WO2016129029A1

WO2016129029A1 - Automatic analyzer

Info

Publication number: WO2016129029A1
Application number: PCT/JP2015/053484
Authority: WO
Inventors: 興子山本; 足立　作一郎; 匡章平野
Original assignee: 株式会社日立製作所
Priority date: 2015-02-09
Filing date: 2015-02-09
Publication date: 2016-08-18

Abstract

The present invention has: a reaction disk in which cells containing a reaction solution mixed from a sample and a reagent are held on a circle, and which is repeatedly rotated and stopped; and a scattered light measurement unit for illuminating the cells with illuminating light from a light source while the reaction disk rotates, and measuring scattered light subsequent to interaction with the reaction solution in the cells. The scattered light measurement unit receives scattered light of angles of less than 15° from the optical axis of the illuminating light, and measures the change over time in the quantity of light received by the scattered light measurement unit using, as the reagent, a latex reagent having a particle diameter of 450 to 875 nm, inclusive, obtained by sensitizing an antibody that recognizes a measured substance. The scattered light measurement performs the measurements by way of reaction process data of an agglutination reaction of a reaction solution mixture of the latex reagent and a sample containing the measured substance, and quantifies the measured substance present in the sample.

Description

Automatic analyzer

The present invention relates to an automatic analyzer that measures the concentration of a substance to be measured contained in a sample.

Latex aggregation turbidimetry is a method for highly sensitive determination of proteins, hormones, viruses, etc. contained in biological samples (samples) such as blood and urine. The latex agglutination turbidimetry is a technique for quantifying the agglutination reaction of latex particles, which are insoluble carriers contained in a reagent, as an optical change. The latex particle surface in the reagent is sensitized with an antibody that reacts immunologically with the antigen to be measured in the sample. When the sample and the reagent are mixed, latex particles are aggregated through the antigen to be measured by the antigen-antibody reaction in the mixed reaction solution. The concentration quantification is performed by measuring a sample whose concentration of the antigen to be measured is known in advance and comparing the signal change amount with a plotted calibration curve. Here, if the aggregation change of latex particles can be grasped as an optically large signal change amount, it leads to high sensitivity.

In recent years, technological development has been made on the enhancement of sensitivity using scattered light. For example, Patent Document 1 discloses an apparatus configuration that performs scattered light measurement on an automatic analyzer. Patent Document 2 discloses a high-sensitivity configuration and a reagent composition plan using scattered light measurement. Patent Document 3 discloses an increase in sensitivity by measuring scattered light using a polarization scattering intensity difference measurement (PIDS) method.

Patent 5318296 JP 2013-64705 A Japanese translation of PCT publication No. 2004-536302

In order to increase the sensitivity, it is necessary that the change when the particles contained in the reagent aggregate to form an aggregate is optically indicated by a large amount of signal change. In an automatic analyzer used for measuring protein in a sample, an absorptiometer is mainly used. Although the scattering photometer is more sensitive than the absorptiometer, the dynamic range is narrow. Therefore, an apparatus configuration using both the absorptiometer and the scattering photometer is desirable. In such a case, using a conventional absorptiometer, it is possible to increase the sensitivity by receiving scattered light at any angle with a reagent using latex particles of any particle size in the scattering photometer. I didn't know what would happen. In particular, a reagent using latex particles is prepared for an absorptiometer, which is composed mainly of components having a particle size of latex particles less than 300 nm. Therefore, the apparatus configuration achieves high sensitivity within a range that assumes a latex reagent for an absorptiometer.

Patent Document 1 shows a configuration in which light receivers are arranged at many angles in a latex agglutination reaction. However, it is assumed that the amount of components contained in the sample is small and only slightly aggregates, and only the change rate of the amount of scattered light when the particle size of the latex particles changes by 1% was considered. In particular, it is considered that the same tendency is shown for any particle size on the low angle side where the light receiving angle is smaller than 20 °. Therefore, on the low angle side, the sensitivity is increased up to a light receiving angle of about 20 °, and no further sensitivity has been achieved.

Patent Document 2 discloses the conditions of the apparatus and the reagent regarding the particle diameter and density in the latex agglutination reaction. However, since the noise may increase at the scattered light receiving angle of less than 15 °, only the light receiving angle of 15 ° or more is assumed, which is limited. For this reason, the calculation is generally performed at a light receiving angle of 20 ° (± 2.5 °). As a result, it is concluded that the latex particle diameter of 300 nm to 430 nm is a suitable condition, but no further increase in sensitivity has been achieved.

