a result,
The fluorescence antibody method and the enzyme antibody method using such a microscope are the fields where the automation is the slowest in the fact that many other tests are automated.

Under these circumstances, an example of an attempt to improve the conventional classical method is a chamber slide manufactured by Nunc. This chamber slide was developed to enable the culture of cells on a slide plate, and has a plurality of through holes integrally formed on the slide plate attached via silicone rubber. The plurality of through holes can be removed,
The once removed hole could not be used again for efficient inspection using automated equipment because it could not be attached again.

The present inventors have arrived at the present invention as a result of earnest research on an inspection plate that can be used for efficient inspection using an automated device.

[0013]

According to the present invention, there is provided a slide plate for observation with a microscope, and an upper member detachable on the slide plate, and the upper member is provided with a plurality of through holes. The inspection plate is characterized in that, when the upper member is mounted on the slide plate, a plurality of through holes provided in the upper member form independent sections.

Further, according to the present invention, there is provided a slide plate on which an antigen to be observed with a microscope is fixed, and an upper member detachable on the slide plate, and the upper member is provided with a plurality of through holes. The present invention relates to a test plate having a slide plate on which an antigen is fixed, wherein a plurality of through holes provided in the upper member form independent sections when the upper member is mounted on the slide plate.

Further, in the present invention, the antigen is attached to the slide plate in the through hole of the inspection plate with the upper member mounted on the slide plate, and then the slide plate is removed to wash, fix the antigen, and air dry. The method for producing a test plate having a slide plate on which an antigen is fixed, characterized in that the slide plate on which the antigen is fixed is attached to the test plate again after performing.

Further, a test sample is dispensed and washed into a through hole of a test plate having a slide plate on which an antigen is fixed with an upper member mounted on the slide plate, a labeled antibody is dispensed and washed, The present invention relates to an inspection method characterized in that a staining substrate is dispensed and washed as needed, an encapsulant is dispensed, and then a slide plate removed from the inspection plate is observed with a microscope.

Further, according to the present invention, the slide plate is removed from the test plate, the test sample is attached or fixed on the slide plate, the slide plate is attached to the test plate, and the inside of the through hole of the test plate is marked. Dispense and wash the antibody or staining solution and, if necessary, the staining substrate and then wash the mountant, then remove the slide plate from the inspection plate and observe under a microscope. The present invention relates to a characteristic inspection method.

The present invention will be described in detail below with reference to the drawings. 1 is an exploded perspective view showing an example of an inspection plate having a lower member according to the present invention as seen obliquely from above, FIG.
4 is an assembled perspective view in which the inspection plate shown in FIG. 1 is set, FIG. 3 is a plan view of the inspection plate shown in FIG.
3 is a bottom view of the inspection plate shown in FIG. 1, FIG. 5 is a cross-sectional view taken along the line AA in FIG. 3, and FIG. 6 is B in FIG.
-B is a cross-sectional view taken along the line B, FIG. 7 is a cross-sectional view taken along the line C-C in FIG. 3, and FIG. 8 is a cross-sectional view corresponding to FIG. 5 in which the upper member is opened with the upper member supporting portion as a fulcrum. 9 is a sectional view corresponding to FIG. 6 in which the upper member is opened with the upper member supporting portion as a fulcrum, and FIG. 10 is a plan view showing a slide plate and a state in which the upper member is removed and the slide plate is set to the lower member. 11 is a plan view showing a slide plate and a state in which the upper member is removed and the slide plate is set on the lower member, and FIG. 12 shows another example of the inspection plate having the lower member according to the present invention as seen obliquely from above. It is an exploded perspective view.

FIG. 13 is an exploded perspective view showing an example of the inspection plate according to the present invention which does not have a lower member as seen obliquely from above, and FIG. 14 is a plan view of the inspection plate shown in FIG. 15 is a bottom view of the inspection plate in which the slide plate shown in FIG. 13 is not set, FIG. 16 is a cross-sectional view taken along the line DD in FIG. 14, and FIG.
4 is a sectional view taken along line EE in FIG. 4, FIG. 18 is a bottom view showing a slide plate, and a state in which the slide plate is set on the upper member, and FIG.
It is a bottom view showing the state where the slide plate was set to the upper member.

The inspection plate shown in FIGS. 1 to 11 is
It has an upper member 1, a slide plate 2 or a slide plate 3, and a lower member 4. Upper member 1
Is usually in the shape of a quadrangle, and can be molded with thermoplastic resins such as polyethylene, polypropylene, polystyrene, acrylic resin, polycarbonate, ABS resin, polysulfone, etc., automatic washing machine, automatic dispensing machine, EL
It is preferable to have a plurality of through holes 5 having a pitch similar to that of a 96-well microplate capable of supporting various inspection devices such as an ISA processor and a multi-pipette. Through hole 5
Is usually 96 holes, but the number may be increased or decreased by changing the inspection equipment or the like. Further, such through holes have, for example, a pitch from the through holes 5 to the through holes of 9.0 mm, an upper diameter of 7.0 mm, a lower diameter of 6.0 mm, and a depth of 8.0 m.
It is preferably m.

