JPH03216553A - Method and apparatus for immunoassay due to particles - Google Patents

Abstract

PURPOSE:To measure many items with high sensitivity by bringing a reaction container, a specimen to be measured and fine particles into contact with each other to bond the fine particles of different kinds corresponding to the kinds of substances to be measured and measuring the kinds of the fine particles and the number thereof. CONSTITUTION:A reaction container 1 to which substances 2, 3 specifically bondable to substances 4, 5 to be measured are fixed, a plurality of the substances 4, 5 to be measured in a specimen to be measured and the substances 6, 8 specifically bondable to the substances 4, 5 to be measured fixed to fine particles 7, 9 are brought to a contact state to be bonded. Next, a reaction container 17 having fine particles 18 caught thereby is placed on the stage of a fluorescence microscope 10 and a filter converting apparatus 13 is set to observe the fluorescence microscopic image of the fine particles 18 by a TV camera 11 and this image is processed by an image processor 12 to compare the fluorescence intensities of all of the fine particles 18 in the image and the kind of the fine particles 18 is discriminated to count the total number of the fine particles and concn. is calculated from the number of the fine particles on the basis of a calculation curve preliminarily prepared from a specimen of known concn. By this method, many items can be measured and the concn. of an antigen can be quantified with high sensitivity.

Description

[Detailed description of the invention] [Industrial application field]

The present invention relates to an immunoassay method using microparticles and an apparatus therefor.

[Prior art 1] As an immunoassay method using microparticles, latex particles having antibodies bound to their surfaces are reacted with an antigen, and the aggregation state of the latex particles generated by the antigen-antibody reaction is measured by absorbance or scattered light intensity. A method for measuring antigen concentration is known (Bunseki, 16, 605 (19
87)). However, since the aggregation state of latex particles is not constant but has a distribution, these methods, which measure the average value of the entire reaction solution, have accuracy problems in calculating the antigen concentration, making it difficult to quantify the amount of antigen at extremely low concentrations. 6 Therefore, a method was developed in which the reaction solution was introduced into a flow cell and the scattered light or fluorescence intensity of the particles flowing inside the cell was measured (Inspection and Technology, 16, 607 (1988), JP-A No. 1986-81567, Journal of Immunological Methods (J. I++ munol. Method
s), 18. 33 (1977)). According to this method, since the size of each aggregate can be measured, the accuracy of calculating the antigen concentration can be improved. In addition, fluorescent particles are used to cause antigen-antibody reactions,
There is also a method of analyzing the agglutination state by image processing the agglutination image and calculating the antigen concentration (Japanese Patent Application Laid-Open No. 64-3537
3). The conventional method described above uses a homogeneous system in which antibodies (or antigens) on the surface of latex particles react with antigens (or antibodies) to cause agglutination. Therefore, the so-called brozone phenomenon, in which the antigen-antibody reaction is suppressed in the antigen-excess region, cannot be avoided. Furthermore, it is difficult to completely eliminate the effects of scatterers, absorbers, and fluorescent substances such as dyes that coexist in the sample. Furthermore, since antigens and antibodies do not necessarily bind one-to-one, the conventional method of counting the number of aggregates has the problem that errors in counts are likely to occur, especially in extremely low concentration regions. As a method that is not affected by the prozone phenomenon or coexisting substances, there is a heterogeneous reaction method such as that disclosed in JP-A-56-151357.However, although this method is simple and allows multi-item measurement, it has high sensitivity. No consideration has been given to the OBJECTS OF THE INVENTION An object of the present invention is to solve the problems of the above-mentioned conventional techniques and to provide an immunoassay method and an apparatus thereof that can perform multi-item measurements with high sensitivity using microparticles.

[Means to solve the problem]