Patent Document 3 describes a method for quantifying a substance to be measured in a sample with higher sensitivity using a polarization scattering intensity difference measurement (PIDS) method, but does not show specific reagents or angle conditions. . In particular, conditions on an analysis system with a limited reaction time and optical measurement time such as an automatic analyzer are not shown.

The automatic analyzer according to the present invention holds a cell containing a reaction mixture in which a sample and a reagent are mixed on the circumference, and repeatedly rotates and stops a reaction disk, and irradiates light from a light source while the reaction disk is rotating. A scattered light measuring unit that measures the scattered light after irradiating the cell and interacting with the reaction liquid in the cell. The scattered light measuring unit emits scattered light at an angle of less than 15 ° from the optical axis of the irradiated light. Using a latex reagent having a particle size of 450 nm or more and 875 nm or less that has been sensitized with an antibody that recognizes the substance to be measured as a reagent, a sample containing the substance to be measured and latex Measured as reaction process data of the agglutination reaction of the reaction liquid mixed with the reagent, and the substance to be measured in the sample is quantified.

As an example, the scattered light measurement unit receives scattered light at an angle of 7.5 to 12.5 ° from the optical axis of the irradiated light.

According to the present invention, it is possible to measure a low-concentration substance to be measured with higher sensitivity than measurement with a conventional scattering photometer.

Issues, configurations, and effects other than those described above will be clarified by the following description of embodiments.

The figure which shows the particle size dependence of the amount of scattered light of a latex particle. The figure which shows the normalization signal of the amount of scattered light of a latex particle. The figure which shows the example of whole structure of an automatic analyzer. Schematic which shows the structural example of an absorbance measurement part. Schematic which shows the structural example of a scattered light measurement part. The figure which shows the example of reaction process data. The figure which shows the signal variation | change_quantity for every reagent particle size and light reception angle. The figure which shows the particle size dependence of the signal variation | change_quantity in a final concentration 1.5abs solution. The figure which shows the particle size dependence of the normalized signal variation | change_quantity. The figure which shows the particle size dependence of noise amount. The figure which shows the particle size dependence of S / N ratio. The figure which shows the particle size dependence of the signal variation | change_quantity in final concentration 2.0abs solution.

In the present invention, theoretical examination and reaction measurement experiment were conducted when the assumption of the scattered light receiving angle and the reagent particle size range of the apparatus was expanded. As a result, by receiving scattered light having a light receiving angle lower than 15 °, for example, a light receiving angle of 10 °, high sensitivity in conventional scattered light measurement is obtained, for example, at a conventional light receiving angle of 20 ° as in Patent Document 2. The present invention has been completed by obtaining a method with higher sensitivity than when using a reagent having a particle diameter of 300 nm to 430 nm or less. In addition, the angle described in this specification is an angle in air.

The reagent according to the present invention contains, for example, latex particles having an average particle diameter of 450 nm or more and 875 nm or less, and the latex particles are sensitized with an antibody that recognizes the antigen to be measured. Particle aggregation occurring between the antibody sensitized to the latex particles and the antigen to be measured is measured as a signal change amount of scattered light.

The optical system conditions of the scattering photometer suitable for high sensitivity were selected from the particle size dependence of the optical signal of the particles, that is, the absorbance and the amount of scattered light. Here, it is assumed that the aggregate is generally spherical, and that the aggregate exhibits the same properties as particles having a particle size twice that of a single particle.

Figures 1 (A) and (B) show polystyrene in water calculated according to "C. F. Bohren, D. R. Huffman, Absorption and Scattering of Light by Small Particles," J. Wiley & Sons, 1983 "(references). The particle size dependence of the amount of scattered light generated when a single particle (refractive index: 1.59) is irradiated with light having a wavelength of 700 nm is shown for each light receiving angle of 5 °, 10 °, 20 °, and 30 °. FIG. 1B is an enlarged view of the vertical axis of FIG. The light receiving angle is defined by the center angle of the light receiving range with respect to the optical axis of the irradiation light. The light receiving range is about ± 2.5 ° with respect to the center angle. These calculations were performed using Mie scattering theory.