A groove 6 is formed on the lower surface of the upper member 1, and the protruding portion 8 of the sealing material 7 enters into the groove 6, whereby the sealing material 7 is formed on the lower surface of the upper member 1 around the through hole 5. Is attached. The sealing material 7 is preferably made of silicon rubber, ethylene rubber, propylene rubber or the like having physical properties such as fluidity, adhesiveness and appropriate elasticity, and has a hardness of 18 to 50 degrees and a thickness of 1.0 to 2.0 mm
Is desirable. The sealing material 7 may be independently fired to enhance the cushioning property. In order to attach the seal material 7 to the lower surface of the upper member 1, for example, a silicon rubber plate is punched or is injection molded to have 96 holes with a diameter of 5.5 mm at the same pitch as the through holes 5 of the upper member 1. Forming a plate-like body, filling the groove 6 of the upper member 1 with the curable liquid silicone rubber, and then laminating the plate-like body, and curing the curable liquid silicone rubber to form the protruding portion 8 of the sealing material 7. The seal member 7 may be attached to the lower surface of the upper member 1 by the above method, or the upper member 1 may be set in a mold and insert-molded when the seal member 7 is injection molded. The upper member 1 and the upper member 1 may be simultaneously molded, or an adhesive sheet may be attached only to the lower surface of the upper member 1 excluding the opening of the through hole 5, and then the sealing material 7 may be attached thereto.

The upper member 1 is supported by the lower member 4, and preferably has a support shaft 11 on its left and right so that one end of the upper member 1 is pivotally supported by the lower member 4. . The upper member 1 may be structured so that it can be detached from the lower member 4 without being axially supported by the lower member 4, as shown in FIG. In this case, the upper member 1 has a shape in which the support shaft 11 is not formed.

The lower member 4 has an inner slide plate 2
Alternatively, it is a frame-shaped body having a supporting portion 9 for supporting the slide plate 3, and can be molded with a thermoplastic resin such as polyethylene, polypropylene, polystyrene, acrylic resin, polycarbonate, ABS resin, or the like, and if necessary, glass fiber or the like. The reinforcing material may be mixed.

A support shaft receiving hole 10 is formed on both left and right sides of one end of the lower member 4, and the support member 11 of the upper member 1 is fitted into the support shaft receiving hole 10 so that the upper member 1 can be attached to the lower member 4. It can be opened and closed repeatedly. The lower member 4 has a protrusion 12 formed at the end opposite to the support shaft receiving hole 10. The upper member 1 is closed to the lower member 4, and the protrusion 1
By fitting 2 into the lock hole 13 provided in the upper member 1, the upper member 1 can be attached to the slide plate 2 or the slide plate 3 placed on the supporting portion 9 of the lower member 4. By pressing the sealing material 7 of the above to the slide plate 2 or the slide plate 3, the through holes 5 of the upper member 1 are independently defined on the slide plate 2 or the slide plate 3, and the diluting liquid or the cleaning liquid is supplied to the through holes 5. It is sealed so that the diluent, the cleaning liquid, etc. do not leak from the through hole 5 even if the liquid etc. is injected. When the upper member 1 is not pivotally supported by the lower member 4, the lower member 4 has a shape in which a support bearing hole is not formed.

The slide plate can be made of glass and synthetic resin such as polyester, polymethyl terpene, ethylene-methacrylic acid copolymer, polystyrene, acrylic resin, polycarbonate, and cyclic olefin copolymer, and can be used for antigen or test sample. If the area 14 to be fixed is, for example, an area having a diameter of 4.5 mm and provided at the same pitch as the 96-well microplate, the 16-hole type split slide plate 2 provided at 16 locations may be used, or 96 locations may be provided. Alternatively, the slide plate 3 may be a single 96-hole type slide plate, or may be the 8-hole or 24-hole slide plate 2 as required. Except for the portion 14 of the slide plate on which the antigen or the test sample is fixed, it is preferable to use a printing ink having Teflon or the like as a binder for coloring and water repellent treatment. This makes it possible to clearly identify the area on which the antigen or the test sample is fixed, and to repel it when the antigen or the test sample protrudes from the site 14.

The slide plate 2 and the slide plate 3 are set on the support portion 9 of the lower member 4. That is, when the slide plates 2 are used, one to six pieces are set within the pitch of the dividing ribs 15 on the supporting portion 9 of the lower member 4 in order to divide and fix each of them, and It is preferable that the size and the size c correspond to the size e and the size g of the support portion 9 with substantially the same size. Also, when using the slide plate 3,
It is practically preferable that one piece is set inside the tips of the dividing ribs 15 and the b dimension and the d dimension of the slide plate 3 correspond to the f dimension and the h dimension of the support portion 9 in substantially the same dimension. By determining the dimensions of the slide plate 2 and the slide plate 3 in this manner, the two types of slide plates 2 and 3 can be reliably set on the support portion 9.

