WO2021199178A1

WO2021199178A1 - Latex agglutination method-mediated target substance measurement method, and reagent therefor

Info

Publication number: WO2021199178A1
Application number: PCT/JP2020/014610
Authority: WO
Inventors: 佐藤　良克; 康紀皆川; 建治宮田
Original assignee: デンカ生研株式会社; 株式会社日立ハイテク
Priority date: 2020-03-30
Filing date: 2020-03-30
Publication date: 2021-10-07
Also published as: CN115516312A; KR20220159382A

Abstract

The invention provides a latex agglutination method-mediated target substance measurement method, and a reagent therefor, allowing for an accurate measurement in both a low concentration region and a high concentration region. The latex agglutination method-mediated target substance measurement method comprises reacting a sensitized latex particle suspension with a target substance, and then measuring, from the amounts of optical changes, the agglutination of the sensitized latex particles. In the method, the volume-based mean particle diameter of the sensitized latex particles prior to sensitization is 80 nm to 335 nm, the final concentration of the sensitized latex particles in the reaction system is 0.005 to 0.10 w/v%, the final concentration, in the reaction system, of particles having a particle diameter of 80 nm or smaller for the sensitized latex particles prior to sensitization, is 0.09 w/v% or lower, and the amounts of optical changes comprise the amount of change in absorbance and the amount of change in scattered ray.

Description

Measurement method of target substance by latex agglutination method and its reagent

The present invention relates to a method for measuring a target substance by a latex agglutination method and a reagent thereof. More specifically, the present invention relates to a latex particle-enhanced immunoaggregation measurement method that combines scattered light intensity measurement and absorbance measurement, and a reagent thereof.

The immunoassay method using latex particles is applied to clinical tests as a method for quantifying the target substance to be measured contained in body fluids such as serum, plasma, and urine, and can be measured easily and quickly by using an automatic analyzer. Therefore, it is widely used.

In recent years, applied technologies aimed at further improving measurement performance have been proposed. For example, the absolute value of the signal is suppressed by using a short wavelength at which a strong signal is obtained when the target substance is measured in the low concentration range, and by using a long wavelength when measuring the high concentration range so as not to exceed the upper limit of the signal detection range of the analyzer. (Patent Document 1). However, it is a known fact that the turbidity of the latex fine particle solution is wavelength-dependent, and the signal decreases as the wavelength becomes longer. For a certain optical change, the magnitude of the signal is adjusted by selecting the wavelength. The method, which is only adapted to the analyzer, cannot be expected to have a dramatic improvement effect enough to compensate for the shortcomings in the accuracy and dynamic range of the particle-enhanced immunoaggregation measurement method.

In addition, in the particle-enhanced immunoaggregation measurement method, there is also a method in which high-sensitivity and wide dynamic range measurement can be performed by combining scattered light intensity measurement and absorbance measurement in one measurement (Patent Document 2). However, although the scattered light intensity measurement can increase the range of the low concentration region, it is described in the literature that the correct particle size must be selected in order to obtain the effect (Patent Document 3). It is described that the effect of measuring the scattered light intensity cannot be obtained when the particle size is 300 nm or less. In other words, selecting a particle size that makes use of the characteristics of scattered light intensity measurement that can increase the range of the low concentration range as in the method narrows the design range of absorbance measurement that can measure the high concentration range. The dramatic effect of increasing the dynamic range cannot be achieved.

Furthermore, according to a measurement method (Patent Document 4) in which an antibody is sensitized to two or more types of latex particles having different average particle sizes, measurement over a wide concentration range becomes possible. According to a method (Patent Document 5) in which a polyclonal antibody and a monoclonal antibody are used in combination with latex particles having a single average particle size, the same effect can be obtained. Further, a measurement method using particles having a small particle size coated with a low-reactivity antibody and particles having a large particle size coated with a highly reactive antibody (Patent Documents 6 and 7) has been proposed. .. However, the methods described in these documents are designed only for the absorbance measurement, and are not suitable for the scattered light intensity measurement that increases the range of the low concentration region. In other words, a true reagent design that increases the dynamic range that can enjoy both the advantages of scattered light intensity measurement that can increase the range of low concentration range and the advantage of absorbance measurement that can measure high concentration range is specified. Not.

Further, in order to achieve the measurement of a desired low concentration region by the latex particle-enhanced immunoaggregation method, there is a tendency to increase the sample amount ratio (referred to as "sample amount concentration" in the present specification and claims) to the amount of reagent. It has been pointed out as a problem. By increasing the sample amount concentration, the antigen concentration in the reaction reagent can be apparently increased. As a result, the effect of improving the measurement accuracy of the low-concentration sample can be obtained. However, as the sample volume concentration increases, the components other than the target substance also increase, which increases the possibility of a (non-specific) reaction that measures other than the target substance, which causes an erroneous judgment (false positive). sell.

Japanese Unexamined Patent Publication No. 08-043393 WO2014-192963 Japanese Unexamined Patent Publication No. 2013-64705 Japanese Patent No. 2588174 Japanese Unexamined Patent Publication No. 10-90268 Japanese Unexamined Patent Publication No. 11-108929 Publication of Patent No. 3513075

An object of the present invention is to provide a method for measuring a target substance by a latex agglutination method and a reagent thereof, which enables accurate measurement in both a low concentration region and a high concentration region.