In the Mie scattering theory, it is known that the larger the size of the scatterer, the more the scattered light is emitted forward (in the traveling direction of the irradiation light). Therefore, the received light quantity tends to increase to a larger particle size as the received light angle becomes smaller, and the scattered light quantity is also larger. The situation can be seen from FIGS. If the aggregation reaction of particles is measured with scattered light, it is understood that a larger amount of light can be obtained by receiving a larger aggregate with scattered light at a lower angle by utilizing this tendency. However, since the low angle side such as 10 ° is easily affected by large bubbles and dust, the S / N ratio is often disadvantageous. For this reason, as described in Patent Document 2, a light receiving angle of about 20 ° is often used in practice, and a light receiving angle lower than that is hardly studied.

Also, in order to make the measurement range highly sensitive by measuring the scattered light, it is necessary to select the first latex reagent condition by comparing the relationship between the signal change amount of the scattered light measurement and the aggregate diameter. Therefore, it is not a change in the amount of light when the particle diameter changes by 1% as in Patent Document 1, but strictly speaking, it is assumed that two latex particles aggregate to form one aggregate, and how much at each angle. It is necessary to select the particle conditions of the first latex reagent from the knowledge of whether it is possible to measure up to the aggregate diameter. 1A and 1B, there is a peak in the particle size dependence of the amount of scattered light, and the amount of scattered light decreases when the particle size becomes too large. For this reason, if the initial latex particles are too large, the aggregate diameter of the aggregates becomes large and measurement as a large signal change becomes impossible.

¡Compare how much particle size (aggregate diameter) can be measured as signal change for each angle. A normalized signal obtained by dividing the scattered light amount by the peak value in FIGS. 1A and 1B (the peak value when the scattered light amount starts to decrease when the particle size is increased from 50 nm). As shown in FIG.

As shown in FIG. 2, by measuring the scattered light at the light receiving angles of 5 °, 10 °, 20 °, and 30 ° in the air, 2.3 μm, 1.9 μm, 1.3 μm, and 0.9 μm are obtained. It was found that even the diameter of the aggregate can be regarded as a signal change.

If the conditions are such that low-angle scattered light is received, the aggregates formed can be made larger. Therefore, particles having a relatively large particle size can be used or mixed as the particle size of the latex particles initially included in the reagent. A method is mentioned. A large amount of signal change associated with a larger size change of the aggregate can be expected by binding the large particle diameter to become a part of the aggregate.

Since the automatic analyzer measures the reaction, the concentration of the latex reagent must be above a certain level in order to proceed with the reaction. A particle concentration corresponding to an absorbance of 0.25 abs to 2.0 abs (converted to an optical path length of 10 mm) is assumed in a reaction solution for final concentration determination using light having a wavelength of approximately 700 nm. The present invention is also applied in a similar concentration range. The signal size dependence as shown in FIG. 2 is applicable because it shows the same tendency in any concentration range. Even when particles of different particle sizes are mixed, if the absorbance of 1/3 or more of the total absorbance of the latex reagent is caused by particles in that range, the effect of increasing the sensitivity is obtained in the same manner. It is possible.

In high sensitivity, since it is a low concentration region, the signal difference between a single latex particle (monomer) and an aggregate (dimer) in which two latex particles are bound is measured as a signal change amount. . Assuming that the dimer exhibits the same characteristics as latex particles having a particle size twice that of the initial latex particles, the light reception angle of 30 ° can measure the signal change amount of the aggregate diameter up to 0.9 μm. It can be seen that particles having a latex particle size of less than 0.45 μm (450 nm) are suitable for high sensitivity. Since the signal change amount of the aggregate diameter up to 1.3 μm can be measured at a light receiving angle of 20 °, it can be seen that as large an initial latex particle size as less than 0.65 μm (650 nm) is suitable for high sensitivity. Since the signal change amount of the aggregate diameter up to 1.9 μm can be measured at a light receiving angle of 10 °, it can be seen that as large particles as possible with an initial latex particle size of less than 0.95 μm (950 nm) are suitable for high sensitivity. Since the light receiving angle of 5 ° can measure the signal change amount of the aggregate diameter up to 2.3 μm, it can be understood that the initial latex particle size is less than 1.15 μm (1150 nm) and that the largest possible particle is suitable for high sensitivity.

In summary, the particle agglomeration reaction is measured using a scattering photometer that measures light at a low angle (an angle smaller than 20 ° (17.5 ° to 22.5 °)). It has become clear that even larger optical signal changes in aggregates can be captured than absorbance measurements that have been used in biochemical analyzers. As a result, by combining the scattered light receiving angle and the particle diameter of the reagent, a larger amount of signal change can be measured as a scattered photometer, leading to higher sensitivity.

