JP2015132634A - Autoanalyzer and analytical method - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a device and a preferable condition of a reagent for highly sensitively measuring a latex immune reaction, by a scattered light measurement method on an autoanalyzer.SOLUTION: Irradiation light having a wavelength in the range of 0.65-0.75 μm is used and scattered light generated from reaction liquid is received during rotation movement of a reaction vessel 8, by a light receiving angle of 15-35° to an irradiation direction of the irradiation light. A reagent contains latex particles which have an average peak particle diameter of 0.3 μm-0.43 μm and to which an antibody is sensitized. The reaction liquid contains the latex particles in concentration in which absorbance of the irradiation light having a wavelength of 0.7 μm is 0.25 abs-1.10 abs. Light quantity change of the scattered light, caused by agglutination of the latex particles through an antigen in a sample is measured to determine the quantity.

Description

  The present invention relates to a method and an analyzer for measuring scattered light of a fine particle agglutination reaction using an antigen-antibody reaction, and more particularly to a method for measuring scattered light on an automatic analyzer.

  Light from the light source is applied to the reaction mixture of the sample and reagent, the absorbance is calculated from the change in the amount of transmitted light at a specific wavelength, and the concentration of the analyte in the blood sample is quantified according to the Lambert-Beer law Automatic analyzers are widely used (for example, Patent Document 1). In these automatic analyzers, a large number of cells that hold the reaction solution are arranged on the circumference of the cell disk that repeats rotation and stop, and the transmitted light measurement unit arranged at a predetermined position during the cell disk rotation. Time series data of the amount of transmitted light that has passed through the reaction solution in the cell for about 10 minutes at intervals of about 15 seconds is acquired as reaction process data, the absorbance is calculated from the change in the amount of light, and the concentration of the substance to be measured is quantified.

  There are mainly two types of reactions measured by an automatic analyzer, ie, a color reaction between a substrate and an enzyme and an immune reaction between an antigen and an antibody. The analysis using the former reaction is called biochemical analysis, and there are LDH (lactate dehydrogenase), ALP (alkaline phosphatase), AST (aminoton raffinase aspartate) and the like as test items. Analysis using the latter reaction is called immunoassay, and test items include CRP (C-reactive protein), IgG (immunoglobulin), and RF (rheumatoid factor). Among the analytes measured in the latter, there are test items that require quantification in low-concentration regions where the blood concentration is low. In such items, antibodies are sensitized (bound) on the surface. Latex immunoassay using latex particles as a sensitizer is used (for example, Patent Document 2).

  In latex immunoassay, antigens to be measured contained in a sample are recognized and bound by the antibody on the latex particle surface contained in the reagent, resulting in latex particles aggregating through the antigen and forming latex particle aggregates. Is done. In the conventional automatic analyzer, light is irradiated to the reaction liquid in which the aggregates are dispersed, and the amount of transmitted light transmitted without being scattered by the aggregates of latex particles is measured. The higher the concentration of the antigen, the larger the size of the aggregate after a certain time, and more light is scattered, so the amount of transmitted light decreases. Therefore, the concentration of the antigen can be quantified from the amount of light measured as reaction process data.

  In recent years, higher sensitivity of latex immunoassay has been desired. Therefore, it has been attempted to measure scattered light instead of measuring transmitted light. For example, a system (Patent Document 3) that separates transmitted light and scattered light using a diaphragm and simultaneously measures absorbance and scattered light is disclosed. Moreover, the reagent particle diameter etc. which are suitable for a scattered light measurement are also disclosed (patent document 4).

U.S. Pat. No. 4,451,433 Japanese Patent No. 16112184 JP 2001-141654 A Japanese Patent No. 16357792

C. F. Bohren, D. R. Huffman, Absorption and Scattering of Light by Small Particles, J. Wiley & Sons, 1983

  Although Patent Document 2 describes a wavelength and particle size for roughly increasing sensitivity, it relates to absorbance measurement, and it is unclear whether it can be applied to scattered light measurement. In addition, Patent Document 3 discloses a configuration that achieves higher sensitivity by performing scattered light measurement simultaneously with transmitted light measurement, but is not considered as a configuration suitable for an automatic analyzer. Furthermore, examination of suitable conditions including a reagent is not made | formed.

