JP2001183377A - Immunological inspection method for many items - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide an immunological analysis method whereby a plurality of items measured at a time can be more clearly identified and a high inspection accuracy is exhibited. SOLUTION: Two or more substances are detected by the immunological analysis method. The method includes a step (1) of mixing a sample and a solid phase carrier particle obtained by solidifying a probe which can form a composite with a substance to be detected for two or more particles of difference content structures and/or surface structures, thereby forming the composite including both the substance to be detected which is included in the sample and the solid phase carrier particle, a step (2) of labelling the object to be detected by a labelling substance, and a step (3) of irradiating the solid phase carrier particle with light and detecting a forward scattering light and a side- way scattering light obtained by the irradiation and an amount of the labelling substance.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to an immunological analysis method.

[0002]

2. Description of the Related Art An immunological analysis is an analysis utilizing an antigen-antibody reaction. Basically, it is a method that uses an antibody or an antigen that specifically binds to a target substance and labels them, thereby enabling accurate detection of a trace substance. At present, it is generally used for detecting various substances in many fields including medicine. However, many improvements have been made in order to further improve the measurement sensitivity and accuracy and to promptly perform the measurement.

[0003] For example, Japanese Patent Publication No. 6-65889 discloses an immunological analysis method for simultaneously measuring multiple items.
In this method, for each carrier particle having a different particle size, a carrier particle on which an antigen or an antibody corresponding to each analysis item is prepared is prepared, and the carrier particle and the sample are reacted in a channel, and further, a labeled substance is used. Labeling, the obtained immunocomplex comprising the substance to be detected, the carrier particles, and the labeling substance, for each complex by a flow system such as flow cytometry, This is a method for determining the amount of the labeling substance. However, the number of items that can be measured by such a method is limited, and it is difficult to identify aggregates and the like, so that there is a problem in measurement accuracy.

[0004]

SUMMARY OF THE INVENTION In view of the above circumstances, an object of the present invention is to provide an immunological analysis method capable of simultaneously measuring more items. A further object of the present invention is to provide an immunological analysis method that can more clearly identify a plurality of items measured collectively and that has high test accuracy.

[0005]

The above objects can be achieved by the present invention described below. That is, an analysis method for detecting two or more substances, wherein (1) a probe capable of forming a complex with a substance to be detected for two or more particles having different content structures and / or surface structures. Mixing a solid support particle obtained by solidifying the above with a sample to form a complex containing the substance to be detected contained in the sample and the solid support particle; (2) A step of labeling the detection target with a labeling substance, and (3) a step of irradiating the solid-phase carrier particles with light and detecting the forward scattered light and side scattered light obtained thereby and the amount of the labeling substance. And a method comprising:

[0006]

BEST MODE FOR CARRYING OUT THE INVENTION DETAILED DESCRIPTION OF DETECTION METHOD The method of the present invention is a method for simultaneously and highly accurately detecting two or more substances to be detected contained in trace amounts in a sample. In addition, the present method uses a binding pair that specifically binds (for example, an antigen and an antibody, a single-stranded DNA and a complementary strand thereof)
This is a method for detecting any one of the above.

The method is based on an antigen-antibody reaction. That is,
In order to detect one of the binding pairs that specifically binds, solid phase carrier particles having the other of the binding pairs fixed to its surface as a probe are used. Probes that can be used include antigens, antibodies, and nucleic acids such as DNA.

[0008] In the method of the present invention, the solid carrier particles may be:
It is composed of two or more particles that can be identified by a difference in content structure and / or surface structure. The desired two or more probes are bound to the two or more identifiable particle surfaces for each type to obtain solid support particles.

In the detection method, the two or more solid-phase carrier particles and a sample containing two or more substances to be detected are mixed, and a detection substance-probe complex is formed, respectively. This is done by identifying the particles.

At this time, after immobilizing the probe for each particle type, a plurality of types of solid phase carrier particles may be mixed, and the mixed solid phase carrier particles may react with the sample. After the solid-phase immobilization, each solid-phase carrier particle and the sample may be reacted for each type of carrier, and the reaction solution may be mixed and supplied to the analyzer.

The identification of the particles is performed based on the difference in the content structure and / or the surface structure of the solid carrier particles. Specifically, the particles are irradiated with light, the resulting forward scattered light and side scattered light are detected, and discrimination is performed based on these differences.