The above purpose is to provide a reaction vessel in which a substance that specifically binds to each of a plurality of analyte substances in a measurement sample is immobilized, and a plurality of reaction vessels in which a substance that specifically binds to each of the analyte substances in a measurement sample is immobilized. A substance that specifically binds to the analyte immobilized in a reaction container using different types of fine particles;
By bringing the analyte in the measurement sample into contact with a substance that specifically binds to the analyte immobilized on the microparticles, the particles are captured in a reaction container and the number of different types of microparticles is counted. Discrimination of fine particles into multiple types can be achieved by using fluorescent fine particles, and by using fine particles with different particle sizes and/or fine particles with different fluorescence wavelengths. For example, we use fluorescent fine particles with diameters of 0.5 μm and 0.8 μm that emit green fluorescence, and fluorescent fine particles with diameters of 0.5 μm and 0.8 μm that emit red fluorescence. By measuring the intensity ratio of fluorescence and red fluorescence, particles can be classified into four types.
Furthermore, it is possible to classify many types of particles by combining particles that do not emit fluorescence. For example, we use non-fluorescent fine particles with diameters of 0.5 μm and 0.8 μm and fluorescent fine particles that emit green fluorescence with diameters of 0.5 μm and 0.8 μm. By measuring the intensity, fine particles can be classified into four types. To measure the type and number of particles trapped in the reaction vessel,
This can be achieved by creating an image of the particles and performing image processing, and can be achieved using a device consisting of a microscope, an image input device (such as a TV camera), and an image processing device. First, the size of each particle is measured based on the image obtained by the image input device. Next, the fluorescence of each particle is identified through a filter (for example, an interference filter) for detecting the target fluorescence. In this way, particles are classified into a plurality of groups based on differences in particle size and fluorescence, and the number of particles in each classification is counted. Another method for measuring the type and number of particles trapped in a reaction vessel is to use a flow cytometer. The particles captured in the reaction vessel are unbound and released from the reaction vessel, and the size and fluorescence intensity of the particles are measured using a flow cytometer, and the number of particles for each type of particle is counted. To unbind particles trapped in the reaction vessel,
Mechanical methods include applying vibration or scraping with a spatula, and chemical methods include denaturing and dissociating antibodies with urea or the like. A typical combination of a substance to be measured in a measurement sample and a substance that specifically binds to the substance to be measured is an antigen (or antibody) and an antibody (or antigen). Other possible combinations include, for example, combinations of hormones and receptors, sugars and lectins, etc. Suitable particles to be counted are particles of a material having a specific gravity of about 1 to 1.4, such as polystyrene, acrylic, styrene-butadiene copolymer, styrene-acrylic acid copolymer, or the like. The reaction is effectively carried out when the particles have a specific gravity of about 1 to 1.4. Moreover, the particle size is 5 μm or less, especially 0.5 μm or less.
A range of 1 to 1 μm is appropriate. In order to bind antibodies (or antigens) etc. to the surface of these particles, commonly known physical adsorption, chemical bonding, etc. can be used. According to the present invention, human α-fetobrotein (AFP), carcinoembryonic antigen (CEA). Various antigens, antibodies, hormones, etc. such as ferritin, rubella antibodies, AIDS virus antibodies, and human chorionic gonadotrobin (HCG) can be measured.