  Latex particles with a particle diameter (hereinafter referred to as particle size) of 500 nm or less are mainly used as latex reagents, and the light scattering characteristics (scattered light intensity and its angular distribution) of each particle depend on the ratio between the particle size and the wavelength. It is thought that it will change greatly (refer nonpatent literature 1). For this reason, in the case of scattered light measurement, the sensitivity can vary greatly depending on the selection of wavelength, particle size, and the like. Patent Document 4 describes scattered light measurement, but it is in the same range as the absorbance measurement in Patent Document 2, and when used for scattered light measurement, the wavelength and particle size are always suitable for high sensitivity. It is speculated that this is not, and there is a concern that the sensitivity may deteriorate depending on the wavelength and the particle size. However, it takes a lot of time and cost to experiment by changing the wavelength, particle size, etc., and it is difficult to grasp a comprehensive trend in practice.

  In addition, conditions have not been studied in consideration of the binding properties of antibodies to be sensitized to the latex surface. That is, when measuring scattered light on an automatic analyzer, the relationship between sensitivity and wavelength and light receiving angle, which are instrument conditions, particle size, density, antigen antibody binding constant, etc., which are reagent conditions, is not clear, No suitable combination of conditions for performing latex immunoassay with high sensitivity has been known.

  The present invention provides suitable conditions on the apparatus and the reagent for measuring latex immune reaction with high sensitivity by the scattered light measurement method on an automatic analyzer.

  In the present invention, as an antigen-antibody reaction, the number of aggregates assumed during a certain reaction time is estimated from the binding constant, and simulation is performed by optically modeling the aggregates. Reproducible reagents and equipment conditions were obtained.

  An automatic analyzer according to the present invention includes a sample container for holding a sample, a reagent container for holding a reagent containing latex particles having an average peak particle size of 0.3 μm to 0.43 μm and sensitized with an antibody, and a sample A reaction container holding a reaction liquid in which the sample in the container and the reagent in the reagent container are mixed, a rotating mechanism for rotating the reaction container, and a reaction liquid in the reaction container having a wavelength of 0.65 to 0.75 μm. A light source unit that irradiates a range of irradiation light, and a photodetector that is disposed at a light receiving angle of 15 to 35 ° with respect to the irradiation direction of the irradiation light and that receives scattered light generated from the reaction solution during the rotational movement of the reaction vessel And have. Here, the reaction solution contains latex particles at a concentration of 0.25 abs to 1.10 abs with respect to irradiation light having a wavelength of 0.7 μm, and the scattered light caused by aggregation of the latex particles via the antigen in the sample. Measure the change in light intensity.

  The concentration of the latex particles contained in the reaction solution is more preferably a concentration at which the absorbance with respect to irradiation light with a wavelength of 0.7 μm is 0.25 abs to 0.50 abs, and further preferably a concentration at which the absorbance is 0.25 abs to 0.31 abs.

  The analysis method according to the present invention is a method for analyzing a substance to be measured based on an antigen-antibody reaction using an automatic analyzer, and the sample and the average peak particle size are 0.3 μm to 0.43 μm in the reaction container. Mixing a reagent containing latex particles sensitized with an antibody to make a reaction solution, and irradiating the reaction solution in the reaction vessel in the wavelength range of 0.65 to 0.75 μm while rotating the reaction vessel A step of detecting the scattered light scattered at an angle of 15 to 35 ° with respect to the irradiation direction of the irradiated light, and the amount of scattered light caused by the latex particles aggregating through the antigen in the sample The reaction solution contains latex particles at a concentration such that the absorbance with respect to irradiation light with a wavelength of 0.7 μm is 0.25 abs to 1.10 abs.