Specifically, the method of the present invention is an analytical method for detecting two or more substances, and (1) detecting two or more particles having different content structures and / or surface structures. By mixing the sample with solid phase carrier particles obtained by immobilizing a probe capable of forming a complex with a substance, the substance to be detected and the solid phase carrier particles contained in the sample are mixed. (2) labeling the detection target with a labeling substance, and (3) irradiating the solid-phase carrier particles with light, thereby obtaining forward scattered light and side light. And a step of detecting the amount of the scattered light and the amount of the labeling substance. Hereinafter, each item will be described.

[Content Structure and Surface Structure] A feature of the method of the present invention is that two or more particles having different content structures and / or surface structures are used as carriers.

"Different content structure" used here
The term “difference” refers to the difference in the content and structure from which different side scattered light can be obtained when light is irradiated to particles having different content structures. In this method, the content structure may be changed depending on the difference in the material used. For example, when an artificial material such as latex or polystyrene is used as the material for the carrier particles, the material may contain colloidal gold, carbon powder, pigment, or the like. In this case, the content structure can be changed depending on the concentration of the contained substance. Figure 1 shows an example of a variation of the content structure
Shown in

For example, FIGS. 1 to 3 show schematic diagrams when the latex particles are uniformly mixed with carbon powder at different concentrations. I in FIG. 1 is the darkest, followed by II the darkest, and III is the particles without carbon powder. As described above, by changing the concentration of the carbon powder, it is possible to obtain the side scattered light weaker as the carbon powder is thicker and to obtain stronger side scattered light as the carbon powder is thinner.

Further, IV and V in FIG. 1 are schematic views of particles obtained by uniformly mixing colloidal gold with latex particles.
IV in FIG. 1 contains more colloidal gold than V.
Side scattered light obtained from these two types is different. Further, the size of the gold colloid may be changed.

As used herein, "different surface structures"
Refers to the difference in surface structure from which different side scattered light can be obtained when light is applied to particles having different surface structures.

In the present method, the surface structure can be formed by subjecting the surface to various processing.
FIG. 2 shows an example of a variation of the surface structure. For example,
I in FIG. 2 is a cross-sectional view of the particles. By processing the surface in a bar-like manner, the side scattered light becomes stronger than that without processing. Variations can be increased by changing the shape and density of the projections. II in FIG. 2 is a schematic view of the particles as viewed from the front, and shows particles having hemispherical protrusions on the surface. As in I of FIG. 2, it is possible to change the shape and density of the projection. Further, a groove may be formed by scratching the surface as shown in III of FIG. Changes can be obtained depending on the shape and density of the groove.

As the material of the carrier particles that can be used in the present invention, any material whose content structure or surface structure can be changed can be used, and any combination of different materials having different content structures or surface structures can be used. Good. For example,
Examples include particulate cells such as blood cells, latex, plastic, gelatin, cellulose, and liposomes, and preferably, latex, polystyrene, gelatin, and the like.

Further, the size of the carrier particles of the present invention is not more than 0.1.
It can be selected from the range of 05 μm to 100 μm, and from the viewpoint of increasing the measurement accuracy to some extent, it is preferably 1 μm to 50 μm, more preferably 3 μm to 20 μm. In this method, the particle size can be changed within the above-mentioned range. By changing the particle diameter, it is possible to increase the number of substances to be detected that can be measured simultaneously, and it is also advantageous when setting an inspection series.

[Probe and object to be detected] The solid-phase carrier particles used in the present method are obtained by solid-phase of a probe on carrier particles having the above-mentioned characteristics. A probe that can be used in the method of the present invention is a substance that can specifically bind to a desired substance to be detected. For example, antigens and antibodies, single-stranded DNA and its complementary strand, and RNA
It is preferable that either one of the binding pair constituted by the complementary strand and the like be used as a probe.

In the present invention, the detectable substance is one of the above-mentioned binding pairs, and may be, for example, an antigen, an antibody, or a DNA.
And nucleic acids such as RNA, allergens, hormones, enzymes and the like. In addition, samples that can be used in the present method are body fluids such as blood and plasma, and biological substances such as tissues and cells, but are not limited thereto.

