JPH08211061A - Immunological inspection method - Google Patents

Abstract

PURPOSE: To provide an immunological inspection method which can execute antigen antibody reaction as well as measurement with a less number of processes, easily execute a series of processes from reaction to measurement within one reaction container even without using any special reaction container, and has a wide inspection application range. CONSTITUTION: A blood sample and a solid phase carrier 7 coated with a number of luminols is dispensed into a reaction container 1, antigen antibody reaction is performed while maintaining the reaction liquid on a filter 6, and a single-substance solid-phase carrier 7 and a liquid phase which are not subjected to reaction are passed through a filter 6 and is forced to be filtered toward the lower portion, thus leaving a combined solid-phase carrier 7 onto the filter and performing optical measurement using a light reception element 11 while peroxidase and H2 O2 are being dispensed into the reaction container 1 as liquid reagents.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to an immunological test method for detecting a specific antigen or antibody based on the result of an antigen-antibody reaction.

[0002]

2. Description of the Related Art Conventionally, the heterogeneous EIA method has been widely used as an immunological test method. Among them, the sandwich method is the method with the widest application range and high sensitivity. FIG. 4 shows Prior Art 1 as an example thereof. In the method shown in the figure, first, in step 1, a sample is dispensed into the reaction container 1 in which the antibody 2 is immobilized on the inner wall surface. Next, in step 2, the first reaction is performed, and when the target antigen is present in the sample, the antibody 2 and the antigen 3 are reacted by the antigen-antibody reaction.
And combine. Next, in step 3, B / F separation is performed in which the antigen that has not bound to the antibody 2 and the antigen that has not bound to it are separated by a washing operation, and only the antigen that has bound to the antibody 2 remains inside the reaction container 1. Next, in step 4, the enzyme-labeled antibody 4 is dispensed. In step 5, the second reaction is carried out, the antigen 3 bound to the antibody 2 and the enzyme-labeled antibody 4 are bound by the antigen-antibody reaction, and the antibody 2 and the enzyme-labeled antibody 4 are sandwiched with the antigen 3 sandwiched therebetween. The enzyme-labeled antibody 4 forming a bound substance and bound to the antigen 3 is fixed to the inner wall of the reaction container 1.

Here, when the antigen 3 is not present in the sample, the antibody 2 and the enzyme-labeled antibody 4 cannot be linked, so that the enzyme-labeled antibody 4 is not fixed to the inner wall of the reaction tube.
Next, in step 6, the enzyme-labeled antibody that has bound and the enzyme-labeled antibody that has not bound are separated by washing. B /
The F separation is performed, and only the labeled antibody that binds to the antigen 3 and is fixed to the inner wall of the reaction container 1 remains in the reaction container 1.

Next, in step 7, a color-developing substrate that reacts with the enzyme 5 bound to the enzyme-labeled antibody 4 to develop a color is dispensed, and in step 8, the color-developing reaction is carried out.
In step 9, the degree of color development is measured by a colorimetric method. When the antigen 3 is not present in the sample, the enzyme-labeled antibody is washed away by the B / F separation operation in step 6 and does not remain in the reaction vessel, so the color reaction does not occur. The degree of color development in the above color development reaction varies depending on the amount of the bound enzyme. In addition, the amount of the antigen 3 bound to the solid-phased antibody 2 is different depending on the amount of the antigen in the sample, and therefore the amount of the enzyme-labeled antibody bound to the bound antigen 3 is also different. By knowing the degree, the amount of the antigen to be tested in the sample can be known.

Next, FIG. 5 shows a conventional technique 2 (see Japanese Patent Laid-Open No. 63-281053) as another example. In the example shown in the figure, a cylindrical reaction container 1 filled with a filter 6 having fine pores formed of glass fiber or the like contains a large number of solid phase carriers 7 each having an antibody immobilized on the surface of fine particles.
Dispense the reaction reagent and the sample. The pore size of the fine pores of the filter 6 is such that the solid phase carrier 7 cannot pass through the filter 6 either by itself or by washing, and is caught on or inside the filter and remains.