According to the present invention, it is possible to measure the aggregation of latex particles using an antigen-antibody reaction with high sensitivity and to measure to a low concentration. This makes it possible to calculate a low concentration of antigen or antibody.
Problems, configurations, and effects other than those described above will be clarified by the following description of embodiments.

Schematic explanatory drawing of scattered light measurement. The figure which shows the antibody binding rate dependence of the antigen binding rate. The figure which shows the antigen density | concentration dependence of the structure particle number of an aggregate. The figure which shows the light quantity change rate (light quantity change rate magnification) of the scattered light with respect to the light quantity change rate of the transmitted light. Schematic which shows the example of whole structure of an automatic analyzer. Schematic of a scattered light measurement part. The figure which shows the angle dependence of light quantity change rate magnification.

  Embodiments of the present invention will be described below with reference to the drawings. The following apparatus configuration (wavelength of irradiated light and angle for receiving scattered light) was selected as a useful configuration for the scattered light measurement unit incorporated in the automatic analyzer.

  First, the wavelength of irradiation light was examined as follows. Since the scattered light measurement unit mounted on the automatic analyzer is used at the same time as a conventional absorptiometer, it is necessary to use a blood sample with a constant volume suitable for the absorptiometer. May be affected by milk, hemolysis and jaundice. Milk powder, hemolysis, and jaundice are highly absorbed at wavelengths less than 650 nm. For this reason, the wavelength of the irradiation light is preferably 650 nm or more so as not to be affected by the disturbance included in the sample. In addition, the automatic analyzer must perform measurement while the cell is rotating, and it is necessary to align the cell and the irradiated light beam by on-site maintenance adjustment or the like with an accuracy of 1 mm or less so as not to generate stray light. In order to improve maintainability, the presence or absence of stray light must be confirmed visually. Therefore, the wavelength of the irradiation light is preferably in the range of 400 to 750 nm in the visible light region so that alignment can be easily performed. As a wavelength range satisfying the above two conditions, 650 to 750 nm was selected as a suitable wavelength.

  In a dedicated machine that is dedicated to measuring scattered light rather than an automatic analyzer, the cell does not rotate during the measurement, so that the alignment accuracy between the cell and the light beam is not necessary. Therefore, fine adjustment is not required, and it is possible to use a wavelength outside the visible light region for measurement. However, if invisible light is used for scattered light measurement on an automatic analyzer, the maintainability deteriorates and is not suitable for practical use.

  The angle for receiving the scattered light was selected as follows. Since the measurement is performed on an automatic analyzer, the transmitted light must be measured at the same time. For this reason, a rectangular cell having a transparent surface through which transmitted light passes is used. This square cell has a structure suitable for transmitted light measurement, and the measurement angle is limited when scattered light measurement is performed. Specifically, the light-transmitting surface is designed to have the least optical distortion among the surfaces, and the cells are arranged adjacent to each other, so only the light in the range that has passed through the light-transmitting surface is accurate. Can measure well. Therefore, it is necessary to measure scattered light passing through the front surface or the rear surface of the cell. That is, the scattered light needs to be measured in front of or behind the irradiation light traveling direction. Among them, since it is necessary to secure a light amount for noise reduction, it is generally more advantageous to receive scattered light on the front side having a larger light amount than the rear side, and an angle of 35 ° or less is desired. In addition, at an angle of less than 15 °, noise may increase due to the influence of transmitted light that is two orders of magnitude stronger than scattered light. Therefore, the scattered light receiving angle that is 15 to 35 ° in air is selected.

In consideration of the above-described apparatus configuration, a useful reagent composition in the scattered light measurement was examined.
First, in order to ensure a certain amount of scattered light, the average particle size of the particles needs to be 0.25 μm or more. Further, in order to maintain the reagent for a long time without precipitation, the average particle size of the particles needs to be 0.50 μm or less. For this reason, the particle size range of the latex particles was set to 0.25 to 0.50 μm.

