JP2001201505A - Analytical method by using reactive particulate and analytical container - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide an analytical method capable of analyzing by reactive particulates simply in a short time, and an analytical container. SOLUTION: In this analytical method, a biological reaction is analyzed based on a distribution image of a particulate group formed after reaction by liquid to be tested containing a reactive particulate group mutually bondable by the biological reaction. The method by using the reactive particulates is characterized by supplying a striped irregular part having a falling region where the reactive particulates fall with the reactive particulate group after the reaction, by determining the falling state near a falling start part of the irregular part by observation or image analysis along the stripes, and by determining existence of the biological reaction or the like based on the determination result.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to an analysis method and an analysis vessel for analyzing a reaction or the like by reacting a large number of reactive fine particles which bind to each other through a biological reaction. . INDUSTRIAL APPLICATION This invention can be utilized suitably for the determination of the aggregation pattern by an immunological agglutination reaction, for example. Therefore, according to the present invention, it is possible to provide, for example, methods and means advantageous for determining various blood types and diagnosing infectious diseases.

[0002]

2. Description of the Related Art At present, in blood transfusion tests and immunological tests, test methods utilizing antigen-antibody reactions are widely and generally used. As a trend in recent years, detection means using an antigen-antibody reaction has come to be required to automate determination of an agglutination image using an agglutination reaction by agglutination particles with improvement in simplicity and sensitivity. There are various types of agglutination detection, and among them, a microplate having a plurality of wells has been used relatively frequently. However, in the determination of the agglutination image by the well, it took about one hour from the start of the reaction until the state became a determinable state (aggregation pattern formation).
For this reason, it has been required to reduce the time required for judging the aggregation image by the well.

Conventional methods for reducing the determination time include, for example, a method of applying an external force to particles for aggregation, such as a centrifugal method and a magnetic particle method, to promote the formation of an aggregated image. A method of promoting the formation of an aggregated image by narrowing and shallowing the concavo-convex portions of the wells involved in the formation of an aggregated image (Japanese Patent Publication No.
No. 21454) and a method for promoting the formation of an agglutination image by reducing the amount of blood cell and particle agglutination reagents utilizing capillary action (JP-A-4-145947, JP-A-4-20)
No. 8836) and other simple attempts have been made.

[0004]

However, when the centrifugal method is used to shorten the determination time, a new mechanism such as a centrifugal machine must be added to the apparatus, and therefore, an apparatus accompanying the enlargement of the system is required. There are problems such as an increase in the size of the device itself and destruction of the aggregated image due to centrifugation. In view of this, centrifugation is not a suitable method for clearly discriminating both positive and negative patterns. In addition, the magnetic particle method also requires the introduction of a new reagent called magnetic particle reagent to the test items that are usually conducted with high sensitivity using inexpensive reagents such as animal blood cells. The use of this method has not yet been realized because of problems such as the development process of the particle reagent and the cost.

On the other hand, if the well described in Japanese Patent Publication No. 1-21544 is used for a red blood cell blood type front test, it is possible to make the aggregation pattern discriminable state by waiting for about 30 minutes in the shortest possible time. (See FIG. 39). Certainly, if the width of the concavo-convex portion related to the formation of the aggregation image in the well is made narrow and the depth is made shallow, the time required for the formation of the aggregation image tends to be shortened. However, even if the time taken to form an agglutination image can be reduced by taking such a measure, if a sufficient number of blood cell or particle agglutinating reagents cannot be used for the determination, the conventional method is used. The reliability of the determination is inferior to that of the aggregation pattern of the above size. In addition, although not a problem that frequently occurs, the inclusion of foreign matter having a size close to the aggregation of the particles makes it difficult to make a clear distinction between positive and negative patterns, which may cause a problem in the determination. .

[0006] In addition, in the method utilizing the capillary phenomenon, it is necessary to first mix the reagent and the sample 1 uniformly in another container, and then introduce the mixture into the agglutination reaction detecting container utilizing the capillary phenomenon. Therefore, a more complicated determination method may be caused due to the increase in the number of reaction steps.

[0007] As described above, conventionally, there has been a demand for a small well capable of making an aggregation pattern discernable within 30 minutes without lowering sensitivity and specificity. An object of the present invention has been made in view of the above-mentioned circumstances, and an object of the present invention is to provide an analysis method and an analysis container that can perform analysis using reactive fine particles in a shorter time and more easily than in the past.

[0008]

SUMMARY OF THE INVENTION The present invention first arrives at a point where a conventional agglomeration pattern is configured to appear as a distribution of agglomeration particles in a planar shape, and a point-like or linear apex is formed. It was completed based on the discovery that an agglomerated pattern was formed on the formed wall surface, and surprisingly, it was possible to make a clear and clear judgment, and on an inclined surface on which agglomerated particles were deposited. Thus, it has been completed based on the discovery that there is a length at which particles start to collapse (hereinafter, referred to as critical collapse length).