[Function] According to the present invention, since different types of fine particles are bonded to each of a plurality of substances to be measured, many types of substances to be measured can be measured in the same container, and multi-item measurement can be achieved. Furthermore, since one fine particle is bound to one substance to be measured, the substance to be measured can be measured with high sensitivity. [Example 1] Hereinafter, a multi-item measurement method will be described as an example of the present invention, taking two antigens, AFP and CEA, as examples. [Example 1] (Preparation of immobilized antibody) A well of a microplate was used as a reaction container, and AF was placed on the inner surface.
Immobilize antibodies against P and CEA. Add 25 μQ of anti-human AFP antibody solution at a concentration of 10 μg/mQ and anti-CE at a concentration of 10 μg/mQ to the wells of a flat-bottomed microplate.
Inject 25 μQ of A antibody solution and stir intermittently for 2 hours.
The anti-human AFP antibody and anti-CEA antibody are immobilized in the wells by reaction. (Preparation of particle-labeled antibody) Fluorescent latex particles having a carboxyl group on the surface are used as a label. The diameter is 0.5μm, 54
An anti-human AFP antibody was immobilized on the surface of microparticles having a maximum fluorescence wavelength of 0 nm by the carbodiimide method (immobilized amount: approximately 0.7 mg/g). Prepare particulate-labeled anti-human AFP antibody. Furthermore, the diameter is 0.5 μm and 580 nm.
An anti-CEA antibody was immobilized by the carbodiimide method on the surface of microparticles having a maximum fluorescence wavelength of approximately 0.
7mg/g). Prepare particulate-labeled anti-CEA antibody. (Reaction procedure) 50 μQ of sample serum containing AFP and CE'A was added to anti-human AF.
Reaction container (well) with immobilized P antibody and anti-CEA antibody
The antigens to be measured (A F P and CEA) are captured in the reaction vessels (wells) by injecting them into the reaction vessel (well) and reacting for 2 hours. Then, phosphate buffer containing 0.5% BSA (0.5% BSA
-PBS) to remove unreacted antigens, etc. Next, 60 μQ of a particle-labeled anti-human AFP antibody solution and 60 μΩ of a particle-labeled anti-CEA antibody solution prepared so that the particle concentration was 0.5% were injected, and left to react for 5 hours. A particle-labeled antibody is bound to human AFP and CEA captured by Next, 0.5
% BSA-PBS to remove excess particulate-labeled antibody. The state of binding between the antigen and the particle-labeled antibody at this time can be represented schematically as shown in Figure 1. Human AFP4 and CE in the sample serum are added to anti-human AFP antibody 2 and anti-CEA antibody 3 immobilized on reaction container (well) 1.
A5 binds through an antigen-antibody reaction. Furthermore, human AFP
4, 5 μm in diameter via anti-human AFP antibody 6.
Fine particles 7 having a fluorescence peak at 40 nm are bound. Furthermore, fine particles 9 having a diameter of 0.5 μm and a fluorescence peak at 580 nm are bound to CEA 5 via anti-CEA antibody 8 . FIG. 2 shows a schematic diagram of an apparatus for measuring the diameter and number of microparticles captured in the wells of a flat-bottomed microplate. The device includes an (inverted) fluorescence microscope 10, a TV camera 11 that reads the microscope, an image processing device 12 that analyzes images, and a filter conversion device 111 that switches the fluorescence detection filter of the fluorescence microscope depending on the measurement target.3. Image processing device 1112
and a controller 1 that controls the filter conversion device 13 and the like.
4. Consists of a monitor television 15 that outputs images, and an output device 16 that outputs processing results. The well 17 of the microplate in which the particles have been captured is placed on the stage of the fluorescence microscope 10, and the fluorescence microscope image of the particles 18 in the well 17 is observed with the TV camera 11, and the type of particle is identified by image processing. First, a microscope image emitting fluorescence at 540 nm is measured. With filter conversion device l3. Set an interference filter on the microscope that transmits 540 nm light and blocks other light. The image at that time is detected by the TV camera 11, and the intensity of the image is digitized into 8 bits and stored in the image memory of the image processing device 1i12. This operation was performed for 64 frames to improve the S/N of the fluorescent image. From this image, calculate the position of the pixel that shows the maximum fluorescence value and the intensity of that part.
The position of the pixel showing the maximum fluorescence value indicates the location of the particle, and the intensity is an index for determining which type of particle group the particle belongs to. Next, measure the fluorescence microscopic image at 580 nm in the same way.
In the filter conversion device 13. An interference filter that transmits 580 nm light and blocks other light is set on the microscope.6 The image at that time is detected by the TV camera 11, and the intensity of the image is digitized into 8 bits and sent to the image processing unit 12. Store in image memory. This operation was performed for 64 frames in the same manner as above to improve the S/N of the fluorescent image. From this image,
The position of the pixel showing the maximum fluorescence value and the intensity of that part are calculated. From the above, by comparing the fluorescence intensity at 540 nm and the fluorescence intensity at 580 nm at the position of the particle, it is possible to determine which particle group the particle belongs to. For example, if the fluorescence intensity at 580 nm is stronger, it can be determined that the anti-CEA antibody is bound to the fine particles and that the antigen CEA is being detected. A comparison was made for all particles in the image, and human AFP
and count the total number of each microparticle for CEA. In order to quantify the human AFP or CEA concentration using this method, a calibration curve may be prepared in advance using samples whose concentrations are known, and the concentration may be calculated from the number of particles of each. [Example 2] Two types of latex fine particles having a carboxyl group on the surface, diameters of 0.5 μm and 0.75 μm, and both i and large fluorescence wavelengths of 540 nm are used as labeling fine particles. Anti-human AFP antibody was prepared with a diameter of 0 using the carbodiimide method.
・Immobilize on the surface of 5 μm fine particles (immobilization amount approximately 0.
7mg/g). Furthermore, anti-CEA antibody was immobilized on the surface of microparticles with a diameter of 0.75 μm by the carbodiimide method (immobilized amount: approximately 0.7 mg/g). In the same manner as in Example 1, the anti-CEA antibody was immobilized on the surface of fine particles with a diameter of 0.75 μm. Immobilize AFP antibody and anti-CEA antibody, and
Next, a solution of the particle-labeled antibody is reacted to bind the human AFP and CEA captured in the well. Fine particles were measured using the same equipment as in Example 1! (Figure 2). First, a filter conversion device! ! Step 13: Set an interference filter on the microscope that transmits 540 nm light and blocks other light. The image at that time is detected by the TV camera 11, and the intensity of the image is digitized into 8 bits and stored in the image memory of the image processing device 12. Please note that this operation is 64
