JPH0552847A - Immunological quantitative analysis method - Google Patents

Abstract

PURPOSE:To achieve higher speed of an immunological quantitative analysis and higher reliability of analysis data. CONSTITUTION:An antibody 200 is immobilized at a part of at least one of a plurality of passages (channels) formed along the radial direction on a rotatable disc 100. After a body liquor 301 is introduced into the inner circumference of the passages, the disc is turned to develop the body liquor thereon and an antigen 300 to be inspected as analysis object in the body liquor is made to trap the antibody immobilized on the disc by an antigen/antibody reaction (primary immune reaction). Moreover, a non-soluble carrier particle 500 which has an antibody 400 reacting specifically with the antigen 300 immobilized thereon is arranged to act on the antigen 300 so that the non-soluble carrier particle 500 traps the antigen 300 through the antibody 400 (secondary immune reaction). Here, a value N/r of a ratio between an average particle size rmum of the non-soluble carrier particle 500 and a density Nwt.% of a suspension obtained is held within a range of 0.5-<=N/r<=2.0.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to an immunological quantitative analysis method, and more particularly to an immunological quantitative analysis method capable of speeding up an immunological test and improving reliability of analytical data.

[0002]

2. Description of the Related Art In recent years,
In the medical field, for the purpose of early detection of illness,
Quantitative analysis of trace components in body fluids is frequently performed.
For example, although quantification of trace components in blood is performed,
The concentration of body fluid components contained in blood is often very small, on the order of ng (nanogram) / ml, and quantitative analysis of these trace components has become an important issue in the medical field. Conventionally, as immunoassay methods such as radioimmunoassay method (RIA method), enzyme immunoassay method (EIA method) or latex agglutination reaction method (LIA method), are known as methods for quantifying trace components in blood. ing.

Here, the latex agglutination reaction method (LIA
Method) is a method developed by Singer and Plotz et al. In 1965. An insoluble carrier particle (latex) on which an antibody is immobilized is used to cause an antigen-antibody reaction to cause aggregation of latex particles. , A method of obtaining the concentration of the antigen from the turbidity. However, latex aggregation method (LIA method)
Since the turbidity is determined by a visual method, it is semi-quantitative and has a problem of insufficient sensitivity and accuracy.

Further, in JP-A-56-151357, a test liquid is brought into contact with a disk (slide glass) on which an antibody is immobilized (primary immune reaction), and then fine particles on which the antibody is immobilized are reacted. After that (secondary immunoreaction), an analytical method is disclosed in which the number of fine particles captured on a slide glass is counted. However, the method disclosed in JP-A-56-151357 has not been optimized for the immune reaction time and the latex concentration, and cannot be said to have been completed as a quantitative analysis method. In addition, the immune reaction time is long (first reaction time 30 minutes, second reaction time 3 hours),
Since a microscope or the like is used as a latex measuring means, there is a problem that automation and speedup of the measurement are extremely difficult.

On the other hand, the present applicant uses a rotatable antibody-immobilized disc and counts antibody-immobilized fine particles with an optical reader while rotating the disc, thereby automating all steps of immunological quantitative analysis. We have filed an application (Japanese Patent Application No. 3-198784), finding that the analysis time can be shortened and the measurement sensitivity can be improved. However, even in such an immunological quantitative analysis method, it is desired to further shorten the analysis time. Further, although higher sensitivity has been achieved as compared with the technique disclosed in JP-A-56-151357, there is room for study on conditions such as latex concentration and particle size.

The present invention has been made in view of the above circumstances, and an object of the present invention is to provide an immunological quantitative analysis method capable of speeding up an immunological test and improving reliability of analytical data. .

[0007]

Means for Solving the Problems As a result of intensive studies to achieve the above object, the present inventors have found that the average particle size r (μm) of the insoluble carrier particles and the concentration N (wt%) of the suspension thereof. )
It was found that when the ratio value N / r and the time of the secondary immune reaction are within a certain range, respectively, the immunoassay can be sped up and the reliability of analysis data can be improved. Has been completed.