In the example of the inspection plate of the present invention shown in FIGS. 1 to 11, the upper member 1 is opened and closed with one end of the lower member 4 as an axis to place the slide plate 2 or slide on the lower member 4. The upper member 1 can be attached to and detached from the plate 3. FIG. 12 shows another example of the inspection plate of the present invention. This inspection plate does not have a support shaft for the upper member and a support hole for the lower member, unlike the inspection plate shown in FIGS. 1 to 11. The slide plate 2 or the slide plate 3 is placed on the lower member 4, and Upper member 1 on top
And a convex portion 16 formed on the lower member 4 is fitted with a concave portion (not shown) formed on the inner surface of the hanging portion 17 of the upper member 1.

The inspection plate of the present invention is preferably an inspection plate having the lower member 4 as shown in FIGS. 1 to 12, but it is composed of an upper member and a slide plate as shown in FIGS. 13 to 19. It may be one.
The inspection plate without the lower member shown in FIGS. 13 to 19 has a mechanism for holding the slide plate 2 or 3 by the claw portions 17 provided on both side walls of the upper member as shown in FIG. The claw portion has elasticity, and the slide plate can be removed from the inspection plate by slightly opening it outward. The claw portions are formed on the side wall of the upper member so as to form two or more pairs in order to hold the slide plate 2 of the split type. Incidentally, FIG.
The upper member of the inspection plate of Nos. 19 to 19 is the same as the through hole and the sealing material formed by the upper member of the inspection plate of FIGS.

The test plate according to the present invention can be used for tests such as an indirect fluorescent antibody method or an indirect enzyme antibody method and a direct fluorescent antibody method or a direct enzyme antibody method. As an indirect fluorescent antibody method or an indirect enzyme antibody method, autonuclear antibodies such as antinuclear antibody, anti-DNA antibody (Crysidia method), anti-centromere antibody, anti-smooth muscle antibody, anti-mitochondrial antibody, anti-gastric parietal cell antibody, anti-neutrophil antibody, etc. Immune disease diagnosis and chlamydia antibody, toxoplasma antibody, syphilis antibody (FTA-AB
S), ATLV antibody, EB virus antibody, rubella virus antibody, herpes antibody, cytomegalovirus antibody, etc. can be used for diagnosis of infectious diseases. Examples of the direct fluorescent antibody method or the direct enzyme antibody method include diagnosis of infectious diseases such as herpes antigen and chlamydia antigen, cytodiagnosis of cancer, and pathological examination such as IgA nephropathy examination.

A method for producing a test plate (indirect method) having a slide plate on which an antigen is immobilized using the test plate will be described. (1) Adhesion of antigen on slide plate In the indirect method, the antigen is fixed on the slide plate. As an antigen, in autoimmune disease diagnosis, Hep2 cells, chrysidia, chromosome, smooth muscle, renal tissue, gastric wall tissue,
Cells and tissues with self-antigens such as neutrophils are available, and pathogenic microorganisms such as bacteria and viruses are used for diagnosis of infectious diseases. These antigens can be attached to the slide plate by suspending them in an appropriate medium or buffer, dispensing them on the slide plate and incubating. At this time, the antigen suspension can be automatically dispensed from the upper part of each through hole of the upper member of the test plate to which the slide plate is attached by using an automatic dispenser or the like.

(2) Washing After the antigen has adhered to the surface of the slide plate, excess antigen, medium and buffer solution are washed and removed with a washing solution such as physiological saline or PBS solution. Also in this case, the slide plate to which the antigen is attached can be washed with the washing machine from above the through hole of the upper member of the inspection plate with the slide plate set.

(3) Immobilization of antigen and air-drying The antigen attached on the slide plate was treated with ethanol, acetone, glutaric aldehyde, paraformaldehyde, formalin, methanol, or a fixing solution containing a combination thereof. Fixed. In this case, the slide plate on which the antigen is fixed in (2) above is taken out from the test plate, and only the sled plate is immersed in the fixing solution to fix the antigen. The slide plate on which the antigen has been fixed is air-dried.

(4) Set of slide plate on which antigen is fixed The slide plate on which antigen is fixed is placed on the lower member, and then the upper member is covered to form a test plate. By thus setting the slide plate on which the antigen is fixed in advance, it is possible to perform the test immediately without preparing the slide plate on which the antigen is fixed each time when testing a large number of test samples. There is a merit that can be done.

In the case of the method for producing a test plate on which the slide plate on which the antigen is fixed as described above is set, an automatic pipetting machine is used without opening or closing the upper member or removing the slide plate. Since the antigen suspension can be dispensed to allow the antigen to adhere to the slide plate and then washed with the washing solution as it is, the test plate having the slide plate on which the antigen is immobilized can be efficiently manufactured.

