JPH04233462A - Immunological quantitative analysis - Google Patents

Abstract

PURPOSE:To achieve an expansion of measurable density area of an immunoassay, a reduction in the frequency of diluting operation of a sample to be inspected of the inspection and a higher measuring accuracy thereof. CONSTITUTION:Immobilizing areas are formed on a substrate for a plurality of antibodies and antigens different in density and the antigen or antibody in a sample is made to act on the substrate to trap the antigen or the antibody in the sample in the antibody or antigen immobilized in the areas. Then, a latex particle having the antibody or antigen immobilized thereon is made to work to trap the latex particle in the antigen or the antibody in the sample. Then, the number of the latex particles or a physical quantity correlated thereto is measured and a density of the antigen corresponding to a measured value is determined from a calibration curve prepared previously using antigen with a known density.

Description

[Detailed description of the invention]

0001

[Field of Industrial Application] The present invention relates to an immunological quantitative analysis method, and more particularly, to an immunological quantitative analysis method that can expand the measurable concentration range of an immunological test, simplify the dilution operation of a specimen sample, and improve the measurement accuracy. Concerning quantitative analysis methods.

0002

BACKGROUND OF THE INVENTION In recent years, in the medical field, quantitative analysis of trace components in body fluids has been frequently carried out for the purpose of early detection of diseases. For example, trace components in blood are being quantified, but many of the body fluid components contained in blood are extremely small, with concentrations on the order of ng (nanograms)/ml, and it is difficult to quantify these trace components. Analysis has become an important issue in the medical field.

[0003] Conventionally, immunoassay methods for detecting trace blood components using antigen-antibody reactions include latex agglutination method (LIA method), radioimmunoassay method (RIA method),
Alternatively, immunological methods such as enzyme immunoassay (EIA method) are known. The above latex agglutination reaction method (LIA)
This method was developed by Singer and Plotz et al. in 1965, and involves performing an antigen-antibody reaction using insoluble carrier particles (latex particles) on which antibodies (or antigens) are immobilized. After causing agglutination, the antigen (or antibody) is determined from the turbidity of the latex.
This is a method to find the concentration of In addition, the radioimmunoassay method (RIA method) uses an antibody (or antigen) labeled with a radioisotope to perform an antigen-antibody reaction, and the radiation dose of the radioisotope labeled with the antibody (or antigen) is determined by the antigen (or antigen). or antibody) concentration. Furthermore, enzyme immunoassay (EIA method) uses an enzyme-labeled antibody (or antigen) to perform an antigen-antibody reaction, and the degree of color development due to the enzyme reaction labeled with the antibody (or antigen) is determined by the antigen (or antigen). This method determines the concentration of antibodies (antibodies). In each of the above immunoassay methods, a calibration curve is prepared in advance by measuring turbidity, radioactivity intensity, absorbance, etc. using a standard substance with a known concentration, and a calibration curve is prepared in advance when measuring a sample with an unknown concentration. The measured values are applied to a calibration curve to determine the concentration of the target substance in the sample.

0004

[Problems to be Solved by the Invention] However, in the conventional immunoassay method described above, only one calibration curve is created due to its nature, and the concentration range that can be measured by this calibration curve is wide. Since the range is only 2 to 3 digits, it is not possible to cover all the various concentrations of specimens used in clinical tests. Therefore, when measuring a sample with an unknown concentration, it is necessary to dilute the sample many times until it reaches a measurable concentration range, and this dilution operation is complicated and disadvantageous in terms of time and cost. There is. Furthermore, there is a problem in that errors are likely to occur during pipetting associated with dilution operations. Also,
It is generally known that the S/N (signal/noise) ratio improves in proportion to the square root of the number of measurements, but in the conventional immunoassay method described above, there is only one calibration curve.
There is a problem in that only a single piece of data can be obtained in one measurement, and there is a limit to the improvement of measurement accuracy. The present invention has been made in view of the above-mentioned problems.
The purpose of the present invention is to provide an immunological quantitative analysis method that can expand the measurable concentration range of immunological tests, reduce the number of sample dilution operations, and improve measurement accuracy.