That is, in the immunological quantitative analysis method of the present invention, a specimen sample containing a test antigen is brought into contact with a substrate on which an antibody against the test antigen is immobilized, and the test antigen is placed on the substrate by an antigen-antibody reaction. After carrying out a primary immune reaction to cause the immobilized antibody to capture, a suspension of insoluble carrier particles obtained by immobilizing an antibody against a test antigen is brought into contact with the substrate, and the antibody immobilized on the substrate. A second immune reaction is carried out to capture the antibody-immobilized insoluble carrier particles by the antigens captured by, and then the number of insoluble carrier particles captured by the test antigen through the antibody or a physical quantity correlating with the number of particles is measured. In the immunological quantitative analysis method described above, the value of the ratio N / r of the average particle size rμm of the insoluble carrier particles and the concentration Nwt% of the suspension is 0.5 ≦ N / r ≦ 2.0, preferably 0. ．
It is within the range of 5 ≦ N / r ≦ 1.5. Further, the immunological quantitative analysis method of the present invention, in the above method, the substrate is replaced with one in which the antigen for the test antibody in the specimen sample is fixed, and insoluble carrier particles are those in which the antigen for the test antibody is fixed. By substituting it, the test antibody in the specimen sample is quantified. Furthermore, in the immunological quantitative analysis method of the present invention, in the above method, the specimen sample is dropped on the substrate, the substrate is rotated to develop the specimen sample on the substrate, and then the specimen sample is moved in the radial direction of the substrate. It is designed to measure the number of insoluble carrier particles with an optical reader.
Preferably, the average particle diameter r of the insoluble carrier particles is 0.1 <r
≦ 0.3 μm.

The present invention will be described in detail below with reference to the drawings. FIG. 1 is an explanatory view showing the procedure of the immunological quantitative analysis method of the present invention. In the immunological quantitative analysis method of the present invention, the substrate 1 on which the antibody 200 against the test antigen is immobilized
00 is used (FIG. 1 (I)).

Here, the size, thickness, shape, etc. of the substrate 100 are appropriately selected and are not particularly limited. However, when the substrate is rotated to develop a sample and analyze by laser light or the like, it is suitable for rotation. As described above, it is preferable to have a disk shape (disk shape). In this case, as shown in FIG. 2, a large number of ridges 100a are radially formed on the rotatable disc 100, and a large number of sample developing surfaces (channels) 100b and a reaction part 100c to which the antibody 200 is fixed are provided. Analysis of multiple samples can be performed simultaneously. Further, the substrate 100 may have a rectangular shape such as a slide glass.

Materials for forming the substrate 100 include polycarbonate, polymethylmethacrylate, polystyrene,
Examples of the material include plastic materials such as polyvinyl chloride, polyvinyl acetate, polyurethane, and epoxy resin, and transparent materials such as glass. Preferred are transparent plastic materials such as polycarbonate and polymethylmethacrylate.
The reason why the substrate is made of a transparent material in this way is to enable optical analysis by a laser or the like. However, the substrate does not have to be transparent when performing analysis using reflected light.

The antibody 200 fixed on the substrate 100 differs depending on the antigen to be measured, but for example, an immunized animal (eg, an antigen to be measured) to be measured (antigen to be tested) is used.
Polyclonal antibodies and monoclonal antibodies produced by administration to rabbits, goats, sheep, etc.

To immobilize the antibody 200 on the substrate 100,
For example, 0.05M Tris buffered saline (TBS) solution (pH 8.2, concentration 0.5-10 μg / ml) of antibody or 0.05M carbonic acid
A bicarbonate buffer solution (pH 9.6, concentration 0.5-10 μg / ml) is dropped on the substrate and kept at 4 ° C for a day (or 20-30 ° C).
The antibody 200 may be physically adsorbed on the substrate 100 by leaving it for 2 hours. In this case, by using a substrate having a functional group such as a sulfone group, an amino group, a carboxyl group or a derivative thereof on the surface of the substrate, the antibody can be chemically bound to the substrate (P.Tijssen, Translated by Eiji Ishikawa “ Biochemical Experimental Method 11 Enzyme-linked immunoassay, "Tokyo Kagaku Dojin, Tatsuo Iwasaki, Tamie Ando," Monoclonal Antibody Hybridoma and ELISA ", published by Kodansha).