Next, an inspection method (indirect method) using the inspection plate will be described. (1) Dispensing of test sample The test sample is usually serum, but heparin plasma, EDTA plasma, citrate plasma, or any body fluid such as ascites, spinal fluid, saliva, and urine can be the test sample. These test samples were phosphate buffered saline (P
It is diluted with a commonly used buffer such as BS) and physiological saline. These test samples are dispensed from the upper part of the through hole of the upper member of the test plate to which the slide plate on which the antigen is fixed is attached. At this time, this process can be automated by using an automatic pipetting machine. When the test sample is dispensed into the through hole of the test plate, the antibody in the test sample binds to the antigen fixed on the slide plate.

(2) Washing 1 Extra antibody (immunoglobulin) that did not bind to the antigen
Are removed by a washing operation. The washing operation is the dispensing of the washing liquid,
Although suction is repeated, in this step as well, the washing can be automated by using the automatic washing machine without taking out the slide plate from the inspection plate.

(3) Dispensing of labeled antibody After the first washing, the labeled antibody is dispensed, and the antibody bound to the antigen fixed on the slide plate is reacted with the labeled antibody. If the fluorescent antibody method is used as the labeled antibody, anti-immunoglobulin, anti-IgG, anti-IgM, anti-IgA, anti-Ig
Antibodies such as E may be labeled with a fluorescent substance such as fluoroscein, rhodamine, and phycoerythrin. In the case of the enzyme antibody method, enzymes such as peroxidase and alkaline phosphatase, Alternatively, those labeled with biotin can be used. in this case,
The labeled antibody can be automatically dispensed from the upper part of the through hole of the upper member of the inspection plate by an automatic dispenser.

(4) Washing 2 In the same manner as in Washing 1, the labeled antibody that has not bound to the antibody is removed by washing. (5) Dispensing of staining substrate (in the case of enzyme antibody method) In the case of the enzyme antibody method, a staining substrate is dispensed, and the site to which the labeled antibody (enzyme) is bound is stained with the staining substrate. As the staining substrate, when the labeled antibody (enzyme) is peroxidase, diaminobenzidine and the like are used. Dispensing of this dyeing substrate can also be automated by the automatic dispenser as described above.

(6) Washing 3 After dyeing, the excess dyed substrate is removed by washing in the same manner as in Washing 1. (7) Dispensing of mounting medium The mounting medium is dispensed to the antigen on the slide plate for which the above operations have been completed. This has the meaning of preventing the stained antigen and the like from drying out, and then bringing a cover glass placed on the slide plate into close contact therewith. This mounting medium is usually 10-
A 100% aqueous glycerin solution is used, but is not limited to this. This process can also be automated by an automatic pipetting machine without removing the slide plate.

In the above steps (1) to (7),
As an automated device, it is possible to use an automated device corresponding to each process such as test sample dispensing, washing, labeled antibody dispensing, staining substrate dispensing, and mounting medium dispensing. The through hole pitch of the member is 96 hole microtiter
If it is the same as the plate, for example, if an automated device for microplate such as Processor-2, Processor-3 of Bering Co., Biomec of Beckman Co., Ergioart of Kokusai Reagent Co., etc. is used. It is also possible to continuously automate all these steps.

After the above steps are completed, slide slide is prepared.
The slide plate is taken out, covered with a cover glass, and measured and observed with a microscope. In the present invention, since the upper member 1 is attachable / detachable on the slide plate, when measuring and observing a small number of test samples with a microscope, a slide of 8 hole type, 16 hole type or 24 hole type is used. It is economical to use an inspection plate in which the plate is stored. For example, in an inspection plate in which six 16-hole type slide plates 2 are stored, when it is necessary to use two slide plates 2, the upper member 1 is removed from the slide plates 2 and four slide plates 2 are used. The slide plate 2 is taken out, a glass plate is housed in place of the four slide plates 2 taken out, the upper member 1 is mounted on the slide plate 2 and the glass plate, and the operations (1) to (7) are performed. After this, the two slide plates 2 should be taken out for measurement and observation. By using the four slide plates 2 taken out earlier at another time, an expensive slide with the antigen immobilized It is possible to prevent the plate from being wasted.

Next, the direct method will be described. (1) Attachment or Immobilization of Test Sample In the direct method, cells, tissues, sputum, etc. become test samples, and the test samples are attached on a slide plate.
These test samples can be subjected to the next step without being immobilized, and may be chemically immobilized if necessary. For chemical fixation, a fixative using formalin, paraformaldehyde, glutaric aldehyde, methanol, ethanol, acetone, or a combination thereof can be used. This process can also be automated by an automatic pipetting machine without removing the upper member from the slide plate,
It is also possible to fix directly to the slide plate with the upper member removed.