That is, according to the first aspect of the present invention, the test liquid containing the reactive fine particles which can be mutually bonded by the biological reaction is used to reduce the fine particles formed after the reaction to the collapse region where the reactive fine particles collapse. Supplied to the streaky irregularities having, the state of collapse near the collapse start site of the irregularities is determined by observation or image analysis along the streak, and the presence or absence of a biological reaction based on the determination result This is an analysis method using reactive fine particles, which is characterized by being determined.

In the invention according to the first aspect, it is preferable that the uneven portion is a downward concave portion, an upward convex portion, or a step taken in both the up and down directions.

[0011]

Embodiments of the present invention will be described below with reference to the drawings. FIG. 1 is a plan view showing a microplate having wells, and FIG. 2 is a cross-sectional view of the microplate shown in FIG. The microplate 1 shown in FIG. 1 uses a chemical-resistant plastic material such as acryl on the top, and 8 × 12 wells 2 of the same configuration are arranged in a matrix on a substrate made of such a plastic material. Is formed.

Hereinafter, various configurations of the well will be described. FIG. 3 is a cross-sectional view of the well 2 provided in the analysis container according to the first embodiment of the present invention.

The well 2 shown in FIG. 3 is formed so as to have a substantially cylindrical shape. Further, a small upwardly projecting annular convex portion 3 is provided near the center of the flat well bottom surface 2a and the periphery thereof. The book is formed. The annular convex portion 3 has an inverted V-shaped cross section in which the peak portion 3a is sharp, and the inclined surface 3b
Is set to a length that allows each of the aggregated and non-aggregated patterns to be clearly and distinctly observed, and is set to a size shorter than the critical collapse length at which the aggregated particles begin to collapse.

The negative or positive state of the well 2 will be described below with reference to FIGS. 4, 5, 6 and 7. When discriminating aggregation negative in well 2 described above,
In the well 2 having the cross-sectional shape shown in FIG. 3, blood cell or particle reaction reagents (hereinafter, referred to as aggregated particles) do not aggregate, so that aggregated particles do not accumulate on the inclined surface 3b of the projection and the lower end 3c of the projection is flat. Surface (see FIG. 4). In particular, since the non-agglomerated particles collapse at the peak 3a of the convex portion quickly and exhibit a very sharp ring-shaped streak pattern along the peak line, it is possible to quickly determine the presence or absence of aggregation. Become.

When the well 2 shown in FIG. 4 at that time is viewed from above, the non-aggregated aggregated particles fall down from the peak 3a of the annular convex portion 3 on the well bottom surface 2a on a circular thin film of aggregated particles. Thus, one sharp annular collection pattern can be formed (see FIG. 5).

When discriminating the aggregation positive in the well 2 described above, since the aggregated particles in the well 2 generate aggregates, the aggregated particles also accumulate on the convex inclined surface 3b (see FIG. 6). . When the well 2 shown in FIG. 6 at this time is viewed from above, a circular aggregation pattern of uniformly aggregated particles can be seen on the well bottom surface 2a (see FIG. 7).

Further, in the first embodiment, the design may be changed to a convex portion having a step so as to be elevated like a plateau without forming the inclined surface 3b on the center side.

FIG. 8 is a sectional view of one well 2 provided in a reaction vessel according to a second embodiment of the present invention. The well 2 shown in FIG. 8 is formed so as to have a substantially cylindrical shape, and further, one small downwardly facing annular concave portion 4 is formed on the substantially flat bottom surface 2 a of the well 2.

The negative and positive states of the well 2 will be described below with reference to FIGS. When discrimination of aggregation negative in well 2 described above, FIG.
In the well 2 having the cross-sectional shape shown in FIG. 9, the agglomerated particles do not agglomerate, so that the agglomerated particles do not accumulate on the convex inclined surface 4b, but concentrate on the valley bottom portion 4a at the lower end of the convex portion and fall (see FIG. 9). Please see). When the well 2 shown in FIG. 9 at that time is viewed from above, the well bottom surface 2a has a circular thin film made of aggregated particles, a non-agglomerated portion formed by an annular concave inclined surface 4b, and a ring which divides the non-agglomerated portion. That is, it is possible to see an aggregation pattern composed of the dark annular aggregation portion along the valley line due to the aggregation particles that have collapsed at the valley bottom portion 4a (see FIG. 10). In particular, since the non-agglomerated particles collapse quickly at the upper end portion 4c of the convex portion and exhibit two sharp ring-shaped streak patterns, the presence or absence of aggregation can be quickly determined based on the unique pattern. Further, when the aggregation positive is determined in the well 2 described above, since the aggregated particles in the well 2 generate aggregates, the aggregated particles also deposit on the concave inclined surface 4b (see FIG. 11). FIG. 11 at this time
7, a circular aggregation pattern of uniformly deposited aggregated particles can be seen on the well bottom surface 2 a as in FIG. 7 (see FIGS. 11 and 12).