In the method of the present invention, first, on the above-mentioned antibody-immobilized substrate 100, the antibody 200 is added to the antigen 30 in the specimen sample.
0 is captured by the antigen-antibody reaction (primary immune reaction) (FIG. 1 (II)). Here, the sample sample to be analyzed is not particularly limited as long as it contains an antigen.
For example, body fluids such as blood and urine can be mentioned. The test antigen that is an analyte is not particularly limited, for example,
C-reactive protein (CRP), α-fetoprotein (AFP), carcinoembryonic antigen (CEA) and the like can be mentioned.

In order to carry out the antigen-antibody reaction on the substrate 100, the specimen sample containing the test antigen is brought into contact with the antibody-immobilized substrate. Specifically, the specimen sample may be dropped on the antibody-immobilized substrate, or the antibody-immobilized substrate may be dipped in the specimen sample. When a rotatable disk is used, for example, an appropriate amount (for example, about 50 μl) of a specimen sample containing the analyte antigen 300 to be analyzed is dropped on the antibody-immobilized substrate, the substrate 100 is rotated, To develop an antigen-antibody reaction efficiently between the antibody 200 fixed on the substrate 100 and the test antigen 300 in the analyte sample by developing the analyte sample into a thin film on the substrate 100 by centrifugal force. You can In this case, the time required for the antigen-antibody reaction is as short as 1 to 5 minutes. After the antigen-antibody reaction, the remaining sample specimens other than the captured test antigen 300 were phosphate buffered saline (PBS) (pH7.4) or Tris buffered saline (TBS) (0.05M, pH8.2). )
After dropping an appropriate amount (for example, about 1 ml), the substrate 100 which can be tilted or rotated is washed by rotating.

Next, the substrate 10 after the above primary immune reaction
A suspension of insoluble carrier particles 500 on which an antibody 400 that specifically reacts with the test antigen 300 is fixed is brought into contact with 0 (secondary immune reaction) (FIG. 1 (III)). The method of bringing the suspension of insoluble carrier particles into contact is the same as in the case of the first reaction.

Here, the insoluble carrier particles 500 on which the antibody 400 is immobilized are insoluble carrier particles (latex).
(For example, plastic particles, colloidal particles, etc.), an antibody 400 against the analyte 300 to be tested, which is an analyte
It refers to a substance that is physically or chemically adsorbed or bonded and fixed. Specifically, for example, the antibody is put in a 1.0% latex aqueous solution and left in a greenhouse for 2 hours to physically adsorb the antibody to the latex. Then, centrifuge to discard the supernatant, remove the non-adsorbed antibody, pour phosphate buffered saline (PBS) (pH 7.4) into the precipitate, redisperse and suspend the antibody-immobilized insoluble carrier particles. Make a liquid.
(JP-A-62-267298, Applied and Environmental Mi
crobiology, Oct. 1988, P2345-2348).

The insoluble carrier particles are those having fluorescence,
Alternatively, it may be colored. In this case, it is possible to perform analysis using fluorescence or coloring property. In addition to the case where the insoluble carrier particles themselves are formed of the fluorescent substance, the case where the insoluble carrier particles are coated with the fluorescent material and the case where the fluorescent substance is attached to the insoluble carrier particles are also included.

In the present invention, the ratio (N / r) of the average particle size r (μm) of the insoluble carrier particles to the concentration N (wt%) of the suspension is 0.5 ≦ N / It is within the range of r ≦ 2.0, preferably within the range of 0.5 ≦ N / r ≦ 1.5, and particularly preferably within the range of 1.0 ≦ N / r ≦ 1.5. Thereby, the secondary immune reaction can be completed in a short time. On the other hand, when the value of N / r is less than 0.5, the number of insoluble carrier particles trapped decreases, the sensitivity of quantitative analysis is significantly reduced, and reproducibility is reduced in a short time secondary immune reaction. Also gets worse. On the other hand, when it exceeds 2.0, the non-specific adsorption described below increases, the measurement accuracy decreases, and it becomes difficult to measure the number of insoluble carrier particles in the quantification of a high-concentration sample. Therefore, in any case, there is a problem in carrying out the secondary immune reaction in a short time.