The insoluble carrier for sensitizing the antibody recognizing the substance to be measured used in the present invention is preferably polystyrene latex particles having an average particle diameter of 450 nm or more and less than 875 nm. Further, metal colloids, magnetic particles, silica particles and the like can be used instead of latex particles. As a method for sensitizing an antibody to an insoluble carrier, a generally used physical adsorption method or chemical bonding method can be applied.

Examples of the sample containing the substance to be measured to be measured in the present invention include human or animal blood, plasma, serum, urine and the like. The substance to be measured in the present invention may be any substance that can recognize the substance to be measured specifically. For example, proteins such as D-dimer, C-reactive protein (CRP), and prostate specific antigen (PSA).

The antibody used in the present invention may be any antibody that can specifically recognize the substance to be measured. In general, polyclonal antibodies and monoclonal antibodies derived from rabbits, mice, rats, and goats can be used. If specificity is taken into consideration, the use of a monoclonal antibody is preferred.

As the buffer used in the present invention, for example, phosphate buffer, Tris-HCl buffer, glycine buffer, Good buffer, and the like are preferably used.

Next, an analysis system that performs measurement will be described. In the present invention, the signal change of particle aggregation is measured with scattered light.

FIG. 3 is a diagram showing an example of the overall configuration of the automatic analyzer according to the present invention. The configuration of the automatic analyzer shown in FIG. 3 is used only for explaining the basic concept of the present invention. The automatic analyzer according to the present embodiment controls three types of disks, a sample disk 3, a reagent disk 6 and a reaction disk 9, and dispensing

mechanisms

10 and 11 for moving samples and reagents between these disks, and these. Control circuit 23, absorbance measurement circuit 24 for measuring the absorbance of the reaction solution, scattered light measurement circuit 25 for measuring scattered light from the reaction solution, data processing unit 26 for processing data measured by each measurement circuit, data processing unit 26 has an input unit 27 and an output unit 28 which are interfaces with the H.26.

The data processing unit 26 includes a data storage unit 2601 and an analysis unit 2602. The data storage unit 2601 stores control data, measurement data, data used for data analysis, analysis result data, and the like. The input unit 27 and the output unit 28 input / output data to / from the data storage unit 2601. In the example of FIG. 3, the input unit 27 is a keyboard. However, a touch panel, a numeric keypad, or other input device may be used.

A plurality of sample cups 2 that are containers for the sample 1 are arranged on the circumference of the sample disk 3. Sample 1 is as in the sample example shown above. On the circumference of the reagent disk 6, a plurality of antibody reagent bottles 5 that are containers for the antibody reagent 4 are arranged. On the circumference of the reaction disk 9, a plurality of cells 8 that are containers for the reaction liquid 7 in which the sample 1 and the reagent 4 are mixed are arranged.

The sample dispensing mechanism 10 is a mechanism used when a certain amount of sample 1 is moved from the sample cup 2 to the cell 8. The sample dispensing mechanism 10 includes, for example, a nozzle that discharges or sucks a solution, a robot that positions and transports the nozzle to a predetermined position, and a pump that discharges or sucks the solution from the nozzle. The antibody reagent dispensing mechanism 11 is a mechanism used when a certain amount of reagent 4 is moved from the reagent bottle 5 to the cell 8. The reagent dispensing mechanism 11 also includes, for example, a nozzle that discharges or sucks a solution, a robot that positions and transports the nozzle to a predetermined position, and a pump that discharges or sucks the solution from the nozzle.

The stirring unit 12 is a mechanism unit that stirs and mixes the sample 1 and the reagent 4 in the cell 8. The cleaning unit 14 is a mechanism unit that discharges the reaction solution 7 from the cell 8 that has undergone the analysis process, and then cleans the cell 8. After the washing is finished, the next sample 1 is again dispensed from the sample dispensing mechanism 10 and the new reagent 4 is dispensed from the reagent dispensing mechanism 11 and used for another reaction process.

In the reaction disk 9, the cell 8 is immersed in a constant temperature fluid 15 in a constant temperature bath whose temperature and flow rate are controlled. For this reason, the temperature of the cell 8 and the reaction liquid 7 therein is maintained at a constant temperature even during movement by the reaction disk 9. In the present embodiment, water is used as the constant temperature fluid 15 and its temperature is adjusted to 37 ± 0.1 ° C. by the control circuit 23. Of course, the medium and temperature used as the constant temperature fluid 15 are examples.

An absorbance measuring unit 13 and a scattered light measuring unit 16 are arranged on a part of the circumference of the reaction disk 9.