The sample dispensed in step 1 and the solid phase carrier 7
Performs the first reaction in step 2, and when the target antigen is present in the sample, the antibody 2 immobilized on the solid phase carrier 7 and the antigen 3 in the sample cause an antigen-antibody reaction. Antigen 3
Bind to the surface of the solid support 7. Next, in step 3, B / F separation is carried out in which the antigen bound to the solid phase carrier 7 and the antigen not bound are separated by a washing operation. That is, when the washing solution is injected from above the reaction container 1, the antigen or antibody not bound to the solid phase carrier passes through the filter 6 together with the washing solution and flows out downward. As a result, only the antigen bound to the surface of the solid phase carrier 7 remains in the reaction container 1.

Next, in step 4, the enzyme-labeled antibody 4 is dispensed, the second reaction is carried out in step 5, and the antigen 3 and the enzyme-labeled antibody 4 bound on the surface of the solid phase carrier 7 in the reaction container 1
Bind to each other by an antigen-antibody reaction, the antibody 2 and the enzyme-labeled antibody 4 form a sandwich binding product sandwiching the antigen 3, and the enzyme-labeled antibody 4 bound to the antigen 3 is attached to the surface of the solid phase carrier. It will be fixed. Here, when the antigen 3 is not present in the sample, the enzyme-labeled antibody 4 and the antibody 2 cannot be linked, so that the enzyme-labeled antibody 4 is not fixed on the surface of the solid phase carrier.

Next, in step 6, B / F separation is carried out in which the enzyme-labeled antibody immobilized on the surface of the solid phase carrier 7 and the unlabeled antibody are washed and separated in the same manner as in step 3 above. . After B / F separation, in step 7, a fluorescent substrate that reacts with the enzyme 5 labeled on the enzyme-labeled antibody 4 to emit fluorescence is dispensed, and the generated fluorescence is measured by the light receiving element 11, and the amount of light is measured. It is possible to know the amount of the antigen to be tested in the sample.

FIG. 6 shows a prior art 3 as still another example. In the illustrated example, first, in step 1, in a reaction container 1 having an antibody 2 immobilized on the inner wall according to the purpose of inspection, a sample and a labeled antigen obtained by labeling the same antigen as the target antigen 3 in the sample with an enzyme 5 are labeled. When 8 is mixed, in step 2, the antigen 3 and the labeled antigen 8 in the sample competitively react with the antibody 2, and the amount of each bound to the antibody 2 depends on the ratio of the amounts of the antigen 3 and the labeled antigen 8. If the dispensing amount of the labeled antigen 8 is fixed and the amount of the antigen 3 bound to the antibody 2 is determined depending on the amount of the antigen 3 in the sample.

In step 3, B / F separation is performed,
After removing the excess antigen 3 and other coexisting substances that were not involved in the reaction, in step 4 a color-developing substrate that reacts with the labeling enzyme 5 to develop a color is dispensed, and after the color reaction is performed in step 5. In step 6, the amount of the target antigen in the sample can be known by applying the excitation light from the light source 9 and comparing the degree of color development with the light receiving element 11.

[0011]

DISCLOSURE OF THE INVENTION Problems to be Solved by the Invention
In contrast, since the immune reaction was performed twice, the operations such as B / F separation were complicated and the time until the results were obtained was long. In addition, since the solid phase of the immobilized antibody is limited to the inner wall surface of the reaction vessel, if there are many opportunities for contact between the antigen in the test solution and the immobilized antibody, and the reaction is to be performed stably and quickly, There was an inconvenience that the stirring operation had to be performed constantly during the reaction process.

Further, in the prior art 2, since the antibody is immobilized on the surface of a large number of fine particles, the fine particles are uniformly dispersed in the test solution in the reaction step, and the antigen-antibody reaction is carried out, which is a special case. Even if the stirring operation is not performed, there are many opportunities for contact between the antigen in the test solution and the antibody on the surface of the microparticles, and a rapid antigen-antibody reaction can be obtained, but the immune reaction is required twice and B / F separation or the like is required. The operation accuracy is complicated, and it takes time to obtain the inspection result.