FIG. 3 shows the relationship between the value of N / r and the occupied area ratio of insoluble carrier particles. However, the secondary immune reaction time is 5 minutes. The occupied area ratio refers to the ratio of the area (planar area) of the insoluble carrier particles to the area of the measurement region. As can be seen from FIG. 4, the occupied area ratio of the insoluble carrier particles is lower than 10% in the region where the value of N / r is less than 0.5, which effectively covers the measured concentration region of the target component existing in blood. It causes inconvenience. Therefore, it is necessary to set the value of N / r to 0.5 or more. Further, as can be seen from FIG. 4, when the value of N / r is 2.0, the occupied area ratio of the insoluble carrier particles becomes about 27%, and when it exceeds 2.0, the insoluble carrier particles agglomerate more and the optical When using a static reader, it is difficult to measure high density areas. Therefore, it is necessary to set the value of N / r to 2.0 or less.

In the present invention, when an optical reader is used, the average particle size r of the insoluble carrier particles is
It is preferable to set it within the range of 0.1 μm <r ≦ 0.3 μm. If r is 0.1 μm or less, the signal to the laser light becomes weak, the S / N ratio deteriorates, and detection by an optical reading device becomes difficult. Therefore, measurement must be performed by other measuring means such as an electron microscope. I have to. r is 0.3
When it exceeds μm, it is disadvantageous in terms of sensitivity and the like in the secondary immune reaction. In the case of insoluble carrier particles that emit fluorescence,
Since the antigen concentration is measured by converting the fluorescence intensity into the number of particles, the particle size may be small.

In the immunological quantitative analysis method of the present invention, the secondary immune reaction time is preferably within 5 minutes. .. When the secondary immune reaction time is within 5 minutes, the occurrence of non-specific adsorption is reduced, and the variation in data is reduced. Here, nonspecific adsorption means that antibody-immobilized insoluble carrier particles are adsorbed on the substrate by physical adsorption or the like other than the antigen-antibody reaction. If this non-specific adsorption increases,
The correspondence between the number of test antigens and the number of antibody-immobilized insoluble carrier particles is impaired. The more preferable time of the secondary immune reaction is 10 seconds to 5 minutes, particularly 30 seconds to 3 minutes.

In the above-mentioned secondary immune reaction, the antibody 400 immobilized on the insoluble carrier particles 500 is the antigen 300-to-be-tested captured by the immobilized antibody 200 on the substrate 100.
An antibody reaction occurs, and thereby the antibody-immobilized insoluble carrier particles are captured (FIG. 1 (III)). The insoluble carrier particles that have not been captured on the substrate are washed away in the same manner as the above-described specimen sample. In this way, the sample substrate is manufactured. Next, the number of insoluble particles captured by the antigen on the sample substrate or a physical quantity corresponding to the number of particles is measured by the measuring means 600 to obtain the number of antigens 300 (concentration of the antigen) (FIG. 1 (IV)). ).

Here, as the measuring means 600 for measuring the number of particles and the like, an optical measuring means is preferable. Examples of the optical measuring means include measuring means such as a change in reflectance, absorption of light having a specific wavelength, fluorescence intensity or rotation of the polarization plane of polarized light. When directly measuring the number of insoluble carrier particles, it is preferable to use a laser as an optical measuring means. As the laser source used, a generally used semiconductor laser or the like can be used as it is, and an appropriate laser source is selected according to the size of the insoluble carrier particles. As an analyzer equipped with an optical measuring means, for example, an apparatus as shown in FIG. 5 is used (Japanese Patent Application No. 1-0923).
See No. 67).

In the figure, reference numeral 100 is a disk,
It is mounted on the turntable 2. On the outer periphery of the lower portion of the disc 100, a tray 6 for receiving the sample falling from the disc 100 is arranged and is collected in a collection tank 7. The motor 8 is for rotating the disk 100, and the drive control circuit 9 is for controlling the drive of the motor 8. The nozzle 11 drops the sample sent from the sample sending device 12 onto the disc 100.
The optical measuring head 14 reciprocates along a feed screw shaft 15 extending in the disk radial direction. The motor 16 is for driving the head 14, and the drive control circuit 17
Controls the drive of the motor 16. The optical reading device (optical measuring head) 14 has a laser light projecting unit and a light receiving unit. As shown in FIG. 6A, the irradiation light 19 from the light projecting unit passes through the half mirror 20 and the lens 21. After that, the sample on the sample developing surface 5 is irradiated with the converged light, and the reflected light 22 reflected from the sample is received by the light receiving portion. As shown in FIG. 6B, the projection light 19 may be emitted from the back surface side of the disk, or the analysis may be performed using transmitted light instead of reflected light. In these cases, the turntable 2 is made of a transparent material or the turntable 2 is removed.