  Furthermore, the density of the latex particles in the reaction solution for ensuring a constant light amount needs to be 0.25 abs or more in terms of absorbance (wavelength 0.7 μm). In consideration of the measurement range in transmitted light measurement, it is desirable that the absorbance is 1.5 abs or less in terms of absorbance before aggregation (wavelength 0.7 μm). In the case of more than that, there is a possibility of deviating from the measurement range in terms of reproducibility and linearity in transmitted light measurement.

  Consider the evaluation index when evaluating using these. FIG. 1 is a schematic diagram illustrating the time course of scattered light measurement by antigen-antibody reaction. On an automatic analyzer with a scattered light measurement unit, first, a sample to which the first reagent is added is mixed with a second reagent in which latex particles are dispersed (reference state), and the scattered light after a certain period of time (aggregation state). Alternatively, the amount of change in transmitted light is detected. Separately, calibration data obtained by measuring the amount of change using an antigen with a known concentration is prepared, and the concentration of the antigen in the sample is calculated by comparing with the data. At this time, since the amount of change light is also proportional to the intensity of irradiation light, a light amount change rate is defined by dividing the amount of change light by the light amount of the reference state that is also proportional to the intensity of irradiation light. As the light amount change rate is larger, it is possible to capture a slight aggregation change. Furthermore, the ratio of the light amount change rate between the transmitted light and the scattered light was defined as the light amount change rate magnification (scattered light amount change rate / transmitted light amount change rate). Since it is considered that the larger this is, the higher the sensitivity in the scattered light measurement, the light quantity change rate magnification was evaluated as an evaluation index.

Next, how much the latex particles in the reaction solution aggregate was assumed as follows. The concentration of a substance to be measured that can be conventionally measured by latex immunoassay is considered to be a concentration region of about 10 ⁻¹¹ mol / L or more. In order to examine the configuration for increasing the sensitivity, a configuration that enables measurement of antigens at a low concentration of about 10 ⁻¹² mol / L was studied.

Here, when the density of the latex particles contained in the reaction liquid is too thin, the amount of scattered light is reduced without almost irradiating the irradiation light. As a result, relative noise increases in the received signal. Therefore, in order to obtain a certain amount of scattered light, latex particles having a concentration of about 10 ⁶ particles / mm ³ or more are generally dispersed in the reaction solution. The density of the latex particles is sufficiently excessive compared with the antigen concentration of 10 ⁻¹² mol / L (≈6 × 10 ⁵ particles / mm ³ ) which is the detection target. That is, a plurality of latex particles are dispersed per molecule of the antigen to be measured. Therefore, the aggregation ratio of latex particles that aggregate during a certain reaction time (volume ratio of aggregated latex particles in all latex particles) is the antigen bound to the antibody among all antigens in the reaction solution. It is thought that it is proportional to the coupling rate which is the ratio of.

  Hereinafter, assuming that the number of antibodies sensitized to the unit surface area of the particles is constant, the number of antibodies in the reaction solution was estimated, and the antigen binding rate, that is, the particle aggregation rate was evaluated.

In the case of reaction liquids having the same latex particle size and different number densities, the number of antibodies contained in the reaction liquid is proportional to the number density of latex particles. The density of latex particles of 0.25 abs to 1.5 abs in terms of absorbance (wavelength 0.7 μm) is 2.8 × 10 ⁶ to 1.7 × 10 in terms of the number density of particles having a particle diameter of 0.3 μm. It corresponds to ⁷ pieces / mm ³ . Assuming that the ratio of the antibody diameter is 15 nm and that the active antibody is sensitized to the maximum density when the antibody is spread on the particle surface is estimated to be 10%, the antibody density on the particle surface is 510 / μm ^2. It is. Further, assuming that one antibody has two reactive groups, there are about 290 antibody binding sites on the surface of a 0.3 μm particle size particle, so the concentration of antibody binding sites in the reaction solution is about 1 It has a width 6 times in the range of 4 × 10 ^{−9 to} 8.2 × 10 ⁻⁹ mol / L. Here, as shown in FIG. 2, the binding constant of a general antigen-antibody reaction: 10 ⁸ mol / L, antigen concentration: 10 ⁻¹² mol / L, antibody concentration: 1.4 × 10 ^{−9 to} 8.2. In the range of × 10 ^-9 mol / L, the binding rate of the antigen to the antibody is almost proportional to the antibody concentration. In other words, the aggregation rate of latex particles is proportional to the antigen binding rate, the antigen binding rate is proportional to the antibody concentration, and the antibody concentration is proportional to the number density of latex particles. It is almost proportional to the number density.