As a result of diligent research, the inventor of the present application has used both absorbance measurement and scattered light measurement, and has specified latex particles having a volume average particle diameter in a specific range and a concentration of small latex particles of a predetermined value or less. The present invention has been completed by finding that accurate measurement is possible in both a low concentration region and a high concentration region when used in a concentration range.

That is, the present invention provides the following.
(1) A method for measuring a target substance by a latex agglomeration method, which comprises reacting a suspended liquid of sensitized latex particles with the target substance, and then measuring the aggregation of the sensitized latex particles from the amount of optical change. The volume average particle diameter of the sensitized latex particles before sensitization is 80 nm to 335 nm, and the final concentration of the sensitized latex particles in the reaction system is 0.005 to 0.10 w / v%. The final concentration of the sensitized latex particles in the reaction system of particles having a particle diameter of 80 nm or less before sensitization is 0.09 w / v% or less, and the amount of optical change is the amount of change in absorbance and the change in scattered light. The amount, the way.
(2) The amount of change in absorbance and the amount of change in scattered light are measured using light having a wavelength in the range of 1 to 10 times the volume average particle diameter of the sensitized latex particles before sensitization (1). The method described.
(3) As the measurement wavelength of the amount of change in absorbance, two wavelengths selected within the range of 500 to 900 nm are used, and the two further selected measurement wavelengths are a main wavelength and a sub-wavelength longer than the main wavelength. The method according to (1) or (2).
(4) When the lower and upper limits of the measured values to be achieved are stipulated for the target substance, the minimum sample concentration required to achieve the lower limit only by absorbance measurement is 0. The method according to any one of (1) to (3), which is carried out at a sample concentration of 7 times or less.
(5) The method according to any one of (1) to (4), wherein at least one type of scattering is measured within a range of a scattering angle of 10 ° to 30 ° in the measurement of the amount of change in scattered light.
(6) A purpose according to the latex agglomeration method, which comprises reacting a suspended liquid of sensitized latex particles with the target substance, and then measuring the aggregation of the sensitized latex particles from the amount of change in absorbance and the amount of change in scattered light. A reagent for measuring a substance, the volume average particle diameter of the sensitized latex particles before sensitization is 80 nm to 335 nm, and the final concentration of the sensitized latex particles in the reaction system is 0.005 to 0.10 w /. A reagent having v% and having a final concentration of 0.09 w / v% or less in the reaction system of particles having a particle size of 80 nm or less before sensitization of the sensitized latex particles.
(7) The reagent according to (6), wherein the volume average particle diameter of the sensitized latex particles before sensitization is in the range of 1 to 1/10 of the measurement wavelengths of the amount of change in absorbance and the amount of change in scattered light.

According to the latex agglutination method of the present invention, accurate measurement can be performed in both a low concentration region and a high concentration region by using an automatic analyzer.

It is a figure which shows the outline of the automatic analyzer which can be used in the method of this invention.

The method of the present invention is basically a kind of well-known method called a latex agglutination method, and in the method of the present invention, the degree of aggregation of latex particles is measured by measuring the amount of change in absorbance and the amount of change in scattered light. It is something to do. For example, in one embodiment of the invention this can be done as follows.

That is, in the method of one embodiment, a step of mixing a sample solution containing the target substance to be measured and a solution containing latex particles carrying a binding partner with the target substance to prepare a mixed solution;
From the difference in scattered light intensity between the first and second time points, (a) the step of measuring the amount of change in scattered light intensity of the mixed solution;
The step of measuring the amount of change in (b) absorbance of the mixed solution from the difference in absorbance between the third and fourth time points;
The measured amount of (a) change in scattered light intensity and (b) amount of change in absorbance are measured using a calibration curve based on the amount of change in scattered light intensity and a calibration curve based on the amount of change in absorbance. It has a step of associating with; According to the present embodiment, since it has such a step, it is possible to obtain a calibration curve that substantially covers from a low concentration to a high concentration, and perform particle-enhanced immunoaggregation measurement with high sensitivity and a wide dynamic range. be able to.

Here, it is preferable that the first, second, third, and fourth time points are selected from the period from the start of preparation of the mixed solution to 1000 seconds later, respectively. This is because it is possible to satisfy both the desired sensitivity and the desired dynamic range while ensuring the degree of freedom in designing the measurement reagent by setting the mixture within 1000 seconds from the start of preparation. Further, (a) the amount of change in scattered light intensity and (b) the amount of change in absorbance are preferably measured within a wavelength range of 500 to 900 nm.

The latex particle-enhanced immunoagglutination measurement method according to the embodiment will be described in detail below, with explanations of the latex particles and the target substance used in the present embodiment. In the following description, "one measurement" means a series of reactions and measurements performed in one reaction vessel. Taking the measurement with an automatic analyzer as an example, the mixing of the first test solution and the sample, the subsequent addition and mixing of the second test solution (solution containing latex particles carrying a binding partner with the target substance), and the scattered light intensity. It means that the measurement of the amount of change and the measurement of the amount of change in absorbance are performed in one reaction vessel. Further, the "sample solution containing the target substance" in the present invention also includes a sample solution mixed and diluted with the first test solution (buffer solution) as described above.