Frame was performed to improve the S/N of the fluorescent image. From this image, the position of the pixel showing the maximum fluorescence value, that is, the position of the particle, and the intensity of that part are determined. Figure 3 is. I! I is a histogram (quantity versus fluorescence intensity) of determined particles. Since the fluorescence intensity and the particle diameter are correlated, the histogram shows two distributions. The one with lower fluorescence intensity is the one with the anti-human AFP antibody immobilized.
．． These are fine particles with a diameter of 5 μm, and the one with higher fluorescence intensity is the fine particle with a diameter of 0.75 μm on which the anti-CEA antibody is immobilized. Human AFP or CEA was determined by counting the total number of particles with a diameter of 0.5 μm and 0.75 μm from the histogram.
concentration can be calculated. As in Example 1, in order to quantify the concentration of human AFP or CEA, a calibration curve may be prepared in advance using samples whose concentrations are known, and the concentration may be calculated from the number of particles of each. [Example 3] Labeling fine particles had carboxyl groups on the surface, had a diameter of 0.5 μm, and were fluorescent (the maximum fluorescence wavelength was 540 nm).
Non-fluorescent latex fine particles having the same diameter of 0.5 μm as the latex fine particles of m) are used. Anti-human AFP antibody is immobilized on the surface of fluorescent fine particles by the carbodiimide method (immobilized amount is approximately 0.7 mg/g).
). Furthermore, anti-CEA antibody was prepared using the carbodiimide method.
Immobilize on the surface of non-fluorescent fine particles (immobilization amount approximately Q.
7 mg/g) - In the same manner as in Example 1, anti-human AFP antibodies and anti-CEA antibodies were immobilized in the wells of a microplate, and the sample and then the microparticle-labeled antibody solution were reacted, and the human AFP and anti-CEA antibodies captured in the wells were reacted. Bind the particle-labeled antibody to CEA. The measurement of fine particles is performed using the same apparatus 1i (FIG. 2) as in Example 1. First, the particle image inside the well is observed using transmitted light. The image detected by the TV camera 11 is digitized into 8 bits and processed by an image processing device! 12 image memories. This operation is performed for 64 frames to improve the image S/N. Identify particles from the obtained image data. Diameter is 0.4μm~
Images in the range of 0.6 μm are recognized as fine particles, and the total number of particles is measured. In addition, those smaller than 0.4 μm and 0
．． Items larger than 6 μm were judged to be dust and were excluded from counting. The total number of microparticles measured is the total number of fluorescent and non-fluorescent microparticles. Next, in the filter conversion device 13. An interference filter that transmits 540 nm light and blocks other light is set on the microscope. The fluorescent image at that time is detected by the TV camera 11, and the intensity of the image is digitized into 8 bits and stored in the image memory of the image processing device 12. Note that this operation was performed for 64 frames to improve the S/N of the fluorescent image. From this image, the total number of particles emitting fluorescence is calculated. It is thought that the particles emitting fluorescence are bound to human AFP, and the total number of these particles is the human AF.
Corresponds to P concentration. Further, by subtracting the total number of particles emitting fluorescence from the total number of particles measured and counted using transmitted light, the number of particles corresponding to the CEA concentration can be calculated. As in Example 1, in order to quantify the concentration of human AFP or CEA, a calibration curve may be prepared in advance using samples whose concentrations are known, and the concentration may be calculated from the number of particles of each. [Example 4] Two types of latex fine particles having a carboxyl group on the surface, having diameters of 0.5 μm and 0.75 μm, and both having a maximum fluorescence wavelength of 540 nm are used as fine particles for labeling. Prepare reagents in the same manner as in Example 2. anti-human A
The FP antibody was immobilized on the surface of fine particles with a diameter of 0.5 μm by the carbodiimide method (immobilized amount approximately 0.7 mg/g),
Anti-CEA antibody was 0.75μm by carbodiimide method.
Immobilize on the surface of fine particles of the same diameter (immobilized amount approximately 0.7 mg
/g) - Additionally, anti-human A was added to the wells of the microplate.
The FP antibody and anti-CEA antibody are immobilized, and the sample is reacted with a solution of the particle-labeled antibody to bind the particle-labeled antibody to the human AFP and CEA captured in the well. The particles captured in the wells are measured using a flow cytometer. Therefore, a concentration of 8M was added to the reaction vessel containing the fine particles.
Inject 0.2 ml of urea to remove bonds from the fine particles to obtain a fine particle suspension. This fine particle suspension is analyzed using a flow cytometer. By simultaneously measuring the scattered light intensity and fluorescence intensity, it is possible to identify the presence or absence of microparticles, the size of the microparticles, and whether the microparticles are fluorescent or non-fluorescent. Particles with a diameter of 0.3 μm or less calculated from the intensity of scattered light were likely to be dust, and were excluded from counting. The labeling fine particles used in this example were fluorescent fine particles with a size of 0.
Since the diameters are 5 μm and 0.75 μm, the type of fine particles can be determined based on the intensity of their fluorescence. The histogram showing the quantity of particles detected by the flow cytometer relative to their fluorescence intensity is almost the same as in Figure 3. The histogram shows two distributions. The one with lower fluorescence intensity is anti-human AF.
These are fine particles with a diameter of 0.5 μm on which P antibody is immobilized.
They are microparticles with a diameter of μm. From the histogram, the human AFP or CEA concentration can be calculated by counting the total number of particles with a diameter of 0.5 μm and 0.75 μm. As in Example 2, in order to quantify the human AFP or CEA concentration, a calibration curve may be prepared in advance using samples whose concentrations are known, and the concentration may be calculated from the number of particles of each. In addition, the concentration of fine particles in the fine particle recognition antibody solution in Examples 1 to 4 is suitably about 0.01% to 5%. When using fine particles such as acrylic particles with a rather large specific gravity, the appropriate concentration of the fine particles is about 0.01% to 1%. Regarding other fine particles, the fine particle concentration is determined by the specific gravity, particle size, etc. of the fine particles. According to this example, since the binding ratio between the antigen to be measured and the fine particles as the labeling substance is constant, highly accurate and sensitive quantification is possible. Furthermore, since the so-called prozone phenomenon, in which antigen-antibody reactions are suppressed in areas with excess antigen, does not occur, quantification in high antigen concentration ranges is also possible. [Effects of the Invention] According to the present invention, multi-item measurement becomes possible by combining different types of fine particles depending on the type of substance to be measured. In addition, since microparticles can be bound to individual amounts of a substance to be measured, such as an antigen to be measured, by a specific reaction such as an antigen-antibody reaction, the antigen concentration can be quantified with high sensitivity.