  On the other hand, when the concentration by weight of latex particles in the reaction solution is constant and the particle size of latex particles is different, the number of particles is the inverse of the cube of the particle size. Furthermore, since the surface area of the particles is proportional to the square of the particle size of each particle, as a result, the total antibody concentration contained in the reaction solution is proportional to the inverse of the particle size. Therefore, the antibody concentration range contained in the reaction solution in the particle size range width of 0.25 to 0.5 μm to be considered is 2 times or less, and is smaller than the density consideration range (6 times) of the above particles. In the following optical simulation, the number of aggregated aggregates was made constant.

  Furthermore, it is considered that the aggregate is mainly an aggregate in which two particles are bound under a sufficiently thin antigen concentration. FIG. 3 shows the latex particle number distribution of aggregates for each CRP antigen concentration (0 to 0.3 mg / dL (the reagent measurable range is 0.01 to 42 mg / dL)) of Nanopia CRP (manufactured by Sekisui Medical). It is a figure which shows an example. A calibrator (2.4 μL), a first reagent (120 μL) and a second reagent (120 μL) at each CRP concentration were mixed, and after 300 seconds from mixing, the reaction solution was applied to the substrate and sampled. The sample was observed with a scanning electron microscope, and the distribution was obtained by counting the number of latex particles constituting the aggregate. As the antigen concentration increased, the aggregate was composed of two latex particles. It was actually confirmed that the aggregates increased.

  Based on the above, the particle size of latex particles under the same antigen concentration and the volume ratio of two aggregates for each density were set.

  In Mishchenko MI, Travis LD, Mackowski DW., T-matrix computations of light scattering by nonspherical particles, a review.J Quant Spectrosc Radiat Transfer, 1996 It has been confirmed by electric field calculation that a spheroid approximately the same type as the two aggregates is approximately equal to a true spherical particle having the same cross-sectional area. With reference to this, the two agglomerates were replaced with true spherical particles having a cross-sectional area equal to the sum of the cross-sectional areas of the carrier particles, and optical modeling was performed.

From the range assumed when the scattered light measurement is performed on the automatic analyzer as described above, wavelength: 700 nm, angle: 20 ° (± 2.5 °), antigen-antibody binding constant: 10 ⁸ mol / L or more , Reagent particle size range: 0.25 to 0.50 μm, density: absorbance before flocculation 0.25 to 1.5 abs as parameters, by optical simulation using the Monte Carlo method. The amount of scattered light was calculated. In the optical simulation, the scattered light theory described in Non-Patent Document 1 was used.

From the results obtained by the optical simulation, FIG. 4 shows the light amount change rate magnification obtained by dividing the light amount change rate of the scattered light caused by the aggregation of latex particles by the light amount change rate of the transmitted light. Since it is possible to capture a slight change in aggregation as the light quantity change rate is larger, in FIG. 4, the scattered light measurement is more sensitive than the transmitted light measurement as the particle diameter and density are larger. It can be considered. Thus, the following conditions have been found that are judged to be suitable when the scattered light measurement is performed on the automatic analyzer. The values of antigen-antibody binding constants and antigen concentrations used as parameters in the simulation are general values. Even if the values change slightly, the above assumption of the optical simulation does not change. Does not affect magnification. The relationship shown in FIG. 4 is established even when the antigen-antibody binding constant is, for example, from 10 ⁸ mol / L to 10 ¹¹ mol / L.