Further, in the second embodiment, the design may be changed to a convex portion having a step so as to be lowered like a plateau without forming the inclined surface 4b on the center side.

FIG. 13 is a sectional view of a well 2 provided in a reaction vessel according to a third embodiment of the present invention. The well 2 shown in FIG. 13 is formed to have a substantially cylindrical shape, and further, two small upward conical protrusions 5 are formed on the substantially flat well bottom surface 2a of the well 2.

In the third embodiment, the concave portion may be inclined so as to have a point-like bottom portion. In this case, the inclined portion near the top surface of the concave portion where the non-aggregated aggregated particles start to collapse. , A small-diameter annular pattern is observed.

The streak pattern in the above embodiment means that the container surface exposed by the collapse of the non-aggregated particles forms a linear pattern of two or more lines. Therefore, it may be two or more independent linear concavo-convex portions (convex portions and / or concave portions) or one continuous linear concavo-convex portion such as one-stroke drawing (for example, meandering shape, spiral shape, or the like). Further, the streak pattern may be partially crossed or intermittent.

The negative and positive states of the well 2 will be described below with reference to FIGS. 14, 15, 16 and 17. In the case of determining the aggregation negative in the well 2 described above, the aggregated particles do not aggregate in the well 2 having the cross-sectional shape shown in FIG. 5c collapses into a flat portion (see FIG. 14). When the well 2 shown in FIG. 14 at that time is viewed from above, two small circular non-aggregated portions formed by two conical convex portions 5 are formed on a circular thin film of aggregated particles on the well bottom surface 2a. The aggregate pattern can be seen (see FIG. 15).

In particular, since the non-agglomerated particles collapse quickly at the convex vertex portion 5a and exhibit two very small dot-like patterns, it is possible to determine the presence or absence of aggregation in a very short time.

When discrimination of aggregation positive is performed in the well 2 described above, since the aggregated particles in the well 2 generate aggregates, the aggregated particles also deposit on the convex inclined surface 5b (see FIG. 16). . When the well 2 shown in FIG. 15 at this time is viewed from above, the well bottom surface 2a has the same shape as in FIGS.
A circular aggregation pattern with uniformly deposited aggregated particles can be seen (see FIG. 17).

The protrusions and / or recesses for the distribution of the aggregated particles described above are of such an extent that the aggregated aggregated particles do not reach the above-mentioned collapse critical level at which the aggregated aggregates begin to collapse under their own weight, and are not aggregated. The groove is completed by forming a groove having an inclined surface enough to confirm that the aggregated particles have started to collapse. The configuration of such an inclined surface depends on the characteristics (diameter, shape, specific gravity, material, etc.) of the aggregated particles to be used, the physicochemical state of the well surface or the aggregated particle surface, and the properties of the liquid during the reaction (viscosity, specific gravity, etc.) It can be set experimentally as appropriate. The well 2 shown in FIG.
FIG. 18 shows a fourth embodiment of the present invention, and FIG.
FIG. 19 is a cross-sectional view of each of the wells 2, and FIG. 19 is a view further enlarging a portion of the bottom surface of the well 2a shown in FIG.

The appearance of the well 2 shown in FIG. 18 is formed so as to be substantially cylindrical, and a plurality of concentric grooves 6 having a similar convex shape are formed on the horizontal surface at regular intervals on the well bottom surface 2a. In this case, both the upward convex portions and the downward concave portions are provided continuously and alternately.
A sawtooth-shaped cross-sectional shape as shown in FIG.

The groove 6 has a depth such that, when the aggregation is negative, the aggregated particles can be quickly deposited on the valley bottom 6c, and when the aggregation is positive, the aggregated particles remain on the inclined surfaces 6b when falling, and do not collapse due to gravity or the like. Preferably, width and angle. Here, for general natural or artificial agglomerated particles (about 2 to 8 μm in diameter), the groove at the bottom of the well having an inner diameter (D) of 6 mm has a depth (h) of 0.1 to 0.15 mm.
, The groove width (d) is set in the range of 0.3 to 1.0 mm, preferably (d) is set in the range of 0.4 to 0.6 mm, and the inclination angle (θ) is set with respect to the horizontal. 5 to 60 °, preferably 10 to 45 °, particularly preferably 20 to 35 °, and the number of grooves may be appropriately determined according to the size of the well. The length of the slope is 0.05 to 1.5 mm for the aggregated particles having a diameter of 1 to 25 μm, and the diameter is 2 to 10 μm.
0.1 to 0.7 mm for agglomerated particles with a diameter of 3 to
0.18 to 0.5 mm is preferable for the 8 μm aggregated particles. Here, it is assumed that the number of grooves is plural.