[Brief explanation of drawings]

FIG. 1 is a schematic diagram showing the binding state of an antigen and a particle-labeled antibody as described in Example 1. FIG. 2 is a schematic diagram of the configuration of the apparatus for microscopic image processing described in the first embodiment. FIG. 3 is a histogram showing the relationship between the fluorescence intensity of particles and the number of particles, which was explained in Example 2 (and Example 4). Explanation of symbols 1... Reaction container (well) 2... Anti-human AFP
Antibody 3...Anti-CEA antibody 4...Human AFP
5...CEA 6...Anti-human AFP
Antibodies 7, 9,... Fine particles 8... Anti-CEA antibody lO... Fluorescence microscope l1... TV camera 12... Image processing device l3... Filter conversion device l4... Controller 15...・Monitor TV l6...output device! 17...Reaction vessel Figure 1

Claims (1)

[Scope of Claims] 1. A reaction container in which a substance that specifically binds to each of a plurality of analyte substances in a measurement sample is immobilized, and a substance that specifically binds to each of the analyte substances in a measurement sample is immobilized, respectively. A particle characterized by using a plurality of different types of microparticles, and by bringing the microparticles into contact with the reaction container, a measurement sample, and the microparticles, the microparticles are captured in the reaction container, and the types of microparticles and the number of microparticles are counted. immunoassay method. 2. The immunoassay method using particles according to claim 1, characterized in that fluorescent fine particles are used. 3. Claim 1, which uses fine particles that can be distinguished into a plurality of types depending on the size of the fine particles and/or the fluorescence wavelength of the fine particles.
An immunoassay method using particles according to item 1 or 2. 4. A particle-based immunoassay device comprising an image input device and a device for image processing an image of particles obtained by the image input device. 5. The method for immunoassay using particles according to claim 1, 2, or 3, characterized in that the microparticles are analyzed by flow cytometry. 6. Claim 4, characterized in that the size and/or fluorescence wavelength of each fine particle is measured, the particles are classified into a plurality of types, and the number of fine particles of the same type is counted. An immunoassay device using the particles described in 2. 7. A process of measuring the size and/or fluorescence wavelength of individual fine particles, classifying them into a plurality of types, and counting the number of fine particles of the same type is performed. Immunoassay method using particles described in Section 2.