(2) Dispensing of labeled antibody or staining solution An antibody against a specific antigen to be tested, for example, CEA, AFP, PSA, CA-1 in the case of tissue diagnosis of cancer
For detection of cancer-specific antigens such as 9-9, tissue-specific antigens such as keratin and vimentin, and immune complexes in the kidney, labeled antibodies such as IgG, IgM and IgA are used. Fluorescein, rhodamine, phycoerythrin, etc. can be used for labeling of these antibodies in the case of the fluorescence method, and peroxidase, alkaline phosphatase, etc. can be used in the case of the enzyme method, and biotin can be used. Indirect labeling with is also possible. Further, instead of the labeled antibody, Papanicolaou stain hematoxylin solution 100, Papanicolaou stain solution OG-100,
A staining method using a staining solution such as Papanicolaou staining solution EA-100 can also be used. In this step, labeled antibody, staining solution, etc. are dispensed onto the test sample on the slide plate, but the dispensing with the upper member attached to the slide plate is performed by an automatic dispenser or the like. It can be automated.

(3) Washing 1 The labeled antibody that did not bind to the antigenic substance in the test sample and the excess staining solution are removed by a washing operation. The washing is performed by repeatedly dispensing and aspirating the washing solution, but it can be automated by an automatic washing machine without removing the slide plate from the inspection plate.

(4) Dispensing of Staining Substrate (In Case of Enzyme-Labeled Antibody) In the case of the enzyme antibody method, the staining substrate is dispensed in order to stain the site to which the enzyme is bound. As the staining substrate, when the enzyme is peroxidase, a buffer solution containing diaminobenzidine is used. Further, in the case of labeling with biotin, an amplification method such as PAP method or ABC method can also be used.

(5) Washing 2 After dyeing, washing is performed to remove excess dyed substrate. Dispensing and washing of this staining substrate can also be supported by automated equipment with the slide plate set on the inspection plate.

(6) Dispensing of mounting medium After the above operation is completed, the mounting medium is dispensed to the antigenic substance on the slide plate. This means preventing the stained antigen and the like from drying out, and then bringing a cover glass to be covered on the slide plate into close contact therewith. This step can also be automated by an automatic pipetting machine by attaching the upper member on the slide plate.

After the above steps are completed, the upper member is removed from the slide plate, covered with glass, and measured by a microscope. When the test plate according to the present invention is used, the slide plate is removed from the test plate to attach or fix the test sample on the slide plate, and then the slide plate is attached to the test plate and labeled using an automated instrument. The antibody or the staining solution is dispensed, washed, the staining substrate is dispensed, and the like, and then the slide plate is removed from the inspection plate and the measurement is performed with a microscope, whereby the measurement can be efficiently performed.

The test plate according to the present invention may be a slide plate on which an antigen is fixed in advance, or a slide plate on which an antigen is not fixed. Well, each can be used depending on the application.

[0050]

[Example 1]

Manufacture of the Upper Member Polystyrene is used, and injection molding is used to perform the process shown in FIGS.
The upper member 1 shown in FIG. In the upper member 1, a pitch from the through holes 5 to the through holes 5 was set to 9.0 mm, and a total of 96 through holes 5 were formed with 12 rows on the long side and 8 rows on the short side. The through hole 5 has an upper diameter of 7.0 mm, a lower diameter of 6.0 mm, a depth of 8.0 mm, and a capacity of about 0.31 ml.
And A groove 6 having a width of 1.0 mm and a depth of 1.0 mm was formed on the lower surface of the upper member 1 in order to fit the sealing material 7.
A support shaft 11 is formed at one end of the upper member 1.

Fixing the seal material A press-processed silicone rubber plate (heat-vulcanizing type silicone rubber SH841V manufactured by Toray Dow Corning Silicone Co., Ltd.) having a thickness of 1.0 mm and a hardness of 40 degrees corresponds to the lower surface of the upper member 1. After punching into the outer shape
96 holes with a diameter of 5.5 mm were formed at the same pitch as the above.
A curable liquid silicone rubber (Toray Dow Corning Silicone coating material SE9187RTV) is filled in the groove 6 formed on the lower surface of the upper member 1,
The lower surface of the upper member 1 is formed by sticking a silicone rubber plate to the lower surface of the upper member 1 while aligning the through holes 5 with the holes and holding the liquid silicone rubber at 25 ° C. for 12 hours to cure the liquid silicone rubber to form a protrusion. The sealing material 7 was fixed to the.

Manufacture of Lower Member A lower member 4 shown in FIGS. 1 to 11 was molded using polycarbonate containing 15% by weight of glass filler. The lower member 4 has eight dividing ribs 15 each having a height of about 5.0 mm, a length of about 2.0 mm, and a wall thickness of about 0.8 mm on the supporting portion 9 at intervals of about 18.0 mm. 16 in total were formed. One end of the lower member 4 has a support hole 1
Formed 0.