(Latex particles)
As the latex particles used in the method of the present invention, conventionally widely used, for example, polystyrene, styrene-butadiene copolymer, styrene-styrene sulfonate copolymer and the like can be used. Since such latex particles are commercially available, commercially available products can be preferably used. In the commercially available product, the particle size of the latex particles is extremely uniform, and in the case of the latex particles on the market with the specified particle size specified, it can be roughly considered that all the particles have the specified particle size. When two or more types of latex particles having different particle sizes are used, the following volume average particle size can also be calculated based on this approximation.

The latex particles used may be composed of a plurality of particle sizes, but before the binding partner is sensitized (in the present specification and claims, it may be simply referred to as "pre-sensitization"). The volume average particle diameter a is in the range of 80 to 335 nm, preferably in the range of 100 to 300 nm. The volume average particle diameter a is calculated by the following formula 1, and has the particle diameter a ₁ (nm) of the first component, the intraparticle ratio n ₁ (%) of the _{same component, and the particle diameter a 2} (nm) of the second component. the intragranular ratio _n 2 (%) of the component is determined from the particle diameter _a n of the n component particles in a ratio of (nm) and the component _n n (%). If the volume average particle diameter a is less than 80 nm, the size of the agglomerates of the latex particles generated by the antigen-antibody reaction becomes small, and sufficient accuracy cannot be obtained in the measurement of the low concentration range of the target substance. On the other hand, if the volume average particle size exceeds 335 nm, the agglomerates become too large and the particles settle, making it difficult to measure the high concentration range.

The final concentration of the sensitized latex particles in the reaction system is in the range of 0.005 to 0.10 w / v%, preferably in the range of 0.010 to 0.090 w / v%. If it is less than 0.005 w / v%, the number of particles will be insufficient and it will be difficult to assume a high concentration range. On the contrary, when the particle concentration exceeds 0.10 w / v%, the optical amount (absorbance) in the initial state becomes large and it is difficult to take a difference from the measurement upper limit of the optical change amount, so that it is difficult to measure the high concentration range. Become.

Further, the latex particles having a particle size of 80 nm or less before sensitization are 0.09 w / v% or less, preferably 0.05 w / v% or less. If particles of 80 nm or less exceed 0.09 w / v%, multiple scattering will occur and the measurement accuracy of the amount of change in scattered light will be reduced.

(Sample)
The method of the present invention can measure various biological samples, and is not particularly limited to body fluids such as blood, serum, plasma, and urine.

The lower and upper limits of measurement of the target substance are not only for clinical significance but also for technical reasons of each measurement method (immunosturbation method, latex agglutination method, chemiluminescent immunity method, fluorescence immunization method, etc.). It may be determined as the industry standard measurement range for species. For example, in the latex agglutination method CRP (C-reactive protein) measured in the following examples, the lower limit value of 0.01 mg / dL and the upper limit value of 32 mg / dL are industry standards. When measuring using each test kit sold by each company, it is necessary to set the sample amount concentration so that the measured value is equal to or more than this lower limit value and satisfies the upper limit value. When the target substance is measured by the method of the present invention, the sample amount concentration (sample amount) is 0.7 times or less of the minimum sample amount concentration (sample amount concentration B) required to achieve the lower limit value only by the absorbance measurement. It is preferable to carry out at the concentration A). Achievement of the lower limit value and achievement of the upper limit value are contradictory, and the lower limit value can be easily achieved by increasing the sample amount concentration, but if the sample concentration is increased in order to achieve the lower limit value, the upper limit is increased depending on the sample. There is a high possibility that some products will not satisfy the value. According to the method of the present invention, it is possible to perform the measurement at the sample amount concentration A which is 0.7 times or less of the minimum sample amount concentration B required to achieve the lower limit value only by the conventional absorbance measurement. Lowering the sample amount concentration in this way is advantageous because the upper limit value can be improved. That is, by setting the sample amount concentration to 0.7 times or less, it is possible to achieve the effect of significantly improving the upper limit of the high concentration measurement range while enjoying the effect of improving the minimum detection sensitivity by the highly sensitive scattered light detection. If it exceeds 0.7 times, the minimum detection sensitivity is improved, but the effect of improving the upper limit of the high concentration measurement range may not be sufficiently obtained.

(Target substance to be measured)
The target substance to be measured by the method of the present invention is not particularly limited as long as it is a molecule that can be theoretically measured by the latex particle-enhanced immunoaggregation measurement method, such as protein, peptide, amino acid, lipid, sugar, nucleic acid, and hapten. .. For example, CRP (C-reactive protein), Lp (a) (lipoprotein (a)), MMP3 (matrix metalloproteinase 3), anti-CCP (cyclic citrulylated peptide) antibody, anti-phospholipid antibody, anti-pyorrhea antigen antibody, RPR, type IV collagen, PSA, AFP, CEA, BNP (brain natriuretic peptide), NT-proBNP, insulin, microalbumin, cystatin C, RF (rheumatic factor), CA-RF, KL-6, PIVKA-II, Examples include FDP, D dimer, SF (soluble fibrin), TAT (thrombin-antithrombin III complex), PIC, PAI, XIII factor, pepsinogen I / II, phenitoin, phenobarbital, carbamatepine, valproic acid, theophylline and the like. ..