[0005]

[Means for Solving the Problems] In order to achieve the above object, the immunological quantitative analysis method of the present invention immobilizes a plurality of antibodies (or antigens) with different measurable concentration regions on a solid phase. An antigen (or antibody) that specifically reacts with the antibody (or antigen) is provided in this antibody (or antigen) immobilization region.
After the antigen (or antibody) is captured by an antigen-antibody reaction by reacting with a sample containing the antigen, insoluble carrier particles are further immobilized with an antibody (or antigen) that specifically reacts with the antigen (or antibody). The antigen (or antibody) concentration is measured by measuring the number of insoluble carrier particles captured by the antigen (or antibody) or a physical quantity correlated with this number of particles. Preferably, the antigens and antibodies are immobilized on each region on the solid phase using solutions of antibodies and antigens having different concentrations, and the regions on which a plurality of antigens and antibodies immobilized with different measurable concentration regions are fixed on the solid phase. The insoluble carrier particles are latex particles. Furthermore, if necessary, a flat substrate or a rotatable disk-shaped substrate may be used as the solid phase.

[0006] Here, antigen refers to immune response (antibody production).
A general term for substances that induce immunogenicity (immunogenicity) or bind to antibodies. Antibody is a general term for the resistance that occurs in individuals who have acquired immunity to a certain antigen and acts specifically on the antigen.

The present invention will be explained in detail below with reference to the drawings. Here, a case will be described in which the configuration is substrate/antibody/antigen/antibody/insoluble carrier particles. FIG. 1 is an explanatory diagram showing the procedure of the immunological quantitative analysis method of the present invention. In the immunological quantitative analysis method of the present invention, first,
A plurality of antibody immobilization regions with different measurable concentration regions are provided on the solid phase. Here, the solid phase is not particularly limited as long as it can immobilize the antibody, but a substrate is usually used. For example, as shown in FIG. 1(I), an antibody 2 is immobilized on a substrate 1. The antibody 2 immobilized on the substrate 1 varies depending on the antigen to be measured, but for example, it may be a polyclonal antibody produced by administering the antigen to be measured to a certain immunized animal (e.g., rabbit, goat, sheep, etc.). Examples include monoclonal antibodies.

[0008] Here, the size, thickness, shape, etc. of the substrate 1 are appropriately selected and are not particularly limited, but it is preferably in the shape of a flat plate (plate) or a rotatable disk (disc). A flat substrate is suitable for analysis using an electron microscope or the like. Furthermore, it is preferable to form the substrate into a rotatable disk shape, since this allows the sample to be developed and analyzed using laser light or the like easily and automatically. Materials for forming the substrate 1 include plastic materials such as polycarbonate, polymethyl methacrylate, polystyrene, polyvinyl chloride, polyvinyl acetate, polyurethane, and epoxy resin, transparent materials such as glass, metal materials with good light reflectivity, and other silicon materials. Examples include inorganic materials such as single crystals. If the substrate is formed of a material with good light transmittance or light reflectivity in this way, optical analysis using laser light or the like becomes possible. To provide multiple antibody immobilization regions with different measurable concentration regions on the above solid phase (substrate), for example, use antibody solutions with different concentrations, drop them onto the substrate, and immobilize them by physical adsorption or chemical adsorption. do. The concentration of the antibody solution to be dropped varies depending on the antigen (or anti-antibody) that specifically reacts with the antibody, but is generally between 10-2 and 10-7.
A concentration on the order of g/ml is used.

Regarding the specific conditions for immobilizing antibodies,
Conditions similar to those for general antibody immobilization methods are adopted (Oxygen immunoreaction method, written by Eiji Ishikawa, published by Igaku Shoin 19
(see 1987). For example, a 0.05M Tris-buffered saline (TBS) solution of the antibody (pH 8.2, antibody concentration 10-2
10-7 g/ml) or 0.05 M carbonate/bicarbonate buffer solution (pH 9.6, antibody concentration 10-2 to 10-7 μg)
/ml) onto the substrate and left at 20 to 30°C for 2 hours (or overnight at 4°C) to physically adsorb antibody 2 onto substrate 1. In this case, the antibody can be chemically bonded to the substrate by using a substrate having functional groups such as amino groups, carboxyl groups, or derivatives thereof on the surface of the substrate.