The method for immobilizing the desired probe on the surface of the carrier particles can be performed by any method known per se.

The method of labeling the substance to be detected can be performed, for example, using an antibody (so-called secondary antibody) to which the label is bound. Specifically, an antibody capable of selectively binding to the substance is selected, and a labeling substance is bound to the antibody by a method known per se. The obtained labeled antibody is bound to the detection substance-probe complex, and the detection substance is detected. The labeling substance that can be used in the present method includes any commonly used labeling substance such as a mixed fluorescent substance, a luminescent substance, a radioactive substance, and a dye.

[Measurement Apparatus] The apparatus for carrying out the present method is an apparatus for individually analyzing or analyzing the reaction components present in the solution after the reaction, such as a flow cytometer, a laser scanning cytometer, and an immunoagglutination measurement apparatus. It refers to a device having a configuration in which optical information can be individually obtained by scanning or the like. The flow cytometer is advantageous because it can obtain information on the forward scattered light, the side scattered light, and the amount of the labeling substance for each of the particles.

Hereinafter, an example of the flow cytometer will be described. As already known, a flow cytometer is a dedicated machine for analyzing cells. First, the flow system will be described with reference to FIG. A sample containing a substance to be detected is collected by the sample collection device 1, and reagents (solid carrier particles and labeled antibodies, etc.) are injected by the reagent injection device 2, and then a complex formation reaction is performed in the reaction tank 3. . In this addition, the sample, the solid phase carrier particles, the labeled antibody and the like may all be added simultaneously, or the labeled antibody solution may be added sequentially after the sample and the solid phase carrier particles are reacted. After that, the reaction solution is thrown into the separation device 4, and the remaining labeled antibody that has not been involved in the complex formation is discharged to the drainage tank 5, and BF separation is performed.
For the separation device, for example, a porous ceramic or the like can be used. By diversifying the pore size of the porous ceramic,
The desired BF separation can be performed.

After the BF separation, the complex containing the carrier is diluted with a buffer or the like by the diluting device 6 and then flown into the flow cell 7 of the flow cytometer. Here, when the scattered light generated by the irradiation of the laser beam 8 and the labeling are performed with a fluorescent substance, the fluorescence is detected by the angular detectors 9 and 10, and the output thereof is processed by the data processing device 11 and the sample is processed. A predetermined detection substance in the substance is analyzed.

This will be further described with reference to FIG. 4 which is a schematic view of the periphery of the flow cell. After the complex formation reaction, the BF separated solution 12 flows through the needle 11 in the flow cell 7. This flow is irradiated with a laser beam 8. The laser light 8 irradiates each and every particle present in the stream, resulting in information about the forward scattered light and the labeling substance, for example,
Measure fluorescence etc. The forward scattered light is detected by a detector 9 positioned substantially horizontally with the laser incident light 8 and is mainly used for measuring the size of particles. Further, the side scattered light due to reflection on the surface of the particle is measured by the detector 10 located in a direction perpendicular to the laser incident angle. Further, the detector 10
Can be used for measurement of fluorescent substances and the like on the surface of particles.

2. Examples [Example 1] Identification of 11 types of carrier particles Using a flow cytometer, forward scattered light and side scattered light were measured for 11 types of particles having different particle diameters and different content structures. The results obtained are shown in FIG.

In the latex, carbon powder is used for I to V
Particles with a concentration gradient in this order. Furthermore, they have a larger particle size in the order of a, b, and c. The horizontal axis of the graph shown in FIG. 5 indicates the intensity of forward scattered light (indicated by FS in the figure), and the vertical axis indicates the intensity of side scattered light (indicated by SS in the figure). Here, the intensity of forward scatter and the intensity of side scatter in the figure are indicated by circles. The pattern in each circle is shown simulated in order to clearly show the concentration of the carbon powder.
Also, although not shown in the figure, due to the difference in the surface structure,
Similar results can be obtained.

Example 2 Measurement of Fluorescence Intensity Regarding 11 Kinds of Carrier Particles When a fluorescent substance is bonded to the carrier particles used in Example 1 and the fluorescence is measured, a graph shown in FIG. 6 is obtained. In the graph of FIG. 6, the x-axis indicates the intensity of forward scattered light, the y-axis indicates the intensity of side scattered light, and the z-axis indicates the fluorescence intensity. FIG. 7 shows the result of the conventional method, that is, the case where particles having the same content structure and surface structure but different particle sizes are used. The horizontal axis of the graph in FIG. 7 is the forward scattered light intensity, and the vertical axis is the fluorescence intensity.