(Join partner)
Examples of the binding partner used in the particle-enhanced immunoaggregation measurement method of the present invention include proteins, peptides, amino acids, lipids, sugars, nucleic acids, haptens and the like as substances that bind to the target substance of interest, but their specificity and affinity. From sex, antibodies, antigen-binding fragments thereof, and antigens are generally used. In addition, there are no particular restrictions on the size of the molecular weight, the origin such as natural or synthetic.

(Measuring reagent)
The composition of the measuring reagent used in the method of the present invention is not particularly limited, but when considering the use in an automatic analyzer widely used in the field of clinical examination, the first reagent solution (R1) composed of a buffer solution and the purpose. A measurement reagent composed of two liquids of a second reagent solution (R2) containing latex particles carrying a binding partner to a substance is common.
The measuring reagent of the present invention can also measure the amount of change in absorbance and the amount of change in scattered light independently.

(Components of measurement reagents)
The components of the measurement reagent used in the method of the present invention include latex particles sensitized (supported) by a binding partner, which is the main component of the reaction, as well as components that buffer the ionic strength and osmotic pressure of the sample, for example. It may contain acetic acid, citric acid, phosphoric acid, tris, glycine, boric acid, carbonic acid, and Good's buffers, their sodium salts, potassium salts, calcium salts, and the like. Further, a polymer such as polyethylene glycol, polyvinylpyrrolidone, or a phospholipid polymer may be contained as a component for enhancing aggregation formation. In addition, as a component that controls the formation of aggregates, one type or a combination of a plurality of general-purpose components such as polymer substances, proteins, amino acids, sugars, metal salts, surfactants, reducing substances and chaotropic substances is used. It may be included. It may also contain a defoaming component.

(Analysis equipment)
The method of the present invention is suitable for using a quick and simple automatic analyzer with a total reaction time of 10 minutes or less, and in particular, scattered light intensity as disclosed in Japanese Patent Application Laid-Open No. 2013-64705. An automatic analyzer that can measure the absorbance at almost the same time is suitable. However, the method of the present invention is not limited to the method using an automatic analyzer.

FIG. 1 shows an overall schematic configuration of a suitable example of an automatic analyzer that can be used in the method of the present invention. In FIG. 1, the automatic analyzer 1 includes a sample disk 10, a reaction disk 20, a reagent disk 30, a sample dispensing mechanism 41, a reagent dispensing mechanism 42, a computer 100, an interface circuit 101, and the like. The sample disk 10 is provided with a drive unit 12. The reaction disk 20 includes a drive unit 22. The reagent disk 30 includes a drive unit 32. Further, the reaction disk 20 is provided with two types of photometers, an absorptiometer 44 and a scattered photometer 45. Further, the reaction disk 20 is provided with a constant temperature bath 28. Further, the reaction disk 20 is provided with a stirring unit 43, a cleaning unit 46, and the like.

The computer 100 includes an analysis control unit 50, a storage unit 70, an output unit 71, an input unit 72, and the like. The analysis control unit 50 is connected to each drive unit and each mechanism through an interface circuit 101 including a signal line or the like. The computer 100 is composed of, for example, a PC, but is not limited to this, and may be composed of a circuit board such as an LSI board or a combination thereof. The storage unit 70 is composed of a storage device such as a ROM, a RAM, and a non-volatile storage device.

A plurality of sample cups 15 are installed and held on the sample disk 10. The sample cup 15 is a sample container that houses the sample 2. Each sample cup 15 is arranged and held side by side on the disk body 11 of the sample disk 10 so as to be separated from each other along the circumferential direction.

The drive unit 12 of the sample disk 10 drives and controls the sample disk 10 according to the control from the analysis control unit 50. At that time, the drive unit 12 rotates the disk body 11 to move the plurality of sample cups 15 along the circumferential direction. In the sample disk 10, one of the plurality of sample cups 15 installed in the disk body 11 is arranged at a predetermined position along the circumferential direction by the drive control of the drive unit 12. The predetermined position is, for example, a sample inhalation position by the sample dispensing mechanism 41 or the like.

In the configuration example of FIG. 1, in the sample disk 10, a plurality of sample cups 15 are arranged on the disk body 11 in a line of circumferences along the circumferential direction. Not limited to this, the sample cups 15 may be arranged in a plurality of rows concentrically of the disk body 11. Further, in the configuration example of FIG. 1, the sample disk 15 of the disk type is provided, but the present invention is not limited to this, and a rack type may be used. In the rack method, a sample rack in which a plurality of sample containers are arranged and held in one or two dimensions is used.

The reagent disk 30 is installed next to the reaction disk 20. A plurality of reagent bottles 35 are installed and held in the disk body 31 of the reagent disk 30. The reagent bottle 35 is a reagent container that houses the reagent 4. The reagent bottles 35 are arranged and held side by side so as to be separated from each other along the circumferential direction of the disc body 31. The reagent bottle 35 contains a reagent 4 of a type corresponding to the target component substance of the inspection item in the automatic analyzer 1. Each type of reagent 4 is contained in a separate reagent bottle 35.