  From FIG. 4, the transmitted light measurement is performed under the conditions that the particle size of the latex particles in the reagent is 0.3 μm to 0.43 μm, and the density before aggregation of the latex particles in the reaction solution is converted to absorbance and is 0.25 to 1.10 abs. It was found that the scattered light measurement can achieve more than twice as high sensitivity. Further, the transmitted light measurement is performed under the conditions that the particle size of the latex particles in the reagent is 0.3 μm to 0.43 μm and the density of the latex particles in the reaction solution before aggregation is 0.25 to 0.50 abs in terms of absorbance. It has been found that the scattered light measurement can achieve a sensitivity of 4 times or more. Furthermore, the transmission is performed under the conditions that the particle size of the latex particles in the reagent is 0.3 μm to 0.43 μm, and the density before aggregation of the latex particles in the reaction solution is 0.25 to 0.31 abs in terms of coagulation absorbance. It has been found that the scattered light measurement can achieve a sensitivity 6 times higher than the light measurement.

  It was found that the apparatus configuration and reagent composition described above enable highly sensitive scattered light measurement when measuring the change in the amount of light accompanying the latex particle agglutination reaction.

  FIG. 5 is a schematic diagram showing an example of the overall configuration of the automatic analyzer according to the present invention. The automatic analyzer of this embodiment can execute scattered light measurement simultaneously with transmitted light measurement. The automatic analyzer according to this embodiment includes three types of disks, a sample disk 3, a reagent disk 6 and a cell disk 9, a dispensing mechanism for moving samples and reagents between these disks, a control circuit 23 for controlling these, A transmitted light measurement circuit 24, a scattered light measurement circuit 25, a data processing unit 26 such as a PC (computer) that processes the measured data, an input unit 27 that is an interface for inputting / outputting data to / from the data processing unit 26, and an output unit 28 Have The data processing unit 26 includes an analysis unit that analyzes data, and a data storage unit that stores control data, measurement data, data used for analysis, analysis result data, and the like.

  A plurality of sample cups 2 containing samples 1 are arranged on the circumference of the sample disk 3. A plurality of reagent bottles 5 containing the reagents 4 are arranged on the reagent disk 6. A plurality of cells 8 are prepared on the circumference of the cell disk 9 to mix the sample 1 and the reagent 4 and make the reaction solution 7 inside. The sample dispensing mechanism 10 moves the sample 1 from the sample cup 2 to the cell 8 by a certain amount. The reagent dispensing mechanism 11 moves the reagent 4 from the reagent bottle 5 to the cell 8 by a certain amount. The stirring unit 12 stirs and mixes the sample 1 and the reagent 4 in the cell 8. The cleaning unit 14 discharges the reaction solution 7 from the cell 8 after the analysis and cleans it. The next sample 1 is again dispensed from the sample dispensing mechanism 10 into the washed cell 8, and a new reagent 4 is dispensed from the reagent dispensing mechanism 11 and used for another reaction. The cell 8 is immersed in a constant temperature fluid 15 in a constant temperature bath whose temperature and flow rate are controlled, and the cell 8 and the reaction liquid 7 in the cell 8 are moved while being maintained at a constant temperature. For example, water is used as the constant temperature fluid 15, and the constant temperature fluid temperature is adjusted to 37 ± 0.1 ° C. by the control circuit. A transmitted light measuring unit 13 and a scattered light measuring unit 16 are provided on a part of the circumference of the cell disk.

  The transmitted light measurement unit 13 can be configured, for example, to irradiate the cell 8 with light from a halogen lamp light source, split the transmitted light with a diffraction grating, and then receive the light with a photodiode array. The wavelengths received are, for example, 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. The transmitted light amount data received by the photodiode array is sent to the data storage unit in the PC through the transmitted light measurement circuit 24.