Further, in the prior art 3, the immune reaction is carried out only once, but as in the prior art 1, there is the trouble that a stirring operation is always required during the reaction process, and in addition, the reaction vessel is preliminarily loaded with an antibody or the like. Since there is an extra step of fixing, there is a drawback that it cannot be applied to a wide range of test items because the types of applicable antigens or antibodies are limited. The present invention solves the above-mentioned drawbacks of the prior art, can stably carry out the antigen-antibody reaction to the measurement in a small number of steps, and can perform the reaction to the measurement in one reaction container without using a special reaction container. It is an object of the present invention to provide an immunological test method which can easily carry out a series of steps and has a wide range of test applications.

[0014]

The immunological test method of the present invention which has achieved the above object is a solid phase in which an antigen or an antibody according to the purpose of the test is immobilized on a sample to be tested and fine particles which can be optically measured. The step of supplying the carrier with a carrier into a reaction vessel having a filter capable of holding a liquid phase for at least a fixed time as a bottom surface, and a reaction solution containing the sample and the solid phase carrier after the reaction while the antigen-antibody reaction is performed. What is provided with a step of keeping in a container, a step of forcibly filtering the liquid phase retained on the filter by negative pressure, and a step of optically measuring fine particles remaining in the filter after filtration. Is.

Here, it is preferable that the surface of the fine particles is coated with a labeling substance, and in particular, the fine particles are coated with a luminescent substance, and the presence of a liquid reagent that causes luminescence in the optical measurement step. It is preferably carried out below. The labeling substance used in the present invention includes a chromogenic substance, a fluorescent substance, an enzyme, a radioactive substance, a luminescent substrate and a fluorescent substrate.

[0016]

EXAMPLE 1 This example is a method suitable for testing hormones, viruses, tumor markers, drugs, etc. in serum, which will be described below with reference to FIGS. 1A and 1B. Diameter 3 with fluorescent material coated on the surface
Solid phase carrier 7 is prepared by sensitizing fine particles of about μm with antibody 2.
Used as

FIG. 1A shows the case where an antigen for inspection is present in the sample, and FIG. 1B shows the case where the antigen for inspection is not present in the sample. First, in FIG. 1A, in step 1, one solid phase carrier 7 is allowed to pass, but a plurality of bound substances cannot be passed, and in this example, a cylindrical reaction vessel 1 filled with a microporous filter 6 of about 5 μm is used. A reaction liquid containing a large number of the sample and the solid phase carrier 7 was dispensed and mixed on the filter 6. In FIG. 1A, the antigen 3 to be inspected in the sample is the antigen-antibody reaction in step 2, and the filter 6 holds the reaction solution in the reaction container 1, with the antigen 3 as an intermediary between the solid phase carriers 7. Aggregation due to a so-called agglutination reaction in which the agglomerates are bound to each other is stable, and aggregates are formed. On the other hand, in FIG. 1B, the target antigen is not present in the sample, and there is nothing that mediates and binds the solid phase carriers to each other, so that no aggregation occurs. Next, in step 3, a washing operation was performed. In this example, the cleaning solution is injected from above the reaction container 1 after the reaction time and is forcibly sucked by the negative pressure from below the reaction container 1 at the same time, but the cleaning solution is not absorbed. Various methods are conceivable, such as a method in which the cleaning solution injected from the upper part of the filter 6 is brought into contact with the lower part of the filter 6 filled with the substance in the same reaction container 1 and the cleaning liquid is sucked from the lower part of the filter 6.

Here, in FIG. 1A, an agglutination reaction occurs and a plurality of solid phase carriers 7 are bound to each other, and the bound solid phase carriers remain on the filter. The phase carrier and other reaction reagents in the sample passed through the filter 6 and were forcedly filtered downward. Figure 1B
In (1), since the target antigen does not exist in the sample, the agglutination reaction does not occur, and all the solid phase carriers are simple substances, the solid phase carrier does not remain on the filter 6 by the washing operation.