[0010] Various embodiments are possible regarding the shape and arrangement of the plurality of antibody-fixing regions. For example, as shown in Figure 2, each antibody immobilization area A to D has a circular shape (Figure 2(b),
(d), (e), (f)) and rectangles ((a), (c))
). In addition, as for the arrangement of each antibody immobilization region, when arranged in the longitudinal direction of a rectangular substrate (plate) 1 (see Figures (a) and (b)), when arranged in the longitudinal direction of a rectangular substrate (plate) 1, and when arranged in the longitudinal direction of a rectangular substrate (plate) 1,
((c), (d) in the same figure), and in the circumferential direction of a disk ((e) in the same figure).
) etc. In this case, the regions A to D may be arranged consecutively ((a), (c) in the figure) or may be arranged at intervals ((b), (d), (in the figure)). e), (f)
). In addition, as shown in FIG. 2(f), the substrate 1 is a disc-shaped disk with a large number of radially formed protrusions 1a to provide a large number of sample development surfaces 1b, and antibodies are immobilized on each of these sample development surfaces 1b. By providing regions A to D, a large number of specimen samples may be analyzed simultaneously.

Note that the number of antibody immobilization regions is not limited to four as shown in FIG. 2, but can be any number. Figure 2
, the concentration of each antibody immobilization region A to D is region A:
10-3 g/ml, area B: 10-4 g/ml, area C
10-5 g/ml, Region D: 10-6 g/ml, so that the concentration decreases toward the right or downward in the diagram, but it is configured so that the concentration is in the opposite order. Good too.

[0012] As shown in FIG. 1 (II), the substrate on which a plurality of antibody immobilization regions A to D obtained as described above were formed,
In order to prevent non-specific adsorption, it is preferable to cover the surface with blocking agent 3 and perform blocking treatment. Here, as the blocking agent, bovine serum albumin,
Examples include casein and skim milk. In the method of the present invention, next, the specimen sample 5 is placed on the antibody immobilized substrate 4.
The antigen 6 inside is captured by an antigen-antibody reaction (Figure 1
(III)). Here, the specimen sample 5 to be analyzed
There are no particular restrictions on the antigen as long as it contains an antigen. Examples include body fluids such as blood, pleural fluid, ascites fluid, heart fluid, joint fluid, and urine. The antigen to be analyzed is not particularly limited, but for example, C-reactive protein (CRP)
, α-fetoprotein (AFP), carcinoembryonic antigen (C
EA), etc. Here, CRP is C-rea
It is an abbreviation for active protein, and it is one of the plasma proteins that increases significantly in pathological conditions such as inflammatory diseases and necrosis of body tissues, and is a so-called acute phase reactive protein.
It is a typical component of te phase proteins.

In order to carry out an antigen-antibody reaction on the substrate 1, for example, an appropriate amount (for example, about 50 μl) of the specimen sample 5 containing the antigen 6, which is the analyte, is applied to each of the antibody-immobilized regions A to D on the substrate. ) just drop it. This allows an antigen-antibody reaction to occur between the antibodies 2 in the antibody immobilization regions A to D formed on the substrate 1 and the antigen 6 in the specimen sample 5. Alternatively, the antigen-antibody reaction may be caused to occur on the substrate 1 by rotating the disk-shaped substrate 1 and spreading the specimen sample on the substrate 1 in the form of a thin film using centrifugal force. In this case, the time required for antigen-antibody reaction is 1~
It only takes about 5 minutes. After the antigen-antibody reaction, the remaining components 7 and 8 other than the captured antigen 6 are treated with phosphate buffered saline (PBS) (pH 7.4) or Tris buffered saline (TBS) (pH 8.2). Appropriate amount (for example, 1
ml) and rinse it off, or after dropping it, further rotate the substrate 1 and rinse it off.

Next, insoluble carrier particles 10 having immobilized antibodies 9 that specifically react with the antigen 6 are applied to the substrate 1 after the antigen-antibody reaction (FIG. 1 (IV)). Here, the insoluble carrier particles 10 on which antibodies 9 are immobilized are particles on which, for example, latex particles are immobilized with antibodies 9 against antigens 6, which are the analyte, physically or chemically adsorbed or bonded to them. say. In this case, the latex particles need only have a uniform particle size, and may be any of plastic particles (eg, polystyrene, etc.), inorganic particles, or metal particles. The insoluble carrier particles may be fluorescent or colored, thereby enabling analysis using fluorescence or coloring. In this case, the insoluble carrier particles themselves may be formed of a fluorescent material, may be coated with a fluorescent material, or the fluorescent material may be attached to the insoluble carrier particles. In order to obtain insoluble carrier particles on which antibodies are immobilized, for example, 1.0% with pH and salt concentration adjusted using TBS.
Add the antibody to the latex suspension and leave it in a greenhouse for 2 hours to physically adsorb the antibody onto the latex particles. Thereafter, the supernatant was discarded by centrifugation to remove unadsorbed antibodies, and the precipitate was filled with phosphate buffered saline (PBS) (pH 7.
4) and redispersing it (Japanese Patent Application Laid-Open No. 62-2
No. 67298, Applied and Enviro
nmental Microbiology, Oct.
．． 1988, P2345-2348).