FIG. 4 is a schematic diagram illustrating a configuration example of the absorbance measurement unit 13. 4 absorbs the light emitted from the halogen lamp light source 29 during the rotation of the reaction disk 9 to the cell 8, and the light 30 transmitted through the reaction liquid in the cell 8 is spectrally separated by the diffraction grating 31. The photodiode array 32 receives light. The wavelengths received by the photodiode array 32 are 340 nm, 405 nm, 450 nm, 480 nm, 505 nm, 546 nm, 570 nm, 600 nm, 660 nm, 700 nm, 750 nm, and 800 nm. Light reception signals from these light receivers are transmitted to the data storage unit 2601 of the data processing unit 26 through the absorbance measurement circuit 24. Here, the absorbance measurement circuit 24 acquires a light reception signal in each wavelength region at regular time intervals, and outputs the acquired light amount value to the data processing unit 26.

FIG. 5 is a schematic diagram illustrating a configuration example of the scattered light measurement unit 16. In the present embodiment, for example, an LED light source unit is used as the light source 17. The irradiation light 18 emitted from the light source 17 is irradiated to the cell 8 located on the optical path, and the transmitted light 19 transmitted through the reaction solution 7 in the cell 8 is received by the transmitted light receiver 20. For example, 700 nm is used as the wavelength of the irradiation light. In scattered light measurement, it is necessary to use irradiation light with a wavelength of 600 nm to 800 nm, considering that it is less susceptible to the influence of scatterers (milky milk, hemolysis, jaundice) contained in the sample and visible light. preferable. In this embodiment, an LED light source unit is used as the light source 17, but a laser light source, a xenon lamp, a halogen lamp, or the like may be used.

The scattered light measuring unit 16 irradiates the cell 8 with the irradiation light 18 from the light source 17 while the reaction disk 9 is rotating, and the scattered light after interacting with the reaction liquid 7 in the cell 8 is scattered light receiver 22a, Measurement is performed at 22b and 22c. The scattered light receiver 22a receives scattered light 21a in a direction away from the optical axis of the irradiation light 18 or the transmitted light 19 by an angle of 10 ° in the air. The scattered light receiver 22b receives scattered light 21b in a direction away from the optical axis of the irradiation light 18 or the transmitted light 19 by an angle of 20 ° in the air. The scattered light receiver 22c receives the scattered light 21c in a direction away from the optical axis of the irradiation light 18 or the transmitted light 19 by an angle of 30 ° in the air. The light receiving angles of the scattered

light receivers

22a, 22b, and 22c are 10 °, 20 °, and 30 °, respectively, and specifically, 7.5 to 12.5 °, 17.5 to 22.5 °, and 27, respectively. .5 to 32.5 °. The scattered

light receivers

22a, 22b, and 22c are constituted by photodiodes, for example. The light reception signals of these scattered

light receivers

22a, 22b, and 22c are transmitted to the data storage unit 2601 of the data processing unit 26 through the scattered light measurement circuit 25. The scattered light measurement circuit 25 also acquires three light reception signals having different light reception angles at regular time intervals, and outputs the acquired light amount values to the data processing unit 26.

The scattered

light receivers

22 a, 22 b, and 22 c are arranged in a plane that is substantially perpendicular to the moving direction of the cell 8 due to the rotation of the reaction disk 9. Here, the reference position (starting point of scattering) of the light receiving angle is set at the center of the optical path of the light passing through the cell 8.

In FIG. 5, the case where the scattered

light receivers

22a, 22b, and 22c are arranged so as to correspond to the light receiving angles of 10 °, 20 °, and 30 ° has been described. May be configured to receive scattered light at a plurality of angles at a time. By using a linear array, the choice of the light receiving angle can be expanded. Further, instead of the light receiver, an optical system such as an optical fiber or a lens may be disposed near the cell 8, and the light may be guided to the scattered light receiver disposed at another position.

Quantification of the concentration of the substance to be measured contained in sample 1 is performed according to the following procedure.

First, the control circuit 23 cleans the cell 8 in the cleaning mechanism 14. Next, the control circuit 23 dispenses a certain amount of the sample 1 in the sample cup 2 into the cell 8 by the sample dispensing mechanism 10. Next, the control circuit 23 dispenses a predetermined amount of the reagent 4 (first reagent) in the reagent bottle 5 to the cell 8 by the reagent dispensing mechanism 11.