The drive unit 32 of the reagent disk 30 rotates the disk body 31 according to the control from the analysis control unit 50 to move the plurality of reagent bottles 35 along the circumferential direction. In the reagent disk 30, one of the plurality of reagent bottles 35 installed in the disk body 31 is arranged at a predetermined position on the reagent disk 30 by the drive control of the drive unit 32. The predetermined position is, for example, a reagent inhalation position by the reagent dispensing mechanism 42.

The reagent disk 30 is provided with a reagent cool box 38 provided with a cooling mechanism. Even if the disk body 31 rotates, the plurality of reagent bottles 35 arranged on the disk body 31 are cooled while being constantly held in the cooling environment of the reagent cool box 38. As a result, deterioration of the reagent 4 is prevented. As the cooling mechanism of the reagent refrigerator 38, for example, a method of circulating low-temperature water, a method of cooling in the gas phase by a Pelche element, or the like is used.

The reaction disk 20 is installed between the sample disk 10 and the reagent disk 30. A plurality of reaction vessels 25 are installed and held in the disc body 21 of the reaction disc 20. The reaction vessel 25 is a vessel in which the reaction solution 3 is produced. The reaction solution 3 is a mixed solution of the sample 2 and the reagent 4. The sample 2 is dispensed into the reaction vessel 25 by the sample dispensing mechanism 41, the reagent 4 is dispensed by the reagent dispensing mechanism 42, and the reaction solution 3 is prepared by the mixed solution of the sample 2 and the reagent 4. The reaction vessels 25 are juxtaposed and held side by side so as to be separated from each other along the circumferential direction of the disc body 21. The reaction vessel 25 is made of a translucent material for measurement by the absorptiometer 44 and the scattered photometer 45. The drive unit 22 of the reaction disk 20 rotates the disk body 21 in accordance with the control from the analysis control unit 50 to move the plurality of reaction vessels 25 along the circumferential direction. The reaction disk 20 arranges one of the plurality of reaction containers 25 at a predetermined position provided along the circumferential direction by rotating the disk body 21. The predetermined position is, for example, a sample discharge position by the sample dispensing mechanism 41, a reagent discharge position by the reagent dispensing mechanism 42, or the like.

Each of the plurality of reaction vessels 25 arranged on the disk body 21 of the reaction disk 20 is constantly immersed in the constant temperature bath water (also referred to as constant temperature fluid) in the constant temperature bath 28. As a result, the reaction solution 3 in the reaction vessel 25 is maintained at a constant reaction temperature (for example, about 37 ° C.). The temperature and flow rate of the constant temperature bath water in the constant temperature bath 28 are controlled by the analysis control unit 50, and the amount of heat supplied to the reaction vessel 25 is controlled.

In addition to the sample dispensing mechanism 41 and the reagent dispensing mechanism 42, the stirring unit 43, the absorptiometer 44, the scattered photometer 45, and the cleaning unit are located on and near the circumference of the reaction disk 20 at different positions. 46 etc. are arranged.

The sample dispensing mechanism 41 is installed between the sample disk 10 and the reaction disk 20. The sample dispensing mechanism 41 performs a sample dispensing operation, which is an operation of sucking the sample 2 from the sample cup 15 at the sample inhalation position of the sample disk 10 and discharging the sample 2 into the reaction vessel 25 at the sample discharge position of the reaction disk 20. The sample dispensing mechanism 41 includes a movable arm and a dispensing nozzle. The dispensing nozzle consists of a pipette nozzle attached to a movable arm. The sample dispensing mechanism 41 moves the dispensing nozzle to the sample suction position on the sample disk 10 during the sample dispensing operation, and from the sample cup 15 arranged at the sample suction position, a predetermined amount is contained in the dispensing nozzle. Specimen 2 is inhaled and contained. After that, the sample dispensing mechanism 41 moves the dispensing nozzle to the sample discharging position on the reaction disk 20, and discharges the sample 2 in the dispensing nozzle into the reaction vessel 25 arranged at the sample discharging position.

The reagent dispensing mechanism 42 is installed between the reagent disc 30 and the reaction disc 20. The reagent dispensing mechanism 42 performs a reagent dispensing operation, which is an operation of sucking the reagent 4 from the reagent bottle 35 at the reagent inhalation position of the reagent disk 30 and discharging the reagent 4 into the reaction vessel 25 at the reagent discharge position of the reaction disk 20. The reagent 4 to be dispensed is a reagent used for quantification of a target component substance, which is an analysis item (also called a test item or the like) set corresponding to the target sample 2. The reagent dispensing mechanism 42 also includes a movable arm and a dispensing nozzle. The reagent dispensing mechanism 42 moves the dispensing nozzle to the reagent suction position on the reagent disk 30 during the reagent dispensing operation, and a predetermined amount of reagent is injected into the dispensing nozzle from the reagent bottle 35 arranged at the reagent suction position. Inhale and contain 4. After that, the reagent dispensing mechanism 42 moves the dispensing nozzle to the reagent discharging position on the reaction disk 20, and discharges the reagent 4 in the dispensing nozzle into the reaction vessel 25 arranged at the reagent discharging position.