[0015] An appropriate amount (for example, 50 μl) of the aqueous solution containing the insoluble carrier particles on which the antibody prepared as described above is immobilized is applied to the substrate 1 (FIG. 1 (III)) after the antigen-antibody reaction described above. It is dropped (or it is further spread out by rotating the substrate 1 after being dropped). As a result, substrate 1
The antigen 6 captured by the antibody 2 and the antibody 9 immobilized on the insoluble carrier particles 10 cause an antigen-antibody reaction again, and the insoluble carrier particles 10 are captured via the antibody 9 (see )). Insoluble carrier particles not captured on the substrate are washed away in the same manner as for the analyte sample described above. In this way, a sample substrate is produced.

Next, the number of insoluble carrier particles 10 captured by the antigen 6 on the sample substrate or a physical quantity correlated with the number of particles is measured by the measuring means 11 to determine the number of antigens 6 (
(Figure 1 (IV)). Here, as the means 11 for measuring the number of particles, etc., an optical measuring means is preferable. An example of the optical measuring means is a measuring means that measures the number of particles by analyzing an image obtained with an optical microscope via an image analysis device. In addition, other optical measurement means include light sources such as lasers, LEDs, and halogen lamps, photodetectors, and CCDs (including line sensors).
Various measurement means are exemplified in combination with a light receiving system such as. In this case, the number of particles may be counted directly from changes in reflectance using laser light, etc.
Alternatively, the number of particles may be determined by measuring a physical quantity that correlates with the number of particles, such as the absorbance due to coloring or the intensity of fluorescence due to a fluorescent substance, and converting this into the number of particles (Patent Application No. 2-270
(See No. 900).

The following can be said about the relationship between the optical measuring means and the particle size of the insoluble carrier particles. When using a measuring means that combines an optical microscope and an image analysis device, the particle size of the insoluble carrier particles is preferably about 0.2 μm. Furthermore, when a laser is used as an optical measurement means, it is not preferable that the particle size of the insoluble carrier particles becomes smaller, since the signal to the laser beam becomes weaker and the S/N ratio deteriorates. On the other hand, when insoluble carrier particles that emit fluorescence are used, the antigen concentration is measured by converting the fluorescence intensity into the number of particles, so the particle size may be small. Furthermore, if an electron microscope is used, insoluble carrier particles of 0.2 μm or less can also be used. Above aspects and antigens-
Considering the reactivity of the antibody, the particle size of the insoluble carrier particles is 0.
It is preferably within the range of 0.01 to 10 μm.

To determine the antigen concentration from the number of insoluble carrier particles measured above, the relationship between the antigen concentration and the number of insoluble carrier particles is determined in the same manner as described above except that a sample with a known antigen concentration is used. , create a calibration curve in advance, and then determine the antigen concentration from this calibration curve. In this case, a calibration curve is created for each of the antibody immobilization regions A to D, which have different measurable concentration regions (therefore, four calibration curves are created). For example, six types of samples with known antigen concentrations (10-2 to 10-7) with different antigen concentrations were prepared.
g/ml), and each of these six types with known antigen concentrations is applied to each antibody-immobilized region A to D. The number of insoluble carrier particles is measured in the same manner as described above, and each antibody-immobilized region is The relationship between the antigen concentration and the number of carrier particles in A to D is determined. Then, plot these relationships on one graph to create calibration curves A' to D' (see Figure 3(a)).
.

Next, a method for determining the antigen concentration of the unknown concentration (Ca) from the calibration curve shown in FIG. 3(a) will be specifically explained. For example, suppose that when a specimen sample with an unknown concentration (Ca) is applied to each antibody-immobilized region A to D, latex numbers a1, a2, and a3 are measured in the antibody-immobilized regions A to C (in D, the calibration curve D' Calibration curve A for each antibody fixation region in Figure 3(a)
By '~C', the concentration Ca corresponding to the latex number
1, Ca2, and Ca3 are found. Then, their average value Ca is taken as the antigen concentration (see Figure 3(b))
. Similarly, the antigen concentration is defined as an unknown concentration (Cb), and the number of latex in each concentration region A to D is b1 (region C) b2
(Region D) (The number of latex is too large in regions A and B and it is outside the effective measurement area, so the number of latex cannot be obtained).
b1 and Cb2 are found. Then, their normal value Cb is taken as the antigen concentration (see FIG. 3(c)).