At the time of dispensing each solution, the control circuit 23 drives the sample disk 3, the reagent disk 6 and the reaction disk 9 to rotate by the corresponding drive unit. At this time, the sample cup 2, the reagent bottle 5, and the cell 8 are positioned at predetermined dispensing positions according to the driving timings of the dispensing mechanisms corresponding thereto. That is, the sample disk 3, the reagent disk 6, and the reaction disk 9 are repeatedly rotated and stopped under the control of the control circuit 23.

Subsequently, the control circuit 23 controls the stirring unit 12 to stir the sample 1 and the reagent 4 dispensed in the cell 8 to generate a reaction solution 7. As the reaction disk 9 rotates, the cell 8 containing the reaction solution 7 passes through the measurement position where the absorbance measurement unit 13 is arranged and the measurement position where the scattered light measurement unit 16 is arranged. Each time the cell 8 passes through the measurement position during the rotation of the reaction disk, the transmitted light or scattered light from the reaction solution 7 is measured via the corresponding absorbance measurement unit 13 and scattered light measurement unit 16, respectively. In this embodiment, each measurement time is about 10 minutes. Measurement data representing the temporal change in the amount of light received by the absorbance measurement unit 13 and the scattered light measurement unit 16 is sequentially output to the data storage unit 2601 and accumulated as reaction process data. During the accumulation of the reaction process data, typically, after 5 minutes, another reagent (second reagent: a reagent containing latex particles sensitized with an antibody recognizing the substance to be measured) is used as reagent 4. The reagent is further dispensed into the cell 8 by the reagent dispensing mechanism 11 and stirred by the stirring unit 12 to cause an agglutination reaction, and further measured for a certain time (about 5 minutes). Thereby, the reaction process data of the agglutination reaction acquired at regular time intervals is stored in the data storage unit 2601.

FIG. 6 is a diagram showing an example of reaction process data acquired by a scattering photometer. The photometric points shown on the horizontal axis in FIG. 6 represent the order in which the reaction process data was measured. On the other hand, the vertical axis of FIG. 6 indicates the amount of scattered light measured by the scattered light measurement circuit 25. FIG. 6 shows reaction process data corresponding to a certain light reception angle. From the scattered light measurement circuit 25 according to the present embodiment, reaction process data corresponding to a light reception angle of 10 ° and light reception angle of 20 ° are shown. Reaction process data and reaction process data corresponding to a light receiving angle of 30 ° are output separately. In addition, reaction process data acquired with an absorptiometer is also output separately.

The analysis unit 2602 calculates a change in the amount of light within a predetermined time specified through an analysis setting screen (not shown) as a calculation value. Here, the fixed period used for calculation value calculation is defined by designating a calculation start point and a calculation end point from the photometric points. The calculated value is calculated as the difference between the light amount at the calculation start point and the light amount at the calculation end point. This calculated value corresponds to the signal change amount in this specification.

In the data storage unit 2601, in addition to the calculated value here, a calculated value when a measured substance having a known concentration is measured in advance is held as calibration curve data. The analysis unit 2602 collates the calculated value with the calibration curve data, and quantifies the concentration of the substance to be measured. The quantified concentration value is displayed through the output unit 28.

Note that data necessary for control and analysis of each unit is input from the input unit 27 to the data storage unit 2601. Various data, measurement results, analysis results, alarms, and the like stored in the data storage unit 2601 are displayed by the output unit 28.

[Example]
A CRP measurement reagent with a changed particle size was prepared, and the absorbance and scattered light were measured with the automatic analyzer shown above. The light receiving angles of the scattered light from the irradiation optical axis are 10 °, 20 °, and 30 °. A 150 mM glycine solution (pH 7.0) was used as the first reagent, and a latex reagent prepared below was used as the second reagent. A 30 mM glycine solution (pH 7.0) and an antibody that recognizes CRP as a substance to be measured are mixed, and four kinds of latex particle solutions (manufactured by Polyscience, particle sizes 200 nm, 350 nm, 500 nm, 750 nm, and 1000 nm) are respectively mixed therewith. Mix and stir for 1 hour at room temperature. Thereafter, the mixture was centrifuged at 15,000 rpm for 7 minutes at 25 ° C., the supernatant was removed, and a 30 mM glycine solution (pH 7.0) containing 0.1 to 0.2 mg / mL BSA was mixed and stirred at room temperature for 1 hour. Thereafter, the mixture was centrifuged at 25 ° C. and 15000 rpm for 7 minutes, the supernatant was removed, and the concentration was adjusted to a concentration of about 3.0 abs (700 nm wavelength) with a 150 mM glycine and 20% arginic acid (pH 7.0) solution. The CRP used for the sample was a sample having a concentration of 0.3 mg / dL. Since the signal change amount at 0 concentration is almost 0, the sensitivity comparison on the low concentration side can be made by comparing the signal change amount when the sample of this concentration is used. After mixing and stirring the sample and the first reagent, the second reagent was added. The amount of each liquid was 120 μL of the first reagent, 120 μL of the second reagent, and 2.4 μL of the sample. The amount of signal change after about 45 seconds and about 300 seconds after the addition of the second reagent was calculated. The particle concentration of the final reaction solution after the addition of the second reagent is 1.5 abs, which is about half of the particle concentration of the reagent.