Next, in the case of FIG. 1A, in step 4, when excitation light is applied to the surface of the filter 6 by the light source 9, the fluorescent substance coated on the surface of the solid phase carrier 7 remaining on the surface of the filter 6 is excited. Since it emits fluorescent light, the intensity of this fluorescent light is passed through an appropriate optical filter 10 to a light receiving element 11
It was measured at. Since the fluorescence intensity measured here is related to the number of aggregated solid-phase carriers 7 remaining on the filter 6, and the number of aggregated solid-phase carriers is related to the amount of the antigen in the sample, By knowing the fluorescence intensity, the amount of the target antigen in the sample can be known. The above-mentioned agglutination reaction is carried out in a state in which the test liquid containing a large amount of fine-particle solid phase carriers in a small amount of liquid is spread over the surface of the filter 6 in an extremely thin test liquid layer and the solid phase carriers are densely packed. Since the antigen 3 in the sample and the antibody 2 on the surface of the solid-phase carrier 7 come into contact with each other extremely often, the agglutination reaction is performed quickly, and the test result can be known quickly, and from the reaction to the photometry. It was possible to easily carry out the series of inspection steps described above in one reaction vessel. Example 2 This example is also a method suitable for testing hormones, viruses, tumor markers, drugs, etc. in serum, in which luminol is used as a luminescent substance and the antibody is sensitized to fine particles coated with luminol on the surface. It was used as the solid phase carrier 7.

A description will be given below with reference to FIGS. 2A and 2B. Steps 1 to 3 of the reaction process of FIG. 2A are the same as those in Example 1, but the substance that causes luminol to emit light in the reaction vessel 1 forcibly filtered in Step 4, that is, in this example, peroxidase (POD) is used. ) And H ₂ O ₂ were dispensed as liquid reagents for optical measurement. At this time, since the luminol coated on the surface of the solid-phase carrier that aggregates and remains on the filter 6 emits light in the presence of the liquid reagent, the emission intensity is measured by the light receiving element 11 through the appropriate filter 10. The concentration of the target antigen in the sample could be known accurately.

Further, as shown in FIG. 2B, when the target antigen is not present in the sample, the agglutination reaction does not occur.
Since the solid-phase carrier 7 does not remain on the filter 6 after the washing operation by forced filtration in step 4, even if peroxidase (POD), H ₂ O _{2 and the} like are dispensed into the reaction vessel 1 in step 4, light emission occurs. Since it did not occur, it was found that the target antigen was not present in the sample. As described above, according to this example, as in Example 1, the agglutination reaction is rapidly performed, and even when the liquid reagent for luminescence (or color development) is used, the reaction from the antigen / antibody reaction to the photometry is performed. It has been found that a series of inspection steps can be easily and stably carried out with one reaction vessel.

In the above Examples 1 and 2, if the antigen for the target antibody in the sample is immobilized on the surface of the solid phase carrier 7, the antibody in the sample can be detected. Example 3 This example shows the case of determining the ABO blood group for blood group determination by hemagglutination.

A description will be given below with reference to FIGS. 3A and 3B. In step 1 shown in FIG. 3A, a sample containing red blood cells (whole blood or red blood cell suspension) and anti-A serum anti-serum were dispensed into reaction container 1 and mixed. Next, in step 2, an antigen-antibody reaction is carried out, a stable agglutination reaction occurs with the blood group antigen 16 corresponding to the antibody 2 in the dispensed antiserum on the surface of the red blood cells 15 in the sample, and the red blood cells are separated from each other. The antibody 2 was mediated and aggregated to form an aggregate on the filter 6.

On the other hand, in the case shown in FIG. 3B, when the blood group antigen 16 corresponding to the antibody 2 in the dispensed antiserum does not exist on the surface of the red blood cells 15 in the sample, the agglutination reaction does not take place, and therefore Only the single red blood cells 15 are present on or in the filter 6. Next, in step 3, a washing operation was performed. The washing method is the same as in Example 1.