As shown in FIG. 3(a), the positions of the calibration curves A' to D' differ depending on the concentration difference of the immobilized antibody, so the dynamic range that could not be covered by one calibration curve is apparently expanded. Become. This eliminates or reduces the need for repeated dilution operations until a measurable range is achieved, which has been conventionally performed. Furthermore, if the antigen concentration is Ca shown in FIG. 3(a), the concentration can be determined from the number of latexes captured in areas A, B, and C (FIG. 3(b)). In this case, only the measured values whose values are within a predetermined measurable region are taken as valid measured values, and the target substance concentration is determined by averaging them (FIG. 3(b)). As a result, it is expected that measurement accuracy will be improved compared to the conventional method using only one calibration curve. This is because it is generally known that the S/N (signal/noise) ratio is proportional to the square root of the number of measurements.

[0021] In the above-described immunological quantitative analysis method of the present invention, for convenience of explanation, a case is shown in which the structure is substrate/antibody/antigen/antibody/insoluble carrier particles. The antigen/antibody/antigen/insoluble carrier particle structure may be used to quantify the antibody concentration.

[0022]

EXAMPLES The present invention will be explained in more detail below based on examples. [Example 1]: Quantification of CRP (C-reactive protein) Creation of antibody-immobilized substrate As shown in Figure 2(b), a rabbit was immunized with CRP antigen on a polycarbonate plate measuring 2 cm in length and 7 cm in width. The CRP antibody obtained is fixed. For antibody fixation,
Antibody concentration 1x10-3, 10-4, 10-5, 10-6
50 μl of TBS (Tris-buffered saline) solution of g/ml was dropped onto regions A to D in the above order so as to have an area of 1 cm diameter, and was allowed to stand at 30° C. for 2 hours to cause physical adsorption. After that, the unadsorbed antibodies were dissolved in TBS (pH 8.2).
The plate was washed with 10 ml of water, and further, in order to prevent non-specific adsorption, a blocking treatment was carried out by standing at 4° C. overnight using a blocking agent (Block Ace, manufactured by Snow Brand). Tween
20 (manufactured by Polyscience) 0.05 wt% Tris buffered saline (hereinafter referred to as TBS-Tween) 10
The remaining blocking agent was washed away with ml and used as an antibody-immobilized substrate.

Preparation of a calibration curve Prepare a standard TBS solution with a known concentration of CRP antigen (for example, 1.0
x 10-3 g/ml) was spread to cover all antibody-immobilized areas A to D on the substrate. After standing at 30°C for 5 minutes to perform an antigen-antibody reaction, the TBS-Twe
The unreacted antigen solution was washed with 10 ml of en. CR in advance
0.1μmφ sensitized (fixed) with P antibody by physical adsorption
A dispersion solution of antibody-sensitized polystyrene latex particles dispersed in TBS (latex concentration: 0.05 wt%) 5
0 μl was spread over areas AD in the same manner as the antigen solution above. After standing at 30°C for 5 minutes to capture latex particles by antigen-antibody reaction, the TBS-Tween1
The unreacted latex solution was washed with 0 ml. The number of latex particles in each region (A to D) was counted using a Hitachi electron microscope S-800 connected to an image analysis device.
Antigen (concentration 1.0 x 10-
3g/ml). Similarly 1.
0×10-4, 1.0×10-5, 1.0×10-7,
1.0 x 10-9, 1.0 x 10-11 g/ml C
The number of latex particles was counted as described above using a TBS standard solution with a known RP antigen concentration, and the number of latex particles was determined for each antigen concentration in each region A to D. The latex numbers for each antigen concentration in each of the above regions A to D were as shown in Table 1. FIG. 4 shows these relationships plotted in one graph. This was used as a standard curve for CRP antigen quantification.