The sample dispensing mechanism 41 and the reagent dispensing mechanism 42 are each provided with a washing tank 46 in preparation for dispensing different types of sample 2 or reagent 4. The cleaning tank 46 is a mechanism for cleaning the dispensing nozzle. Each dispensing mechanism cleans each dispensing nozzle in the cleaning tank 46 before and after the dispensing operation. As a result, contamination between the samples 2 or the reagents 4 is prevented. Further, the dispensing nozzle of each dispensing mechanism is equipped with a sensor for detecting the liquid level of the sample 2 or the reagent 4. Thereby, the measurement abnormality due to the shortage of the sample 2 or the reagent 4 can be monitored and detected. In addition, the sample dispensing mechanism 41 is provided with a pressure sensor that detects clogging of the dispensing nozzle. As a result, it is possible to monitor and detect a dispensing abnormality that occurs when an insoluble substance such as fibrin contained in the sample 2 is clogged in the dispensing nozzle. The analysis control unit 50 can monitor and detect various abnormalities during measurement through a mechanism including these sensors.

The stirring unit 43 stirs the mixed solution of the sample 2 and the reagent 4 in the reaction vessel 25 arranged at the stirring position which is a predetermined position on the reaction disk 20. As a result, the mixed solution in the reaction vessel 25 is uniformly stirred, the reaction is promoted, and the reaction solution 3 is prepared. The stirring unit 43 is provided with, for example, a stirrer equipped with a stirring blade or a stirring mechanism using ultrasonic waves.

In two types of photometers, it has one absorptiometer 44 as a first-class photometer and one scattered photometer 45 as a second-class photometer. Each photometer of the absorptiometer 44 and the scattered photometer 45 has a light source and a light receiving unit as a basic structure. The light source of each photometer is arranged on the inner peripheral side of the reaction disk 20, for example, and the light receiving portion of each photometer is arranged on the outer peripheral side of the reaction disk 20. Each photometer is connected to the analysis control unit 50.

The absorptiometer 44 measures the reaction solution 3 of the reaction vessel 25 arranged at the measurement position (particularly the first measurement position) which is a predetermined position on the reaction disk 20. The scattered photometer 45 measures the reaction solution 3 of the reaction vessel 25 arranged at the measurement position (particularly the second measurement position) which is a predetermined position on the reaction disk 20. In the configuration example of FIG. 1, two photometers, an absorptiometer 44 and a scattering photometer 45, are installed at predetermined positions on the circumference of the reaction disk 20 and diagonally opposed to each other through the rotation center of the reaction disk 20. ing. An absorptiometer 44 is arranged at the first measurement position, and a scattered photometer 45 is arranged at the second measurement position. The stirring unit 43 and the cleaning unit 46 are arranged at predetermined positions on the circumference between the first measurement position and the second measurement position.

The absorptiometer 44 irradiates the reaction solution 3 of the reaction vessel 25 at the first measurement position with light from the light source. At that time, the absorptiometer 44 detects the transmitted light obtained from the reaction solution 3 by the light receiving unit, and describes at least one of the light intensity or the light intensity of the transmitted light having a single or a plurality of wavelengths (light intensity / light intensity). May) be measured. Further, the absorptiometer 44 may obtain a quantitative value such as a concentration by a predetermined calculation based on the measured value. The absorptiometer 44 outputs a signal including a measured value or a calculated value.

The scattered photometer 45 irradiates the reaction solution 3 of the reaction vessel 25 at the second measurement position with light from the light source. At that time, the scattered photometer 45 detects the scattered light obtained from the reaction solution 3 by the light receiving unit, and measures at least one of the amount of scattered light and the light intensity (light amount / light intensity). Further, the scattered photometer 45 may obtain a quantitative value such as a concentration by a predetermined calculation based on the measured value. The scattered photometer 45 outputs a signal including a measured value or a calculated value.

The cleaning unit 46 cleans the reaction vessel 25 arranged at the cleaning position on the reaction disk 20. The cleaning unit 46 discharges the remaining reaction solution 3 from the reaction vessel 25 for which the measurement and analysis have been completed, and cleans the reaction vessel 25. The washed reaction vessel 25 can be reused. That is, the next sample 2 is dispensed from the sample dispensing mechanism 41 again, and the next reagent 4 is dispensed from the reagent dispensing mechanism 42 into the reaction vessel 25.

(Scattering angle)
The scattering angle of the scattered light intensity measurement used in the present invention is not particularly limited, but is preferably 10 ° to 35 °, more preferably 20 ° to 30 °. By setting the scattering angle within the above range, the light receiving portion for detecting the scattered light is not strongly affected by the transmitted light, and the ability to receive the scattered light is also advantageous.

(Measurement of scattered light intensity)
The light source for measuring the scattered light intensity and the wavelength of the irradiation light used in the present invention are not particularly limited, but 1 to 10 times the volume average particle diameter a of the latex particles is preferable. A more preferable range is 1.5 to 5 times. If it is less than 1 times, the measurement accuracy of a high-concentration sample may be significantly reduced. Further, if it is larger than 10 times, the minimum detection sensitivity ability, which is an advantage of the scattered light intensity measurement, may be significantly lowered. There is no particular limitation on the photometric interval at two time points for measuring the amount of change in scattered light intensity, and in general, the longer the interval, the higher the sensitivity. In the above-mentioned automatic analyzer, scattering at any two time points from immediately after mixing the sample solution containing the target substance to be measured and the solution containing latex particles carrying a binding partner with the target substance to a maximum of 1000 seconds. The amount of change in light intensity measurement and the amount of change in absorbance measurement can be measured respectively, but by measuring both the amount of change in scattered light intensity and the amount of change in absorbance at two time points within 300 seconds immediately after mixing, the first reagent solution, The total measurement time per measurement (one sample) through the second test solution can be set to 10 minutes or less, and the benefit of the maximum sample processing speed of various commercially available automatic analyzers can be enjoyed.