The signal processing device 23 has a function of sending a signal to the head 14 to turn on a light source, and a function of processing and analyzing an optical signal received by the head 14. The display device 24 displays the analysis result on the screen, and the recording device 25 prints out and outputs the analysis result. A CPU (central processing unit) 27 controls the sample feeding device 12, the drive circulation circuits 9 and 17, the signal processing device 23, and operates them according to a program. The CPU 27 has a program operation device 28 and a program storage device 29. According to the analyzer having the above structure, all steps of the immunological quantitative analysis can be performed on the disk, and the analysis can be fully automated.

When the insoluble carrier particles emit fluorescence, a photodetector can be used as an optical measuring means. In this case, by interposing a filter, a specific wavelength (for example, 397 nm, 472 nm)
nm, 577 nm, etc.) and convert the fluorescence intensity to the number of particles.

In order to obtain the antigen concentration from the measured number of insoluble carrier particles, the same procedure as described above is performed except that a sample having a known antigen concentration is used, and the relationship between the antigen concentration and the number of insoluble carrier particles is obtained. A calibration curve may be prepared in advance, and the antigen concentration may be determined from this calibration curve. In the above-mentioned immunological quantitative analysis method of the present invention, for the sake of convenience of explanation, the case where the structure is disk / antibody / antigen / antibody / insoluble carrier particles is shown.
Alternatively, the antibody concentration may be quantified by configuring the antigen / antibody / antigen / insoluble carrier particles.

[0029]

The present invention will be described in more detail based on the following examples. Example 1 (1) CR obtained by previously immunizing rabbits with human CRP antigen
Tris buffered saline (TBS) solution of P antibody (concentration 1.69 ×
5 μl of 10 ^-3 g / ml) was dropped on a rotatable polycarbonate disc with a radius of 13.0 cm at a point with a radius of 3.5 cm in a channel, and left still for 2 hours to sensitize (fix) the CRP antibody on the substrate. I made it. Then, the remaining antibody was removed by washing with 200 μl of TBS solution. (2) After that, 10 μl of a blocking agent (Block Ace: manufactured by Snow Brand Co., Ltd.) was added dropwise to the antibody-sensitized site, and left standing at 4 ° C. for 24 hours for blocking treatment. (3) The above-mentioned antibody-immobilized substrate was set on the rotary table of the analyzer shown in FIG. 5, and each CRP sample adjusted by TBS (concentration 0, 1.0 × 10 ⁻⁹ , 1.0 × 10 ⁻⁷ , 1.0 × 10 ^{− 5} g / ml)
10 μl was dropped on the antibody-sensitized site, and an antigen-antibody reaction was performed at room temperature for 10 seconds to capture the antigen on the antibody-immobilized substrate (primary immune reaction). Then turn the rotary table to 500r
The sample on the substrate was removed by rotating at pm and further washed with 200 μl of TBS solution (TBS-Tween) containing 0.1% of a surfactant (Tween-20 manufactured by Polyscience).

(4) 10 μm of a 0.3 wt% anti-CRP latex solution (N / r = 1.0) obtained by sensitizing a CRP antibody to polystyrene latex having a particle size of 0.3 μm
1 was dropped on the antibody-sensitized site, and a secondary immune reaction was performed at room temperature for 300 seconds to capture the latex particles on the substrate. Then, the rotary table was rotated at 500 rpm to remove excess latex, and 200 μl of TBS-Tween was added.
The remaining latex was washed with the solution and further removed with 200 μl of distilled water. (5) While rotating this disk at 1800 rpm, an optical head using a semiconductor laser having a wavelength of 830 nm as a light source was scanned in the radial direction, and the number of latex particles captured on the substrate was measured. When each concentration and the corresponding number were plotted, the good calibration curve shown in FIG. 7 was obtained.