  An outline of the scattered light measurement unit 16 is shown in FIG. As the light source, for example, an LED light source or the like can be used. Irradiation light 18 from the LED light source unit 17 is irradiated to the moving cell 8, transmitted light 19 is received by the transmitted light receiver 20, and scattered light is scattered light. Light is received by the light receiver 22. In the LED light source unit 17, for example, 700 nm can be used as the irradiation light wavelength. In this embodiment, an LED is used as a light source, but a laser, a xenon lamp, or a halogen lamp may be used. The scattered light receiver 22 measures the scattered light 21 in a direction away from the optical axis by an angle θ in the air. The angle θ may be an angle in the range of 15 to 35 °, but in this embodiment, θ = 20 °. The scattered light receiver 22 is disposed in a plane substantially perpendicular to the moving direction of the cell 8 due to the rotation of the cell disk 9. Here, the reference position of the angle θ is the starting point at the center of the length that allows light to pass through the cell 8. As means for receiving scattered light from the reaction solution 7, light receivers having different scattering angles may be provided, and signals may be acquired from the light receivers therein.

  In this embodiment, the photodiodes are arranged as the light receivers 20 and 22 at the respective angles. However, a single linear array holding a large number of light receivers inside is arranged to receive scattered light at a plurality of angles. Also good. Thereby, the choice of a light reception angle can be expanded. Further, instead of the light receiver, an optical system such as a fiber or a lens may be disposed at the scattered light receiving position, and light may be guided to the scattered light receiver disposed at another position.

  The concentration of the substance to be measured in the sample 1 is determined by the following procedure. First, a certain amount of sample 1 in the sample cup 2 is dispensed into the cell 8 by the sample dispensing mechanism 10. Next, a predetermined amount of the reagent 4 in the reagent bottle 5 is dispensed into the cell 8 by the reagent dispensing mechanism 11. At the time of dispensing, the sample disk 3, the reagent disk 6, and the cell disk 9 are rotationally driven by the respective drive units under the control of the control circuit 23, so that the sample cup 2, the reagent bottle 5, and the cell 8 are dispensed. , 11 in accordance with the dispensing timing. Subsequently, the sample 1 and the reagent 4 dispensed in the cell 8 are stirred by the stirring unit 12 to obtain a reaction solution 7. The transmitted light and scattered light from the reaction liquid 7 are measured each time the cell disk 9 rotates and passes through the measurement positions of the transmitted light measurement unit 13 and the scattered light measurement unit 16, and the transmitted light measurement circuit 24, scattered light is measured. Reaction process data is sequentially accumulated from the measurement circuit 25 to the data storage unit of the data processing unit 26. After measurement for a certain time, for example, about 10 minutes, the inside of the cell 8 is cleaned by the cleaning unit 14 and the next inspection item is analyzed. Meanwhile, if necessary, another reagent 4 is added into the cell 8 by the reagent dispensing mechanism 11 and dispensed, stirred by the stirring unit 12, and further measured for a certain time. As a result, reaction process data of the reaction solution 7 having a constant time interval is stored in the data storage unit. From the stored reaction process data at a single received angle or multiple received angles of the scattered light measurement unit, the analysis unit determines the change in the amount of light due to the reaction for a certain period of time, and uses the calibration curve data stored in the data storage unit in advance. Based on this, a quantitative result is calculated and displayed from the output unit. Data necessary for control / analysis of each unit is input from the input unit 27 to the data storage unit of the data processing unit 26. Data, results, and alarms in various storage units are output on the display or the like by the output unit 28.

  The wavelength for scattered light measurement may be any wavelength included in 650 nm to 750 nm. The scattered light receiving angle may be in the range of 15 ° to 35 °. FIG. 7 shows the light quantity change rate magnification at each particle size and particle density (expressed by absorbance at a wavelength of 700 nm) calculated by the optical simulation, but if the forward scattering (15 ° to 35 °), the light quantity change rate. It was confirmed that the magnification was almost unchanged.

  The latex particles contained in the reaction solution 7 have a particle size of 0.3 μm to 0.43 μm. By using latex particles in this particle size range, as shown in FIG. 4, it is possible to obtain a light quantity change rate larger than that of transmitted light, so that it is possible to measure an antigen with a smaller concentration than in the past.

  As the concentration of latex particles contained in the reaction solution 7 is lower, the irradiation light is less likely to be scattered by the reaction solution 7. Therefore, the density of the latex particles contained in the reaction solution 7 is scattered by setting the absorbance to 0.25 abs or more. The amount of scattered light measured by the optical receiver 22 can be made sufficiently large.