Manufacture of slide plate A glass plate having a thickness of 1.0 mm has a longitudinal dimension (a dimension) of 7
6.7 mm, cut to have a lateral dimension (c dimension) of 16.8 mm, and a site for immobilizing an antigen or a test sample (area with a diameter of 4.5 mm provided at the same pitch as a 96-well microplate) The slide plate 2 was manufactured by printing a printing ink other than 14 with a printing ink using Teflon as a binder, and coloring and water-repellent treatment.

Further, a glass plate having a thickness of 1.0 mm is cut so that the longitudinal dimension (d dimension) is 111.3 mm and the lateral dimension (b dimension) is 72.7 mm, and the antigen or the test sample is fixed. The slide plate 3 is manufactured by printing and coloring with a printing ink having Teflon as a binder except the part (area having a diameter of 4.5 mm provided at the same pitch as the 96-well microplate) 14 and subjected to water repellent treatment. did.

Preparation of Inspection Plate After the support shaft 11 of the upper member 1 is fitted in the support hole 10 of the lower member 4, six slide plates 2 are set on the support portion 9 of the lower member 4, or Alternatively, the inspection plate according to the present invention was produced by setting one slide plate 3. Example 2 90 parts by volume of ME at 37 ° C. in a carbon dioxide incubator containing 5% carbon dioxide in air.
Hep2 cells were cultured for 3 days in a medium obtained by adding 10 parts by volume of fetal bovine serum to the ME medium (hereinafter referred to as medium). Hep2 cells attached to the culture vessel were washed with a 10 mM phosphate buffer solution containing 0.15 molar concentration of sodium chloride (hereinafter referred to as PBS solution), and then 0.125 wt% trypsin was added. Hep2 cells were collected by suspending them in a buffer containing 0.01% by weight of ethylenediaminetetraacetate, adding a medium, and then centrifuging. The Hep2 cells were then resuspended in the medium and centrifuged to collect the Hep2 cells for washing. Hep2 cells are resuspended in the medium at 10 ⁴ cells / ml to 10 ⁵ cells / ml, and suspended in the through holes 5 of the test plate of Example 1 equipped with the slide plate 3 by an automatic pipetting machine. The liquid was dispensed in 200 μl aliquots.

The test plate was allowed to stand at 37 ° C. for 12 hours in a carbon dioxide incubator containing 5% carbon dioxide in the air, and Hep2 cells were allowed to adhere to the slide plate 3.

Discard the medium in the through hole 5 of the inspection plate,
Remove the slide plate 3 from the inspection plate, and
After being immersed in a BS solution for washing, it was immersed in acetone to fix Hep2 cells on the slide plate 3.

The slide plate 3 on which the Hep2 cells were fixed was air-dried and mounted in the test plate to prepare a test plate on which the antigen was fixed on the slide plate 3. Example 3 A goat-derived anti-human immunoglobulin antibody was labeled with fluoroscein isothiocyanate to prepare a labeled antibody, and the obtained labeled antibody was diluted with a PBS solution.

50 samples of sera, which are test samples, were diluted 20 times, 40 times, 80 times with PBS solution using an automatic diluter.
Diluted 160-fold, 320-fold, 640-fold and 1280-fold. In the test plate in which the antigen is fixed on the slide plate 3 described in Example 2, after the upper member 1 is mounted on the slide plate 3, an automatic pipetting machine is used in the through hole 5 of the test plate. Then, add the serum dilution solution to 100
After dispensing μl, the mixture was left at room temperature for 30 minutes.

Then, an operation of using an automatic washing machine to dispense 300 μl of the PBS solution into the through hole 5 of the inspection plate, suctioning and washing was repeated 4 times. 100 μl of the labeled antibody diluted solution was dispensed into the through hole 5 of the inspection plate using an automatic dispenser, and then left at 25 ° C. for 30 minutes.

Then, the inside of the through hole 5 of the inspection plate was washed in the same manner as described above. Then, using an automatic dispenser, 10 μl of 80 wt% glycerin aqueous solution was dispensed into the through holes 5 of the inspection plate, the slide plate 3 was removed from the inspection plate, and the slide glass 3 was covered with a cover glass. , Observed with a fluorescence microscope.

In the above measuring method according to the present invention,
Of the 50 sera, 15 were determined to be positive, which was similar to the most widely used assay method using the fluoro-HEPANA test plate of MBL.

Further, the antibody titer (dilution limit magnification) and the staining pattern of 6 cases among the sera judged to be positive are shown in Table 1. According to the method of the present invention, the antibody titer is equal to or higher than that of the conventional method. It was clear that it could be measured with accuracy.

[0064]

[Table 1] [Example 4] A goat-derived anti-human immunoglobulin antibody was labeled with peroxidase to prepare a labeled antibody, and the obtained labeled antibody was diluted with a PBS solution.