The negative and positive states of the well 2 will be described below with reference to FIGS. 19, 20, 21 and 22. When judging the aggregation negative in the well 2 described above, the aggregated particles do not aggregate in the well 2 having the cross-sectional shape shown in FIG. 18, so that the aggregated particles are not deposited on the convex portions of the plurality of inclined surfaces 6 b. , Falls into the valley bottom portion 6c at the lower end of the convex portion (see FIG. 19). When the well shown in FIG. 19 at that time is viewed from above, the well bottom surface 2c has a non-aggregated portion due to the annular convex portion and a ring that divides the non-aggregated portion into two, that is, the aggregated particles that have fallen into the valley bottom portion 6c. (See FIG. 20). In particular, in the peak line shape of the peak portion 6a of the convex portion, not only a light-colored streak pattern due to the collapse of the non-agglomerated particles appears, but also the non- Since a dark streak pattern due to the agglomerated particles is generated, the clearest annular pattern can be quickly obtained by the synergistic effect.

In the case of judging the aggregation positive in the well 2 described above, since the aggregated particles in the well 2 generate aggregates, the aggregated particles also accumulate on the inclined surface 6b of the convex portion (FIG. 21).
Please refer to). At this time, when the well 2 shown in FIG. 21 is viewed from above, the well bottom surface 2a is shown in FIG. 7, FIG.
Similar to FIG. 7, a circular aggregation pattern due to uniformly deposited aggregation particles can be seen (see FIG. 22).

When the well having the above-described structure is used, the time required for all the particles to form an aggregation pattern can be shortened even if the number of particles used is almost the same as that of the conventional method. This leads to a reduction in time.

In addition, since there are a plurality of concentric aggregation points on the bottom of the well, it is easy to confirm the presence or absence of aggregation, and the accuracy of the aggregation image determination is improved.

The shape of the bottom surface of the well according to the present invention is not particularly limited to the cross-sectional shape as described above, and various changes or modifications are possible.

Conventionally, the following method has been employed for the quantitative measurement of antigens and antibodies in a sample by the agglutination method. That is, the sample is diluted twice, and it is determined whether or not agglutination occurs or not in each well for each diluted sample. The amount of the antigen or antibody was determined. Since the accuracy of the quantification was determined by the roughness of the dilution ratio, dilution was performed at a finer ratio in order to perform highly accurate measurement. However, in order to dilute the sample to the magnification of the aggregation limit, it is necessary to prepare a large number of diluted samples, so that a large number of dilution vessels are required for the determination. Therefore, when the above-described principle of detecting the particle aggregation pattern according to the present invention is used, generally, the discrimination accuracy can be variously changed depending on the degree of the inclination angle of the inclined surface of the well bottom surface. It is possible to make adjustments such as to enable detection or to make it difficult to detect even strong agglutination reactions by steepening the slope. A measurement can be made.

Here, by using a reaction vessel having a cross-sectional shape as described later, the number of diluted samples and agglutination determination vessels involved in sample preparation can be reduced, and more accurate quantitative measurement can be performed.

The depth of the groove for each groove on the bottom of the well will be described below.
Examples of capturing samples having different agglutination titers as patterns by changing the width, the inclination angle, and the like will be described with reference to FIGS. 23, 24, 25, 26, 27, and 28.

FIG. 23 shows that the depth of the groove cut in the well becomes shallower toward the center of the well (and
23 shows the container set. The well bottom is formed as a groove as shown in FIG. 23, so that the closer to the center, the smaller the amount of particles deposited on the slope of the groove, and the lower the peripheral part. You can do more as you go.

In this case, even if agglomerated particles having the same agglutination force are deposited, the particle agglomeration pattern is more stable toward the center of the shallower slope, and collapses toward the periphery, accumulates in the groove, and tends to become negative. Become.

Regarding the judgment, irrespective of the strength of the cohesion titer, if the titer is weak, even if there is a slight change, as shown in FIG. 24, particles accumulate from the peripheral groove and a streak-like portion is detected. If the titer is high, the number of detected grooves is small as shown in FIG.

As described above, according to the present invention, it is possible to accurately determine the aggregation pattern of the boundary region which is usually difficult to quantify.

The following FIGS. 25, 26, 27 and 28 are also made based on the principle of the machine, and the groove on the bottom of the well shown in FIG. The case where it is widened is shown.

By forming the bottom of the well with a groove as shown in FIG. 25, a small change can be represented as an aggregation pattern as shown in FIG. 25 regardless of the strength of the aggregation titer.

The groove at the bottom of the well shown in FIG. 27 shows a case where the groove width is constant and the depth of the groove is gradually increased from a shallow state at the center of the well 41 to a deep state.