The measurable area of the particles is usually from 20 μm to 200 μm. Therefore, when a plurality of particle diameters are set within the range, as shown in FIG. 7, it is difficult to clearly separate each data obtained according to the particle diameter for each particle diameter. 4 to 5 types are the limit.

On the other hand, when the present method is used, although there are three types of particle diameter variations,
By diversifying the content structure and / or the surface structure, clear data can be obtained for many items as shown in FIG. As is clear from this graph, in the present method, eleven items can be measured simultaneously and with high accuracy by diversifying the content structure and / or the surface structure. In particular, a combination in which both the content structure and / or the surface structure and the particle size are different (for example, in FIG. 5, Ia and V
(combination of c, combination of Ic and Va)), the result of each item can be more clearly identified. In the case where the particle size is different for each item as described above, as one of the methods, a probe whose particle size is substantially zero (that is, a free type which is not immobilized on carrier particles) may be employed. Good.

[Example 3] Simultaneous detection of three types of antigens By setting the particle size to three types of a, b, and c, and applying a concentration gradient to the latex in the order of I to V using carbon powder. , And three types of particles whose internal structures are set to I, III, and V are used as carriers.

The desired probe is immobilized on the surface of the carrier particles (FIG. 8). Particles having a particle diameter a and an internal structure I (hereinafter, referred to as particles Ia; other particles are similarly represented) are:
The anti-HBs antigen antibody was immobilized on the IIIb particles, and the anti-IP antigen antibody was immobilized on the particles Vc.
In this way, it is possible to construct a particle reagent series dedicated to infectious diseases by immobilizing the solid phase.

A sample is analyzed using the series of particle reagents dedicated to infectious diseases in the flow system shown in FIG.
As a sample, a human serum sample containing HBs antigen, HBe antigen, and IP antigen is used. The labeling substance is FITC
This was bound to an anti-IgG antibody as a secondary antibody and used as a labeled antibody.

Sample 10 μl and solid carrier particle solution 50 μl
is added to the reaction tank 3 and the reaction is carried out at 37 ° C. for 10 minutes. After the reaction, the cells are washed three times with physiological saline and stained by adding a labeled antibody solution. Reaction tank 3 to 30
0 μl of the reaction solution is aspirated, passed through a porous ceramic cylinder, and subjected to BF separation. The immune complex collected by the BF separation is diluted with a certain amount of buffer solution, and is passed through a flow cytometer.

An argon laser having a wavelength of 588 nm is irradiated through a fine channel system of the flow cytometer, and detection is performed by each optical system (FIG. 9). As shown in FIG. 9, clear measurement can be performed.

Example 4 Simultaneous Inspection for Two Series As in the method shown in Example 3, but it is also possible to use a plurality of fluorescent labeling substances. Thereby, a further effect can be obtained.

It is possible to simultaneously measure the tumor-related test series and the hepatitis virus test series. First, carrier particles on which an antibody for detecting a tumor-associated antigen is immobilized and carrier particles on which an antibody for detecting a hepatitis virus are immobilized are prepared. At this time, each series uses a different number of particles corresponding to the number of substances to be measured.

The series of tumor-related tests includes FIT
A C-labeled secondary antibody is used, and a PE-labeled secondary antibody is used in the hepatitis virus test series.

The solid carrier particles for the tumor-related test series and the solid carrier particles for the hepatitis virus test series are reacted with the sample in separate test tubes. Furthermore, each labeled secondary antibody is added to each test tube and stained. Thereafter, the two test tubes are mixed and measured by flow cytometry. By switching the filters of the optical system, two series each consisting of a plurality of detection items can be measured simultaneously.

The tumor-related test series can be obtained from light transmitted through a band-pass filter at 525 nm, and the hepatitis virus-related test series can be obtained from light transmitted from a band-pass filter at 575 nm.