Then, the extra red blood cells 15 and other coexisting substances that were not involved in the agglutination in the case of FIG. As shown in FIG. 3A-a, a clear agglutination image 14 of only agglutinates without blurring due to excess red blood cells that were not involved in agglutination could be obtained, as shown in FIG. 3A-a. In step 4, the pattern is recognized by the image sensor 13 or the like through the optical system such as the lens 12 and the aggregation is determined or visually determined.

On the other hand, in the case of FIG. 3B, when the washing operation is carried out in step 3, since all the red blood cells on the filter are single substances, all the red blood cells and other coexisting substances in the test solution are removed by filtration through the filter 6. Since no red blood cells remain on the filter 6 as shown in FIG. 3B-b,
When this is subjected to pattern recognition by the image sensor 13 or visually observed in step 4, it can be clearly seen that there is no aggregation.

In the above reaction, if anti-A serum and anti-B serum are used as antisera, ABO blood group can be determined, and by using other various antisera, various blood groups can be determined. it can. Also, using serum or plasma as a sample, O for irregular antibody screening
By performing the above operation using type blood cells as a reagent, irregular antibodies in the sample can also be detected.

[0028]

As described above, according to the present invention, 1) the antigen / antibody reaction and the filtration step are completed once,
A series of steps from reaction to measurement can be easily and stably carried out in one reaction vessel. 2) Conventional enzyme immunoassay (EIA), radioimmunoassay (RIA), luminescent immunoassay (LIA or C)
LEIA) and fluorescent immunoassay (FIA) as well as hormones, viral antigens or viral antibodies,
It can be applied to tests for tumor markers, drugs, etc., as well as blood group determination performed by the so-called agglutination method, irregular antibody screening, virus detection, and the like.

[Brief description of drawings]

FIG. 1A is a process diagram of a method according to a first embodiment. FIG.
B is a process drawing of the target method of Example 1. FIG.

FIG. 2A is a process drawing of the method of the second embodiment. Figure 2
B is a process drawing of the target method of Example 2. FIG.

FIG. 3A is a process drawing of the method in Example 3; FIG.
Aa is an explanatory view showing an agglutination image of red blood cells on the filter after being filtered in step 3 of Example 3. FIG. FIG. 3B is a process diagram of the target method of the third embodiment. FIG. 3B-b is an explanatory view showing the state on the filter after filtering in step 3 of the target method of Example 3.

FIG. 4 is a process diagram of a method of Prior Art 1.

FIG. 5 is a process diagram of a method of Prior Art 2.

FIG. 6 is a process diagram of a method of Prior Art 3.

[Explanation of symbols]

 1 Reaction Container 2 Antibody 3 Antigen 4 Enzyme Labeled Antibody 5 Enzyme 6 Filter 7 Solid Phase Carrier 8 Labeled Antigen 9 Light Source 10 Filter 11 Photoreceptor 12 Lens 13 Imaging Device 14 Aggregation Image 15 Erythrocyte 16 Blood Group Antigen

Claims (3)

[Claims] 1. A filter having, as a bottom surface, a sample to be inspected and an optically measurable fine particle on which a solid phase carrier in which an antigen or an antibody according to the purpose of the inspection is immobilized is immobilized for at least a fixed time. The step of supplying the reaction solution into the reaction vessel, the step of maintaining the test solution containing the sample and the solid phase carrier after the supply in the reaction vessel during the antigen-antibody reaction, and the negative pressure of the liquid phase retained on the filter And a step of optically measuring fine particles remaining on the filter after filtration, which is an immunological test method. 2. The immunological test method according to claim 1, wherein the surface of the fine particles is coated with a labeling substance. 3. The immunological test according to claim 2, wherein the labeling substance is a luminescent or chromogenic substance, and the optical measurement step is carried out in the presence of a liquid reagent which causes luminescence or coloring. Law.