By forming the groove on the bottom of the well as shown in FIG. 27, a small change can be represented as an aggregation pattern as shown in FIG. 28 regardless of the strength of the aggregation titer.

The coagulation pattern thus obtained can not only digitize the titer in one well when image processing or the like is performed, but also changes the depth, width, and inclination angle of the groove in the well (that is, changes in the well). Agglomerates that form a thin film in which weak antibodies can be formed by blood cells or particles, by successively increasing the depth toward the center or the angle being acute, or vice versa) However, the agglutination titer of an antibody having a weak titer that cannot be detected and quantified by a normal agglutination method can be compared and quantified in one well from the position of the groove that starts to collapse at the bottom of the groove.

Further, the wells shown in FIGS. 23 to 28 can be quantitatively compared with each other in terms of the agglutination titer. The value can be quantified.

For the above reasons, it is preferable to use the wells shown in FIGS. 23 to 28 for detecting an antibody having a weak agglutination titer (usually, a blood type back test).

In the present invention, the groove on the bottom surface of the well for forming the aggregated image is shallower than in the prior art, so that the aggregated pattern in a distinguishable state can be formed quickly. Can be increased and the sensitivity specificity can be improved.

As described above, by providing a plurality of types of groove angles and depths on the bottom surface of one well with respect to one well, it becomes possible to determine the agglutination titer with a smaller number of wells than usual.

In addition, for example, at present, when determining the agglutination titer of a certain specimen, a dilution series is prepared by sequentially diluting the specimen in a stepwise manner, and the number of wells of the diluted sample solution is used. Each agglutination reaction is performed, and the titer is determined depending on which dilution step agglutination was negative.On the other hand, as described above, if the titer can be quantified in one well The titer can be determined with a smaller number of wells without having to make a large number of dilution series.

In addition, by performing the total differentiation of the bottom of the well with an image analyzer using a CCD camera, more quantitative numerical data can be provided, and highly reliable judgment can be made.

Further, if a flat portion for monitoring the amount of blood cells is provided around the portion where the agglutination reaction is performed, the measured value can be corrected for samples having different blood cell concentrations.
Further, the planar shape of the well bottom surface is not limited to that of the above embodiment, but is shown in FIGS.
1 may be used.

In particular, the shape of the well shown in FIG. 31 is such that, by arranging a plurality of square pyramids regularly, the agglomerated particles when agglutination is negative do not accumulate on the point-like convex portions of the square pyramid. It is also possible to collapse on the inclined surface to form a lattice-like aggregation pattern of the aggregation particles on the bottom surface of the well, or to reverse the above-mentioned shape as it is.

Further, the upward conical convex portion shown in FIG. 13 is not limited to that of the embodiment and is not shown. For example, an upward quadrangular pyramid convex portion or an upward cylindrical convex portion may be obliquely formed. It can also be cut into shapes.

Also, the shape of the groove of the well is not limited to the above-described embodiment, and may be a shape inclined only in one direction as shown in FIGS.
As a result, when observing or analyzing the image from the inclined side,
A striped pattern can be formed.

Further, as shown in FIGS. 34 and 35, the collapse conditions are diversified by alternately arranging the convex portions and the concave portions with an appropriate flat portion interposed therebetween, so that the collapse characteristics of the aggregated particles can be improved. A clear linear pattern can always be obtained without dependence. In FIG. 34, since both the continuous slope and the stepped surface in which the convex portion and the concave portion are continuous are mixed,
It is preferable to design the continuous slope so that it does not become longer than the critical length of collapse.

Further, as shown in FIGS. 34 and 35, one or more sets of convex portions and concave portions which are inclined so as to have a point-shaped converging portion may be formed alternately.

Also, a non-slip small step 7 (depth of 2 to 2), which is formed on the inclined surface in a fine step shape as shown in FIG.
50 μm. (See JP-B-61-44268) to prevent agglomerated particles at the time of agglutination positive from collapsing on the inclined surface 8 and design the length of the radial slope relatively long. Even when the aggregated particles are easily collapsed, the stability of the aggregated pattern can be further increased.

If the interval between the fine step-like grooves is short, the stability can be increased even in the case of weak aggregation.

Further, by making the tops or valleys of each groove coincide with the same height, it is possible to make the temporal change of the aggregation pattern coincide.

On the other hand, if the heights are different, various reaction results can be determined based on the formation speed of the aggregation pattern.

The microplate described above is not limited to the one referred to in the embodiment. For example, when the number of inspection items is small, a microplate having 8 × 12 wells is too wasteful, so that eight microwells are used. Plate formed in a single vertical line or a container having only one well (see JP-A-1-109246)
Can be used, and an efficient inspection can be performed.