(Absorbance measurement)
The wavelength of the absorbance measurement used in the present invention is not particularly limited, but as in the measurement of the scattered light intensity, it is preferably 1 to 10 times the volume average particle diameter a of the latex particles used. A more preferable range is 1.4 to 8 times. If it is less than 1 times, the measurement accuracy of a high-concentration sample may be significantly reduced. Further, if it is larger than 10 times, the minimum detection sensitivity may be lowered. Further, the wavelengths for the absorbance measurement used in the present invention include a main wavelength on the short wavelength side and a sub-wavelength on the long wavelength side as compared with the wavelength used for one wavelength measurement or scattered light intensity measurement within the above range. A combination of two wavelength measurements can be used, the latter being more accurate and preferred. There is no particular limitation on the photometric interval at two time points for measuring the amount of change in absorbance, and in general, the longer the interval, the higher the sensitivity.

(Amount of change)
The amount of change in the amount of light (scattered light intensity and absorbance) used in the present invention is a calculation method applicable to the latex particle-enhanced immunoaggregation measurement method, such as the difference and ratio between two time points and the conversion value per unit time. There are no particular restrictions.

(Step to associate with the abundance of the target substance)
In the latex particle-enhanced immunoaggregation measurement method of the present invention, calibration curves for scattered light intensity measurement and absorbance measurement are individually prepared using a sample containing a target substance having a known concentration, and a low concentration range of the target substance, that is, the minimum The detection sensitivity is calculated based on the calibration curve for highly sensitive scattered light intensity measurement, and the upper limit of measurement in the high concentration range of the target substance is calculated based on the calibration curve for absorbance measurement with a wide dynamic range. For absorbance measurements with a wide dynamic range, a calibration curve can be created over a wider concentration range.

(Detection coefficient of variation ratio)
The detection coefficient of variation ratio is obtained by deriving the degree of variation (coefficient of variation) in the reproducibility measurement of the low-value standard material by the ratio of the absorbance measurement value and the scattered light measurement value.

(High concentration range measurement upper limit)
The upper limit of measurement in the high concentration range means the maximum amount of the target substance that can be measured. The upper limit of measurement in the high concentration range of the method of the present invention is a range in which a change in the amount of light proportional to the concentration of the target substance can be detected. If necessary, the sample can be appropriately diluted to obtain an appropriate concentration.

Hereinafter, the present invention will be described in more detail based on examples. However, the present invention is not limited to the following examples.

(Examples 1 to 3 and Comparative Examples 1 to 9)
(Preparation example: Preparation of CRP measurement reagent by single particle size)
1. 1. Second reagent solution (R2): Preparation of antibody-binding latex solution Anti-CRP rabbit polyclonal antibody (antibody solution containing 17% of specific antibody amount (mg / mL) with respect to total protein amount (mg / mL) in antibody solution) ) Was sensitized to polystyrene latex particles, dispersed and suspended in a glycine buffer, and a reagent was prepared. That is, the reagent is sensitized by mixing a specified amount of antibody and polystyrene latex particles in a buffer solution (glycine 170 mM pH 6.0), stirring sufficiently at room temperature for 60 minutes, and then centrifuging. Excluding Qing, the antibody-insensitive portion on the surface of the polystyrene latex particles was blocked with a glycine buffer solution containing bovine serum albumin, and the antibody-sensitized polystyrene latex particles were collected again by centrifugation, and the latex particles were placed in the glycine buffer solution. It was prepared by nicking and sufficiently dispersing and suspending to obtain a second reagent (antibody-bound latex solution: R2).

2. Preparation of First Test Solution (R1) A 170 mM glycine buffer solution containing 200 mM sodium chloride and 1.0% BSA was prepared and used as the first test solution.

3. 3. Standard sample As a measurement sample, a CRP standard prepared by diluting human CRP purified antigen with normal human serum was used. The CRP standard is priced according to the plasma protein international standard CRM470, and contains 0.25, 0.5, 1, 2, 4, 8, 16, 32, 48, 67, 100 mg / dL. I prepared it. Saline was used at 0 mg / dL.

(Analyzer and measurement conditions)
Both the scattered light intensity and the absorbance were measured for one measurement using the automatic analyzer described in JP2013-64705. Each condition of the scattered light intensity measurement was an irradiation wavelength of 700 nm, a scattering angle of 20 °, and 30 °, and each condition of the absorbance measurement was measured as two wavelengths of a main wavelength of 570 nm and a sub-wavelength of 800 nm. Add 122 μL of R1 to 1.5 μL of the sample containing CRP and incubate at 37 ° C. for about 300 seconds, then add and stir 122 μL of R2 and incubate at 37 ° C. for about 300 seconds. The amount of change in scattered light intensity and absorbance was calculated from the difference between the two.