The 50 sera used in Example 3 were diluted with PBS solution using an automatic diluter at a ratio of 20 times, 40 times, 80 times,
Diluted 160-fold, 320-fold, 640-fold and 1280-fold. In the test plate in which the antigen is fixed on the slide plate 3 described in Example 2, after the upper member 1 is mounted on the slide plate 3, an automatic pipetting machine is used in the through hole 5 of the test plate. Then, add the serum dilution solution to 100
After dispensing μl, the mixture was left at room temperature for 30 minutes.

Then, an operation of using an automatic washing machine to dispense 300 μl of the PBS solution into the through hole 5 of the inspection plate, sucking and washing was repeated 4 times. Then, 100 μl of the labeled antibody dilution was dispensed into the through hole 5 of the inspection plate using an automatic dispenser, and then left at room temperature for 30 minutes.

Then, the inside of the through hole 5 of the inspection plate was washed in the same manner as described above. Then, using an automatic pipetting machine, 100 μl of the staining substrate (0.0
50 mM Tris buffer containing 05% by weight hydrogen peroxide, 0.02% by weight diaminobenzidine,
After dispensing pH 7.6), the mixture was left at room temperature for 10 minutes.

Then, the inside of the through hole 5 of the inspection plate was washed in the same manner as described above. Then, using an automatic dispenser, 10 μl of an aqueous 80% by weight glycerin solution was dispensed into the through holes 5 of the inspection plate, the slide plate 3 was removed from the inspection plate, and a cover glass was placed on the slide plate 3. When covered with a microscope and observed under a microscope, almost the same results as in Example 3 were obtained. [Example 5] Fresh sputum from 10 lung cancer (squamous cell carcinoma) patients and 10 normal subjects were applied to the slide plate 3 removed from the test plate.

Next, the slide plate 3 was immersed in a fixative solution containing 1 part by volume of ethanol and 1 part by volume of diethyl ether to fix sputum on the slide plate 3. Next, the slide plate 3 was attached to the inspection plate, and the upper member 1 was attached to the slide plate 3.

Then, using an automatic washing machine, 300 μl of an aqueous ethanol solution having a concentration of 70% by volume is dispensed into the through hole 5 of the inspection plate and suctioned, and 300 μl of an aqueous ethanol solution having a concentration of 50% by volume is dispensed. The operation of sucking by suction and the operation of dispensing 300 μl of distilled water and suctioning were repeated twice, and the inside of the through hole 5 was washed.

Next, using an automatic pipetting machine, the Papanicolaou-stained hematoxylin solution 10 is placed in the through hole 5 of the inspection plate.
0 (staining solution manufactured by Wako Pure Chemical Industries, Ltd.) was dispensed in an amount of 200 μl and held for 5 minutes for staining.

Then, using an automatic washing machine, 300 μl of distilled water was dispensed into the through hole 5 of the inspection plate and suctioned, an aqueous solution obtained by adding 1% by volume hydrochloric acid to a 70% by volume aqueous ethanol solution. To 300 μl of a 70% by volume ethanol solution, 300 μl of a 70% by volume aqueous ethanol solution containing 3% by volume of ammonia, and 300 μl of a 70% by volume aqueous ethanol solution
Dispensing and sucking and 300 μl of 90% by volume ethanol aqueous solution are dispensed and sucked respectively.
The inside of the through hole 5 was washed by repeating each time.

Then, using an automatic dispenser, 200 μl of Papanicolaou dyeing solution OG-100 (Wako Pure Chemical Industries, Ltd. dyeing solution) was dispensed into the through-holes 5 of the inspection plate and held for 2 minutes for staining. .

Next, the operation of pouring 300 μl of 95% by volume aqueous ethanol solution into the through hole 5 of the inspection plate using an automatic washing machine and suctioning was repeated twice to wash the inside of the through hole 5.

Next, using an automatic dispenser, 200 μl of Papanicolaou stain EA-100 (Wako Pure Chemical Industries, Ltd. stain solution) was dispensed into the through holes 5 of the inspection plate, and held for 3 minutes for staining. .

Then, an automatic washing machine was used to dispense 300 μl of 95% by volume concentration of ethanol aqueous solution into the through hole 5 of the inspection plate, and suctioning was repeated 3 times to wash the inside of the through hole 5. Add 200 μl of xylol to
Let stand for a minute.

Then, 10 μl of an aqueous 80% glycerin solution was dispensed into the through holes 5 of the inspection plate using an automatic dispenser, and then the slide plate 3 was removed from the inspection plate and placed on the slide plate 3. Covered with a cover glass and observed with a fluorescence microscope, lung cancer patient 1
All 0 cases were positive.

[0078]

According to the present invention, a slide plate,
When an inspection plate having a detachable upper member is used for it, it has been difficult to automate it until now. The indirect fluorescent antibody method using a microscope, the indirect enzyme antibody method, the direct fluorescent antibody method, the direct enzyme antibody method, and In immunohistochemistry etc., it becomes possible to perform the steps prior to the step of observing with a microscope very efficiently, and it is possible to manufacture the test plate in which the antigen is immobilized on the slide plate extremely efficiently. Become.