FIG. 7 shows the magnitude of the signal change amount in the scattered light measurement for each particle size (200 nm, 350 nm, 500 nm, 750 nm, 1000 nm) and the light receiving angle (10 °, 20 °, 30 °). FIG. 8 is a diagram in which these data are plotted. It can be seen from FIG. 8 that the signal change amount at the light receiving angle of 10 ° is large.

FIG. 9 shows the normalized signal change amount [A. 1] when the signal change amount that peaks at each light receiving angle (10 °, 20 °, 30 °) in FIG. U. FIG. According to FIG. 9, the signal change amount of the latex reagent having a particle size of 350 nm is obtained at a light receiving angle of 30 °, the signal change amount of the latex reagent having a particle size of 500 nm is received at a light receiving angle of 20 °, and the latex reagent having a particle size of 750 nm at a light receiving angle of 10 °. It can be seen that the amount of signal change is the maximum. This coincides with the high-sensitivity reagent particle size condition considered from the relationship between the particle size and the normalized signal in FIG.

The signal change amount is a guideline, and whether the sensitivity is actually increased is evaluated by the S / N ratio considering the influence of noise. FIG. 10 is a diagram showing the variation (standard deviation) in the scattered light measurement value of the reaction process for each particle size and light receiving angle as the amount of noise. The amount of change in signal varies as much as the variation propagates as a standard deviation. FIG. 11 is a graph showing the particle size dependence of the S / N ratio obtained by dividing the signal change amount by the noise amount. The relationship and tendency between the particle diameter derived from the signal change amount and the light receiving angle are the same, and it was found that a latex reagent particle diameter of 750 nm at a light receiving angle of scattered light of 10 ° is advantageous for high sensitivity.

11 that the S / N ratio is improved at the light receiving angle of 10 ° compared to the light receiving angle of 20 °. The reception of scattered light at a light receiving angle of 20 ° is almost equivalent to the reception of scattered light at 17.5 to 22.5 ° from the optical axis of the irradiated light, so at least less than 17.5 ° from the optical axis of the irradiated light. In other words, it can be seen that measurement with an improved S / N ratio than the light receiving angle of 20 ° is possible by using scattered light having a light receiving angle of less than 15 °. Furthermore, the S / N ratio is improved by using scattered light having a light receiving angle of less than 12.5 °. In particular, by using a particle diameter of a reagent particle diameter range of 375 nm or more and 950 nm or less at a light reception angle of 10 ° (light reception angle of 7.5 ° to 12.5 °), the S / N ratio of 150 (dotted line) at a light reception angle of 20 ° is exceeded. High sensitivity can be obtained. The limitation on the larger particle size side can also be seen from the fact that the scattering angle of 10 ° in FIG. 3 can be measured up to an aggregate diameter of 1.9 μm (the particle size in the initial reaction tends to be halved to 950 nm). In addition, by using a reagent particle diameter of 450 nm or more and 875 nm or less, the S / N ratio is more than twice that in the case of a light receiving angle of 20 °, and high sensitivity can be reliably achieved compared to the conventional case. More preferably, a particle size of 750 nm capable of securing a S / N ratio of 3 times or more is preferable. Further, scattered light reception at a light receiving angle of 10 ° is almost equivalent to receiving scattered light at an angle of 7.5 to 12.5 ° from the optical axis of the irradiated light.