Comparative Example 1 Latex solution concentration was 0.75 wt% (N / r = 2.
The number of traps at each antigen concentration was plotted in the same manner as in Example 1 except that the above was set as 5). As a result, non-specific adsorption increased and the baseline greatly increased, and the number of latexes could not be measured in the high concentration region. No good calibration curve was obtained.

Comparative Example 2 The latex solution concentration was 0.06 wt% (N / r = 0.
The number of traps at each antigen concentration was plotted in the same manner as in Example 1 except that 3), and the number of latex traps did not increase even in the high concentration region, and the reproducibility at each concentration was poor and was good. No calibration curve was obtained.

[0033]

As described above, according to the immunological quantitative analysis method of the present invention, the speed of immunological test can be increased and the reliability of analysis data can be improved.

[Brief description of drawings]

FIG. 1 is an explanatory view showing a procedure of an immunological quantitative analysis method of the present invention.

FIG. 2 is a plan view showing a plurality of discs for performing simultaneous analysis of a plurality of samples.

FIG. 3 is a graph showing the relationship between the value of N / r and the occupied area ratio of insoluble carrier particles.

FIG. 4 is a graph showing a relationship between a secondary immunization reaction time and an occupied area ratio of insoluble carrier particles.

FIG. 5 is a configuration diagram showing an example of an analyzer.

FIG. 6 is a diagram showing a mode of measurement by an optical analysis means.

FIG. 7 is a graph showing a calibration curve in an example.

[Explanation of symbols]

 100: Substrate (disk) 200: Antibody 300: Antigen 400: Antibody 500: Insoluble carrier particles

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Minoru Takase 1660 Kamizumi, Sodegaura-shi, Chiba Idemitsu Petrochemical Co., Ltd.

Claims (6)

[Claims] 1. A primary immunity in which a sample sample containing a test antigen is brought into contact with a substrate on which an antibody against the test antigen is immobilized, and the test antigen is captured by the antibody immobilized on the substrate by an antigen-antibody reaction. After the reaction, a suspension of insoluble carrier particles obtained by immobilizing an antibody against a test antigen is brought into contact with the substrate, and the antibody-immobilized insoluble carrier is trapped by the antibody immobilized on the substrate. In the immunological quantitative analysis method of performing a secondary immune reaction to capture particles, and then measuring the number of insoluble carrier particles captured by the test antigen through the antibody or the physical quantity correlating with the number of particles, the insoluble carrier Average particle diameter rμm and concentration Nw of the suspension
The immunological quantitative analysis method, wherein the value of the ratio N / r to t% is within the range of 0.5 ≦ N / r ≦ 2.0. 2. A primary immunity in which a sample sample containing a test antibody is brought into contact with a substrate on which an antigen against the test antibody is immobilized, and the test antibody is captured by the antigen immobilized on the substrate by an antigen-antibody reaction. After carrying out the reaction, a suspension of insoluble carrier particles obtained by immobilizing an antigen against a test antibody is brought into contact with the substrate, and the antigen-immobilized insoluble carrier is immobilized on the substrate by the antibody captured by the antigen immobilized on the substrate. In the immunological quantitative analysis method of performing a secondary immune reaction to capture particles, and then measuring the number of insoluble carrier particles captured by the test antibody through the antigen or the physical quantity correlating with the number of particles, the insoluble carrier Average particle diameter rμm and concentration Nw of the suspension
The immunological quantitative analysis method, wherein the value of the ratio N / r to t% is within the range of 0.5 ≦ N / r ≦ 2.0. 3. The immunological quantitative analysis method according to claim 1, wherein the secondary immune reaction time is within 5 minutes. 4. The immunological quantitative analysis method according to claim 1, 2 or 3, wherein the value of N / r is 0.5 ≦ N / r ≦ 1.5. 5. The number of insoluble carrier particles is measured by dropping an analyte sample on the substrate, rotating the substrate to develop the analyte sample on the substrate, and then measuring the number of insoluble carrier particles by an optical reading device that moves in the radial direction of the substrate. Claims 1, 2, 3 characterized in that
Or the immunological quantitative analysis method described in 4. 6. The particle diameter r of the insoluble carrier particles is 0.1 <r
The immunological quantitative analysis method according to claim 5, wherein ≦ 0.3 μm.