As described above, the depth and width of the well groove can be determined not only because a streak-like agglutination image can be formed on the bottom of the container, but also because the concentration of the blood cell or particle agglutinating reagent deposited when agglutination is negative under the reaction conditions. The method is not particularly limited as long as it has a width and a depth enough to be observed in a streak shape by an optical method or the naked eye.

Further, the material constituting the microplate is not limited to the chemical-resistant plastic material such as acrylic described in the embodiment, and for example, glass can be used as the material.

The present invention can be applied not only to the reaction by hemagglutination but also to any agglutination reaction that forms an agglutination pattern.

According to the analysis method or the analysis container of the present invention, by providing the above-mentioned uneven portion, a portion where the non-aggregated agglomerated particles start to collapse can be used for determination, whereby the agglutination or The difference in the ease of rolling of the aggregated particles due to the difference in non-aggregation can be expressed as a clear pattern difference at the beginning of the pattern formation process, and an aggregated pattern that is always quick and easy to identify can be obtained. A large number (preferably 4 or more) of streak, dot or ring pattern
This is important in facilitating judgment with the naked eye. The height of the arrangement of a plurality of (for example, two or more, preferably four or more) concaves and / or convexes in the present invention may be on an inclined surface. Since the aggregation pattern needs to be formed independently, the concave portions and / or convex portions at the high position and the concave portions and / or
Alternatively, the setting is made so that the aggregated particles do not flow into the convex portion. As described above, when a plurality of concave portions and / or convex portions are arranged on the inclined surface, a difference in brightness between the aggregation and the non-aggregation appears, so the number of aggregated particles is changed according to the item to be analyzed. When necessary or when the amount of particles is unknown or fluctuates, such as the agglomerated particles of the sample itself (e.g., red blood cells), any recesses and / or protrusions will sediment with the best amount of particles to provide the best S. / N ratio, and data correction according to the particle amount can be eliminated. However, a plurality (for example, 2
The height of the concave portion and / or the convex portion (preferably 4 or more) is more preferably on a horizontal plane. This is because the number of agglomerated particles settling on a horizontal plane is uniform in each concave portion and / or convex portion, so that each concave portion and / or
Alternatively, since the brightness difference between the aggregated and non-aggregated developed in the convex portion is all the largest, the larger the number of concave portions and / or convex portions, the easier the macroscopic judgment such as visual observation becomes. . The difference in lightness between the case where the non-aggregated particles cover the container surface and the case where the non-agglomerated particles do not cover the container surface is most emphasized by the method and the container of the present invention, so that it is easy to identify with the naked eye. Not only CC
The accuracy of quantitative determination is also improved in automatic measurement in which image data of the bottom surface of a container captured by an image capturing means such as D is compared with a brightness difference for each pixel by calculation such as differential analysis. In the uneven portion of the container, the longer and / or more the initial collapsible portion where the non-aggregated aggregated particles start to collide can be determined by a measuring means with lower sensitivity. However, the same effect can be obtained by using a measuring device having a resolution capable of measuring a local area (high-density CCD, laser microscope, etc.) or observing the naked eye with a magnifying lens even when the initial portion of collapse is short or small. You can enjoy the effect.

[0068]

An embodiment using a well according to the present invention will be described below with reference to the drawings.

The well according to the embodiment of the present invention is shown in FIGS.
It has the same configuration as that shown in FIG. 20, and has an inclined groove formed of a concavo-convex portion in which a concave portion and a convex portion are continuous in a saw-like shape on the bottom of an acrylic well having a diameter of about 6 mm. Further, the groove has a depth (h) of 0.1 mm, a width (d) of 0.2 mm, an inclination angle of 30 °, a length of the inclined surface in the inclination direction of 0.17 mm, and a number of grooves of four (that is, four). , Four peak lines and four valley lines). On the other hand, what was used as a comparative example is a well described in Japanese Patent Publication No. 1-21544, in which an acrylic well having a diameter of about 6 mm is provided concentrically at a right angle and has a step without inclination. Approximately 4mm in diameter to form the aggregation pattern
(See FIG. 39). The shape of the bottom surface of the second reduced well is a substantially conical shape that is concave downward like a conventional general well, and has a cross section inclined in a V-shape (Japanese Patent Publication No. 61-42868). No. 2), the depth (h) is about 1 mm, the width (d) is about 2 mm, the inclination angle is about 30 °, and the length of the inclined surface in the inclination direction is about 1.7 mm. It has become.

[Blood type frontal test] In the blood type frontal test of this example, a microplate similar to that described in the embodiment was used, and the blood type was determined for an antibody (hereinafter referred to as an antibody reagent stock solution). Monoclonal antibodies anti-A and anti-B for
The determination is performed using the blood type determination polyclonal antibody anti-D.