[0024]

[Table 1]

Quantification of CRP Concentration in Unknown Samples As shown in Table 2, blood was collected from a subject and serum was separated using a conventional method (centrifugation method) to obtain samples 1, 2, and 3. 50 μl of a solution obtained by diluting the above sample 1 5 times with TBS was added to the antibody immobilized area
~ Spread on D. After standing at 30°C for 5 minutes to react,
Unreacted sample solution was washed and removed with 10 ml of TBS. CRP antibody sensitized latex dispersion solution (0.05wt
%/TBS solution) was spread over areas A to D and 30
The mixture was allowed to stand at ℃ for 5 minutes to react. The latex solution that was not captured by the antigen-antibody reaction was transferred to TBS-Tween1.
It was washed and removed with 0ml. The number of latex particles in each area was counted by electron microscope observation, and the concentration of sample 1 was determined to be 540 (A) ng/ml, 5
The concentrations were 38 (B) ng/ml and 534 (C) ng/ml. Note that the number of latex obtained from area D was outside the measurable range and was ignored. Next, the arithmetic mean of these concentrations was taken, and 540 ng/ml was determined as the CRP concentration of sample 1. Samples 2 and 3 are treated in the same manner, each with 24.
CRP concentrations of 8 μg/ml and 3.5 ng/ml were obtained. The results are shown in Table 2. [Comparative Example 1] Samples 1 to 3 were measured using a conventional method (LIA method). As a result, the CRP concentration of sample 1 was 538 ng/m
l. The CRP concentration of sample 2 was 24 μg/ml, and the CRP concentration of sample 3 was unmeasurable. The results are shown in Table 2.

[0026]

[Table 2]

According to the immunological quantitative analysis method of the present invention described above, since a plurality of antibody immobilization regions with different measurable concentration regions are provided, the calibration curves of each region overlap, resulting in an apparent dynamic The range (measurable region) is expanded (the dynamic range of each region is not necessarily expanded), and the actual measurable concentration region is expanded. Therefore, a complicated dilution operation can be omitted or the number of dilutions can be reduced, thereby shortening the testing time and reducing the cost of testing. Furthermore, since a plurality of pieces of data (detected concentration) can be obtained by one sample measurement, detection accuracy is improved.

[0028]

[Effects of the Invention] As explained above, according to the immunological quantitative analysis method of the present invention, it is possible to expand the measurable concentration range in immunological tests, reduce the number of dilution operations, and improve measurement accuracy. I can do it.

[Brief explanation of the drawing]

FIG. 1 is an explanatory diagram showing the procedure of the immunological quantitative analysis method of the present invention. (I) to (IV) indicate each procedure.

FIG. 2 is a plan view showing an embodiment of an antibody immobilization region.

[Figure 3] (a) is a graph showing the calibration curve, (b), (c)
FIG. 2 is an explanatory diagram showing an example of determining an antigen concentration from a specimen sample with an unknown concentration.

FIG. 4 is a graph showing a calibration curve created in an example.

[Explanation of symbols]

1...Substrate 2...Antibody 3...Blocking agent 5...Specimen sample 6...antigen 9...Antibody 10...Latex particles 11...Measuring means

Claims (6)

[Claims] Claim 1: A plurality of antibody immobilization areas with different measurable concentration ranges are provided on a solid phase, and a sample containing an antigen that specifically reacts with the antibody is applied to the antibody immobilization areas to convert the antigen into an antigen-antibody mixture. After the antigen is captured by reaction, insoluble carrier particles on which an antibody that specifically reacts with the antigen is immobilized are applied to detect the number of insoluble carrier particles captured by the antigen or a physical quantity correlated with the number of particles. An immunological quantitative analysis method characterized by measuring the antigen concentration in a sample. 2. A plurality of antigen immobilization regions with different measurable concentration ranges are provided on a solid phase, and a sample containing an antibody that specifically reacts with the antigen is allowed to act on the antigen immobilization region. - After being captured by an antibody reaction, insoluble carrier particles on which an antigen that specifically reacts with the antibody is immobilized are further applied to detect the number of insoluble carrier particles captured by the antibody or a physical quantity correlated with the number of particles. An immunological quantitative analysis method characterized by measuring the antibody concentration in a sample by detection. Claim 3: Antigens and antibodies are immobilized on each region on a solid phase using solutions of antibodies and antigens with different concentrations, and the regions on which a plurality of antigens and antibodies with different measurable concentration regions are immobilized are fixed on the solid phase. Claim 1 or 2 characterized in that it is formed on
Described immunological quantitative analysis method. Claim 4: Claim 1, wherein the solid phase is a flat substrate.
3. The immunological quantitative analysis method according to 2 or 3. 5. The immunological quantitative analysis method according to claim 1, 2 or 3, wherein the solid phase is a rotatable disk-shaped substrate. 6. The immunological quantitative analysis method according to claim 1, 2, 3, 4 or 5, wherein the insoluble carrier particles are latex particles.