  Latex particles used for antigen-antibody reaction and antibodies sensitized to the carrier account for the majority of reagent costs. Therefore, by increasing the density of latex particles contained in the reaction solution 7 in the range of 0.25 abs to 1.10 abs in terms of absorbance, an antibody sensitized to the latex particles used and the carrier while increasing the light quantity change rate magnification. And the reagent manufacturing cost can be reduced.

  In an antigen-antibody reaction, an antibody having a small binding constant exists. Even in the case of antibodies having a small binding constant, the density of latex particles contained in the reaction solution 7 may be in the range of 0.25 abs to 0.5 abs in order to obtain a higher light quantity change rate magnification.

  In the particle size distribution based on volume, the latex particles contained in the reaction liquid 7 are included within ± 15% of the particle size (peak particle size) at which the full width at half maximum of the particle size becomes the peak of the distribution, and the peak particle You may use what a diameter falls in the range of 0.3 micrometer to 0.43 micrometer. Generally, the particle size of fine particles has a distribution, and the cost increases as the distribution width is suppressed. From FIG. 4, it can be confirmed that the light quantity change rate magnification is not substantially changed by a difference of about several percent of the particle size. Therefore, in the particle size distribution based on the volume, the particle size at which the full width at half maximum of the particle size becomes the distribution peak. By using latex particles that are included within ± 15% of (peak particle size) and that have a peak particle size in the range of 0.3 μm to 0.43 μm, the cost can be reduced while obtaining a high light quantity change rate magnification. Can do. On the other hand, the full width at half maximum of the particle size may be within ± 10% of the peak particle size in order to suppress the scattered light intensity variation from the reaction liquid due to the particle size variation. Moreover, in order to suppress the variation between measurements of the same sample, the full width at half maximum of the particle size may be within ± 7% of the peak particle size.

  In addition, this invention is not limited to an above-described 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. Further, the drawings show what is considered necessary for explanation, and do not necessarily show all the components and functions on the product.

DESCRIPTION OF SYMBOLS 1 ... Sample, 2 ... Sample cup, 3 ... Sample disc, 4 ... Reagent, 5 ... Reagent bottle, 6 ... Reagent disc, 7 ... Reaction liquid, 8 ... Cell, 9 ... Cell disc, 10 ... Sample dispensing mechanism, 11 DESCRIPTION OF SYMBOLS ... Reagent dispensing mechanism, 12 ... Stirring part, 13 ... Transmitted light measuring part, 14 ... Washing part, 15 ... Constant temperature granule, 16 ... Scattered light measuring part, 17 ... LED light source unit, 18 ... Irradiation light, 19 ... Transmission Light, 20 ... Transmitted light receiver, 21 ... Scattered light, 22 ... Scattered light receiver, 23 ... Control circuit, 24 ... Transmitted light measuring circuit, 25 ... Scattered light measuring circuit, 26 ... Data processing unit, 27 ... Input unit , 28 ... Output unit

Claims (1)

In the method for analyzing a substance to be measured based on an antigen-antibody reaction using an automatic analyzer,
Mixing a sample in a reaction vessel with a reagent containing latex particles having an average peak particle size of 0.3 μm to 0.43 μm and sensitized with an antibody to obtain a reaction solution;
While rotating the reaction vessel, the reaction solution in the reaction vessel is irradiated with irradiation light having a wavelength in the range of 0.65 to 0.75 μm, and is 15 to 35 ° with respect to the irradiation direction of the irradiation light. Detecting scattered light scattered at an angle;
Measuring the change in the amount of scattered light caused by aggregation of the latex particles via the antigen in the sample,
The antibody has a binding constant of 10 ⁸ mol / L or more and 10 ¹¹ mol / L or less with an antigen contained in the sample,
The analysis method, wherein the reaction solution contains the latex particles at a concentration such that the absorbance with respect to irradiation light with a wavelength of 0.7 μm is 0.25 abs to 1.10 abs.

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