Example 5 Application to Agglomerates The method of the present invention can be used in an immunoagglutination measuring device. The immunoagglutination measuring device is manufactured and sold by, for example, Sysmex Corporation (PAMIA-30,
Sysmex). This device is a microfluidic device that has an optical system that measures the light scattering generated by a laser after reacting latex carrier particles on the surface of which a substance reacting with a different target substance is immobilized with a subject. It is composed of roads. This device is a device that individually measures the size of the latex particle population after the reaction and detects a substance to be detected based on the presence or absence of aggregation (FIG. 10).

By using a plurality of types of particles whose internal structures are diversified, it is possible to increase the number of items that can be detected by the agglutination measurement. In addition, by combining particles of different materials, for example, agglutination by a patient's red blood cells (blood type determination, especially a frontal test) and agglutination by latex particles (an irregular antibody, particularly a back test)
Can be performed at the same time.

It should be noted that the present invention is not limited to the above-described embodiment, and various changes can be made. For example,
In the above-described example, the solid phase of the probe is always used on the carrier particles.However, due to the binding between the probe and the detection target, both optically forward scatter and side scatter show different values. If it is a probe, it may not be solid-phased on a solid such as carrier particles. In addition, reactions with various probes corresponding to a plurality of types of items may be performed in separate reaction regions, or may be sequentially performed in the same reaction region.

[0047]

According to the present invention, solid-phase carrier particles are identified by both forward scattered light and side scattered light, so that more objects to be detected can be simultaneously detected.

Unlike both the forward scattered light and the side scattered light, which is different from the conventional identification using only the forward scattered light,
The overlap of each data group can be minimized, and each data can be clearly separated. For this reason, it is not necessary to correct the raw data obtained from the plurality of particle populations, and thus the inspection accuracy is improved.

Conventionally, the inspection performed for each item is changed to
Since the reaction can be performed in one container and can be determined simultaneously by one measurement, the size of the apparatus can be reduced. Further, the inspection can be speeded up. Therefore, the cost can be reduced.

As a labeling substance used as a secondary antibody,
Even when one kind of fluorescent labeling substance is used, it is possible to carry out a test of many items, and therefore, the handling of the reagent is simple and the cost can be reduced.

[Brief description of the drawings]

FIG. 1 is a diagram showing an example of the internal structure of diversified particles.

FIG. 2 is a diagram showing an example of the surface structure of diversified particles.

FIG. 3 is a diagram showing an example of a flow system for implementing the method of the present invention.

FIG. 4 illustrates a flow cytometer.

FIG. 5 is a diagram showing identification of particles by forward scattered light and side scattered light.

FIG. 6 is a view showing a result of detection of 11 types of carrier particles.

FIG. 7 is a diagram showing identification of particles by forward scattered light.

FIG. 8 is a diagram showing immobilization of a probe on a particle surface.

FIG. 9 shows the results of simultaneous detection of three types of antigens.

FIG. 10 is a diagram showing an agglutination measuring device.

[Explanation of symbols]

 8. Laser light 9. Detector 10. Detector 11. needle

Claims (4)

[Claims] An analysis method for detecting two or more substances, wherein (1) forming a complex with a substance to be detected with respect to two or more particles having different content structures and / or surface structures. By mixing the sample with solid phase carrier particles obtained by immobilizing a possible probe, forming a complex containing the substance to be detected contained in the sample and the solid phase carrier particles, (2) a step of labeling the detection target with a labeling substance, and (3) irradiating the solid-phase carrier particles with light, thereby obtaining forward scattered light and side scattered light,
And a step of detecting the amount of the labeling substance. 2. An analysis method for detecting two or more substances, wherein (1) it is possible to form a complex with a substance to be detected whose content structure and / or surface structure and particle diameter are optically different. Forming a complex containing the substance to be detected contained in the sample and the solid-phase carrier particles by mixing the two or more probes with the sample; and A method comprising the steps of: labeling; and (3) irradiating the solid-phase carrier particles with light, and detecting the forward scattered light and side scattered light obtained thereby, and the amount of the labeling substance. 3. The method according to claim 2, wherein the two or more probes include a probe immobilized on carrier particles and a non-solid phase probe. 4. A solid carrier particle for use in combination of a plurality of particles having different content structures and / or surface structures, wherein a probe capable of forming a complex with an object to be detected is provided on the surface thereof. Solid support particles for solid phase immobilization.