As antigens, human erythrocytes of type A (Rh +), type B (Rh +), type O (Rh +), type AB (Rh +), type A (Rh−) are used. Is diluted with physiological saline to prepare a 1.5% blood cell suspension.

First, each of the blood cell suspensions as antigens and 3
Each well formed in a matrix on the plate is subjected to 5
To each column, add 25 μL of the diluted antibody reagent stock solution diluted 1: 2 (5 rows (number of antigen types) × 3 columns (number of antibody types)).

To each well to which the addition of the antibody was completed, 25 μL of each antigen was further added in each row (5 rows (number of types of antigen) × 3 columns (number of types of antibody)), and various reaction solutions were added. It is made and left to stand after lightly stirring with a plate mixer to promote the reaction of various reaction solutions.

Next, about 5 minutes after the start of the standing, whether the reaction solution was negative or positive was confirmed by the difference in the sedimentation pattern of each well. After 10 to 15 minutes, the distinction between negative and positive by the naked eye can be determined from the aggregation pattern.

FIG. 37 is a schematic diagram showing a reaction example (about 15 minutes after the start of the reaction) when the above-mentioned red blood cell blood type front test was performed. It can be seen that if the blood cells spread uniformly on the bottom of the well and the blood cells sediment concentrically (ie, spirally) in the negative case, the positive and negative patterns can be clearly distinguished by the naked eye.

According to this well, the entire well is darkened when aggregation occurs, and the entire well is brightened when no aggregation occurs, so that it becomes clear at a glance with the naked eye. On the other hand, FIG. 38 shows a reaction example obtained 35 minutes after the start of the reaction using the conventional well for speeding-up shown in FIG. 39. When the whole bottom surface of the well is darkened and no aggregation occurs, a round dark spot is observed at the center of the well only when non-aggregated aggregated particles are sufficiently collapsed in the center of the well. Until the aggregated particles were sufficiently disintegrated, it was not possible to distinguish between positive and negative, and it was not possible to predict the judgment at least about 30 minutes after the start of the reaction. As described above, in this embodiment, the length of the inclined surface of the groove on the well according to the present invention in the inclined direction is 10 minutes smaller than the length of the reduced well according to the prior art in the inclined direction. By doing 1,
A clear pattern difference is obtained so that the prediction judgment of aggregation or non-aggregation can be made within 5 minutes with the naked eye, and the formation of the aggregation pattern is completed in about 10 to 15 minutes, and a clear pattern difference is obtained. Can be Since the well shown in FIG. 39 is a vertical and conical concave portion, it does not have an inclined portion where non-aggregated aggregated particles start to collapse, and a clear pattern as in the present invention is not observed.

As described above, the time required from the start of the reaction until the state where the aggregation pattern can be determined can be reduced from the conventional 60 minutes to about 15 to 20 minutes.

The same experiment as described above was carried out for 1.6% erythrocytes and a 16-fold dilution of the antibody at different concentrations, and a difference in the aggregation pattern began to appear in 10 minutes.

[0079]

According to the present invention, in a detection system for detecting the presence or absence of an antigen-antibody reaction by using an agglutination reaction, it is possible to make a determination in a shorter time than before.

[Brief description of the drawings]

FIG. 1 is a plan view showing a microplate having wells.

FIG. 2 is a cross-sectional view of the microplate of FIG. 1 taken along line AA ′.

FIG. 3 is a sectional view of a well provided in an analysis container according to a preferred embodiment of the present invention.

FIG. 4 is a cross-sectional view of the well of FIG. 3 in the case of agglutination negative.

FIG. 5 is a diagram showing the bottom surface of the well in FIG. 3 when the aggregation is negative.

FIG. 6 is a cross-sectional view of the well in FIG. 3 when the aggregation is positive.

FIG. 7 is a diagram showing the bottom surface of the well in FIG. 3 when the aggregation is positive.

FIG. 8 is a cross-sectional view of a well provided in an analysis container according to a preferred embodiment of the present invention.

FIG. 9 is a cross-sectional view of the well of FIG. 8 in the case of agglutination negative.

FIG. 10 is a diagram showing the bottom surface of the well of FIG. 8 in the case of agglutination negative.

FIG. 11 is a cross-sectional view of the well in FIG. 9 in the case of positive aggregation.

FIG. 12 is a diagram showing the bottom surface of the well in FIG. 9 in the case of aggregation positive.

FIG. 13 is a sectional view of a well provided in an analysis container according to a preferred embodiment of the present invention.

FIG. 14 is a cross-sectional view of the well of FIG. 13 in the case of agglutination negative.

FIG. 15 is a diagram showing the bottom surface of the well in FIG. 13 in the case of agglutination negative.

FIG. 16 is a cross-sectional view of the well in FIG. 13 in the case of positive aggregation.