[Brief description of drawings]

FIG. 1 is an exploded perspective view showing an example of an inspection plate according to the present invention as seen obliquely from above.

FIG. 2 is an assembled perspective view in which the inspection plate shown in FIG. 1 is set.

FIG. 3 is a plan view of the inspection plate shown in FIG.

FIG. 4 is a bottom view of the inspection plate shown in FIG.

5 is a cross-sectional view taken along the line AA in FIG.

6 is a cross-sectional view taken along the line BB in FIG.

7 is a cross-sectional view taken along the line CC of FIG.

FIG. 8 is a sectional view corresponding to FIG. 5 in which an upper member is opened.

9 is a sectional view corresponding to FIG. 6 in which the upper member is opened.

FIG. 10 is a plan view showing a state in which the slide plate is set on the lower member with the slide plate and the upper member removed.

FIG. 11 is a plan view showing a state in which the slide plate is set on the lower member with the slide plate and the upper member removed.

FIG. 12 is an exploded perspective view showing another example of the inspection plate according to the present invention as seen obliquely from above.

FIG. 13 is an exploded perspective view showing another example of the inspection plate according to the present invention as seen obliquely from above.

FIG. 14 is a plan view of the inspection plate shown in FIG.

FIG. 15 is a bottom view of the inspection plate shown in FIG.

16 is a cross-sectional view taken along the line DD in FIG.

FIG. 17 is a cross-sectional view taken along the line EE in FIG.

FIG. 18 is a bottom view showing a state where the slide plate is set on the upper member.

FIG. 19 is a bottom view showing a state where the slide plate is set on the upper member.

[Explanation of symbols]

 1 Upper member 2 Slide plate 3 Slide plate 5 Through hole

Front page continuation (72) Inventor Naoki Kurai 519-3 Katakura-cho, Hachioji-shi, Tokyo (72) Inventor Tomomasa Matsuda 7-18 Otemachi, Tatebayashi-shi, Gunma Shuttle Court B-203 (72) Inventor Masahide Kawamura 1-18-206, Kashiwadai, Inage-ku, Chiba City, Chiba Prefecture Inventor Takashi Kuroda 456 Neda, Chiba City Chiba Prefecture, 101 Kaneso Heights Kokubun Templedai 101 (72) Inventor Tetsuya Ono 4-4 Tatsumidai Higashi, Chiba City, Chiba Prefecture

Claims (10)

[Claims] 1. A slide plate for observing with a microscope,
An upper member that is removable on the slide plate,
The upper member is provided with a plurality of through holes, and when the upper member is mounted on the slide plate, the plurality of through holes provided in the upper member form independent sections. Plate. 2. The inspection according to claim 1, wherein the inspection plate has a lower member, a slide plate supported on the lower member, and an upper member detachable on the slide plate and supported by the lower member. Plate. 3. One end of the upper member is pivotally supported by the lower member,
The inspection plate according to claim 2, wherein the upper member is attachable to and detachable from the slide plate by opening and closing the upper member. 4. The inspection plate according to claim 1, 2 or 3, wherein a portion of the upper member which is in contact with the slide plate is made of a sealing material. 5. The test plate according to claim 1, claim 2, claim 3, or claim 4, wherein the slide plate is treated to be water repellent except for the portion where the antigen or the test sample is fixed. 6. The test plate according to claim 1, 2, 3, or 4, wherein an antigen or a test sample is immobilized on a slide plate. 7. The test plate according to claim 6, wherein the antigen fixed to the slide plate is an animal cell or tissue for an antinuclear antibody test. 8. The antigen is attached to the slide plate from above the through hole of the upper member of the inspection plate according to claim 1, claim 2, claim 3 or claim 4, and then the upper member is opened and closed to slide. A method for producing a test plate having an antigen-fixed slide plate, characterized in that the plate is removed, washed, and air-dried, and then the slide plate to which the antigen is fixed is attached to the test plate again. 9. A test sample is dispensed and washed into the through hole of the upper member of the test plate having the slide plate on which the antigen obtained in claim 8 is immobilized, labeled antibody is dispensed and washed, and stained. Dispense and wash the substrate and dispense the mounting medium,
Then, the slide plate removed from the inspection plate is observed with a microscope. 10. The slide plate is removed from the test plate of claim 1, claim 2, claim 3 or claim 4, and a test sample is attached or fixed onto the slide plate, and the slide plate is used as a test plate. After mounting, dispensing and washing labeled antibody or staining solution into the through hole of the upper member of the inspection plate, and if necessary staining substrate and washing, then mounting agent is dispensed, then slide plate The inspection method, which comprises observing with a microscope after detaching from the inspection plate.