From the above, by measuring the agglutination reaction of a reagent having a particle size of 750 nm in the direction of a light receiving angle of 10 °, the sensitivity is increased by using a reagent having a particle size of 500 nm at a light receiving angle of 20 ° and a particle size of 350 nm at a light receiving angle of 30 °. It was found that the sensitivity can be increased by 3 times or more. In this measurement, a reagent having a single particle size of 750 nm was used. However, the reagent particle size may be mixed to expand the dynamic range. Even if small particles or large particles are mixed, the S / N is simply three times more sensitive, so in the absorbance, 1/3 or more of the total absorbance is due to particles in the particle size range shown above. Similar effects can be obtained as long as the absorbance is obtained.

When one particle size is specified above, a typical peak particle size is shown, and the same effect can be obtained if the half-value width is in the range of about ± 10%. That is, in the case of a particle size of 750 nm, the same effect can be expected if the particle size of the reagent is actually in the range of 675 nm to 825 nm. In this experiment, the reagent uses a concentration corresponding to 1.5 abs corresponding to the final concentration in the reaction solution at a wavelength of 700 nm, but the same effect can be obtained at a concentration of 0.25 to 2.0 abs in the final reaction solution.

FIG. 12 shows the magnitude of the signal change obtained when the reagent concentration is adjusted to 4.0 abs, that is, at the final reaction solution concentration of 2.0 abs after the addition of the second reagent, the particle size (200 nm, 350 nm, 500 nm, 750 nm). , 1000 nm) and light receiving angles (10 °, 20 °, 30 °). The same tendency as in FIG. 8 could be confirmed, indicating that high sensitivity could be maintained up to a final reaction solution concentration of 2.0 abs.

In addition, this invention is not limited to the above-mentioned Example, Various modifications are included. For example, the above-described embodiments have been described in detail for easy understanding of the present invention, and are not necessarily limited to those having all the configurations described. Further, a part of the configuration of one embodiment can be replaced with the configuration of another embodiment, and the configuration of another embodiment can be added to the configuration of one embodiment. Further, it is possible to add, delete, and replace other configurations for a part of the configuration of each embodiment.

1 Sample 2 Sample Cup 3 Sample Disc 4 Reagent 5 Reagent Bottle 6 Reagent Disc 7 Reaction Solution 8 Cell 9 Reaction Disc 10 Sample Dispensing Mechanism 11 Reagent Dispensing Mechanism 12 Stirring Unit 13 Absorbance Measuring Unit 14 Washing Unit 15 Constant Temperature Fluid 16 Scattered Light Measuring unit 17 Light source 18 Irradiated light 19 Transmitted light 20 Transmitted

light receivers

21a, 21b, 21c Scattered light 22a, 22b, 22c Scattered light receiver 27 Input unit 28 Output unit 29 Light source 30 Transmitted light 31 Diffraction grating 32 Receiver

Claims

A cell containing a reaction mixture in which a sample and a reagent are mixed is held on the circumference, and a reaction disk that repeatedly rotates and stops,
A scattering light measuring unit for irradiating the cell with irradiation light from a light source during rotation of the reaction disk and measuring scattered light after interacting with the reaction liquid in the cell;
The scattered light measurement unit receives scattered light having an angle of less than 15 ° from the optical axis of the irradiation light,
Using a latex reagent having a particle size of 450 nm or more and 875 nm or less sensitized with an antibody that recognizes a substance to be measured as the reagent,
The time-dependent change in the amount of light received by the scattered light measurement unit is measured as reaction process data of the agglutination reaction of the reaction liquid in which the sample containing the substance to be measured and the latex reagent are mixed, and the substance to be measured in the sample is measured. Automatic analyzer characterized by quantification.
The automatic analyzer according to claim 1, wherein
The automatic analyzer according to claim 1, wherein the scattered light measuring unit receives scattered light having an angle of 7.5 ° to 12.5 ° from the optical axis of the irradiation light.
The automatic analyzer according to claim 1, wherein
An automatic analyzer using a latex reagent having a particle size of 675 nm to 825 nm sensitized with an antibody recognizing a substance to be measured as the reagent.
The automatic analyzer according to claim 2,
An automatic analyzer using a latex reagent having a particle size of 675 nm to 825 nm sensitized with an antibody recognizing a substance to be measured as the reagent.
The automatic analyzer according to claim 3,
An automatic analyzer, wherein a latex reagent having a concentration of particles sensitized with an antibody recognizing a substance to be measured as the reagent is 0.25 abs to 2.0 abs at a wavelength of 700 nm in the reaction solution.
The automatic analyzer according to claim 5, wherein
An automatic analyzer characterized in that a latex reagent component of 675 nm to 825 nm in the reaction solution is caused by an absorbance of 1/3 or more of the absorbance of the entire latex reagent.