FIG. 17 is a diagram showing the bottom surface of the well in FIG. 13 when the aggregation is positive.

FIG. 18 is a cross-sectional view of a well provided in an analysis container according to a preferred embodiment of the present invention.

FIG. 19 is a cross-sectional view of the well of FIG. 18 in the case of agglutination negative.

FIG. 20 is a diagram showing the bottom surface of the well of FIG. 18 in the case of agglutination negative.

FIG. 21 is a cross-sectional view of the well of FIG. 18 when the aggregation is positive.

FIG. 22 is a diagram showing the bottom surface of the well in FIG. 18 when the aggregation is positive.

FIG. 23 is a cross-sectional view of a well provided in an analysis container according to a preferred embodiment of the present invention.

FIG. 24 is a diagram showing a bottom surface of a well provided in an analysis container according to a preferred embodiment of the present invention.

FIG. 25 is a cross-sectional view of a well provided in an analysis container according to a preferred embodiment of the present invention.

FIG. 26 is a diagram showing a bottom surface of a well provided in an analysis container according to a preferred embodiment of the present invention.

FIG. 27 is a cross-sectional view of a well provided in an analysis container according to a preferred embodiment of the present invention.

FIG. 28 is a diagram showing a bottom surface of a well provided in an analysis container according to a preferred embodiment of the present invention.

FIG. 29 is a diagram showing a bottom surface of a well provided in an analysis container according to a preferred embodiment of the present invention.

FIG. 30 is a diagram showing a bottom surface of a well provided in an analysis container according to a preferred embodiment of the present invention.

FIG. 31 is a diagram showing a bottom surface of a well provided in an analysis container according to a preferred embodiment of the present invention.

FIG. 32 is a view showing a groove pattern on a bottom surface of a well provided in an analysis container according to a preferred embodiment of the present invention.

FIG. 33 is a view showing a groove pattern on a bottom surface of a well provided in an analysis container according to a preferred embodiment of the present invention.

FIG. 34 is a view showing a groove pattern on a bottom surface of a well provided in an analysis container according to a preferred embodiment of the present invention.

FIG. 35 is a view showing a groove pattern on a bottom surface of a well provided in an analysis container according to a preferred embodiment of the present invention.

FIG. 36 is a view showing a groove pattern on a bottom surface of a well provided in an analysis container according to a preferred embodiment of the present invention.

FIG. 37 is a view showing the results obtained when a red blood cell blood type frontal test is performed using the reaction container of the present invention.

FIG. 38 is a view showing a result obtained when a red blood cell blood type frontal test is performed using a conventional reaction vessel.

FIG. 39 is a cross-sectional view of a well provided in a conventional analysis container.

[Explanation of symbols]

 1. Microplate 2. Well

 ──────────────────────────────────────────────────続 き Continuing on the front page (72) Inventor Noriko Kato 2-43-2 Hatagaya, Shibuya-ku, Tokyo Inside Olympus Optical Industrial Co., Ltd. (72) Inventor Yuji Takamiya 2-43-2 Hatagaya, Shibuya-ku, Tokyo Olympus Optical Co., Ltd. F term (reference) 2G058 AA09 BA01 CC02 CC17 FA03 4B029 AA07 AA08 BB11 CC01 FA05 FA15 GA03 GB06 GB10 4B063 QA05 QA14 QQ03 QQ08 QQ96 QR48 QS33 QS39 QX01

Claims (5)

[Claims] 1. An analysis method for analyzing a biological reaction based on a distribution image of a group of fine particles formed after a reaction using a test liquid containing a group of reactive fine particles that can bind to each other by a biological reaction. The reactive fine particles after the reaction are supplied to an inclined portion having a collapse region where the reactive fine particles collapse, and the collapse state near the collapse start site of the inclined portion is determined by observation or image analysis along a line. And analyzing the presence or absence of a biological reaction based on the determination result. 2. A method according to claim 1, wherein said reactive fine particles are continuously formed on said concave / convex portion having an inclined region and / or a concave portion having a collapse region in which reactive fine particles capable of binding to each other due to a biological reaction collapse. An analysis container using a reactive fine particle having a liquid storage part capable of being held in the container. 3. A method according to claim 1, wherein the reactive fine particles have a collapsed area in which reactive fine particles that can bind to each other due to a biological reaction are collapsed, and an inclined concave / convex part having a convex part and / or a concave part having a dot-like top or bottom. For analysis using reactive fine particles. 4. The analysis container according to claim 2, wherein a plurality of uneven portions are integrally formed. 5. The uneven portion is such that the aggregated particles after the aggregation reaction do not reach a critical level at which the aggregated aggregated particles begin to collapse under their own weight, and the non-aggregated aggregated particles are collapsed. The analytical container according to claim 2 or 3, wherein the analytical container has a diameter slope whose length is such that the start can be confirmed.