(Calibration curve and sample measurement)
The CRP concentration in the sample was calculated using a calibration curve based on each of the scattered light intensity and the absorbance calculated by spline using a CRP calibrator (manufactured by Denka Seiken Co., Ltd.). The concentration range of the calibration curve was selected each time according to the dynamic range under each measurement condition.

(Latex particle concentration and sample volume concentration during measurement)
In this measurement, latex particles having a particle size and a particle concentration as shown in Table 1 were used. The sample volume concentration was 0.62 v%.

The raw materials and the obtained reagents used in Examples 1 to 3 and Comparative Examples 1 to 9 were evaluated by the following methods. The results are shown in Table 2.

[Detection coefficient of variation ratio]
The absorbance and scattered light intensity of the low-value side sample of CRP control serum (manufactured by Denka Seiken Co., Ltd.) used for quality control of measurement diluted with physiological saline as a sample were measured at n = 20 each. .. From the obtained standard deviation of n = 20, the coefficient of variation (CV) was obtained for each measurement method, and the ratio of the coefficient of variation (CV _sc _{) of the scattered light intensity was obtained based on the coefficient of variation of absorbance (CV abs} ), and the same value was obtained. It was used as the detection coefficient of variation ratio (Equation 2). When the detection coefficient of variation ratio is 1 or less, it means that the coefficient of variation of the scattered light intensity is smaller than the coefficient of variation of the absorbance, which is an index expected to improve the detection sensitivity, which is an advantage of the scattered light intensity.

As a result, in the reagents using the latex particle diameters of 60 nm and 70 nm of Comparative Examples 1 to 8, the detection coefficient of variation ratio was 1 or more at any of the adjusted particle concentrations (0.074 to 0.350 w / v%). It was suggested that the reagent composition is not expected to improve the low value sensitivity, which is an advantage of the scattered light detection system.

In the reagent using the latex particles of 210 nm, the detection coefficient of variation ratio was 1 or less in the particle concentration range of 0.008 to 0.033 w / v% (Examples 1 to 3), and the particle size and concentration range were the same. It was suggested that the composition is expected to improve low value sensitivity, which is an advantage of the scattered light detection system. However, even when the same latex diameter of 210 nm was used, the detection coefficient of variation ratio was 1 or more when the particle concentration was 0.004 w / v% (Comparative Example 9).

(Examples 4 to 7)
Similar evaluations were performed with different reagent items for the purpose of verifying whether the results obtained in Examples 1 to 3 and Comparative Examples 1 to 9 were unique to the CRP measurement reagent.

(Example 4)
A second reagent (R2) was prepared in the same manner as in Example 1 except that the binding partner supported on the latex was changed to an anti-cystatin C (Cys-C) polyclonal antibody and changed as shown in Table 3. Measurements were made. The results obtained are shown in Table 4. The detection coefficient of variation ratio is 1 or less, and both the particle size and the particle concentration are equivalent to the ranges obtained in the evaluations in the CRP measuring reagent (Examples 1 to 3 and Comparative Examples 1 to 9), and are consistent with each other. It was confirmed.

(Example 5)
A second reagent (R2) was prepared and measured in the same manner as in Example 1 except that the binding partner supported on the latex was changed to an anti-myoglobin (Mb) polyclonal antibody and changed as shown in Table 3. rice field. The results obtained are shown in Table 4. The detection coefficient of variation ratio is 1 or less, and both the particle size and the particle concentration are equivalent to the ranges obtained in the evaluations in the CRP measuring reagent (Examples 1 to 3 and Comparative Examples 1 to 9), and are consistent with each other. It was confirmed.

(Example 6)
A second reagent (R2) was prepared and measured in the same manner as in Example 1 except that the binding partner supported on the latex was changed to an anti-immunoglobulin E (IgE) monoclonal antibody and changed as shown in Table 3. Was done. The results obtained are shown in Table 4. The detection coefficient of variation ratio is 1 or less, and both the particle size and the particle concentration are equivalent to the ranges obtained in the evaluations in the CRP measuring reagent (Examples 1 to 3 and Comparative Examples 1 to 9), and are consistent with each other. It was confirmed.

(Example 7)
A second reagent (R2) was prepared and measured in the same manner as in Example 1 except that the binding partner supported on the latex was changed to an anti-myeloperoxidase (MPO) polyclonal antibody and changed as shown in Table 3. went. The results obtained are shown in Table 4. The detection coefficient of variation ratio is 1 or less, and both the particle size and the particle concentration are equivalent to the ranges obtained in the evaluations in the CRP measuring reagent (Examples 1 to 3 and Comparative Examples 1 to 9), and are consistent with each other. It was confirmed.

(Examples 8 to 12, Comparative Examples 10 to 17)
Similar to Example 1, except that the binding partner carried on the latex was an anti-CRP polyclonal antibody, two types of latex particles having different particle sizes were used, and the concentration of each particle was changed as shown in Table 5. Two reagents (R2) were prepared and measured. The results obtained are shown in Table 6.

When the concentrations of the two types of latex particles used on the small particle diameter (60, 70 nm) side were in the range of 0.35 to 0.15 w / v% (Comparative Examples 10 to 15), the detection coefficient of variation ratio was 1 or more. It was suggested that the reagent composition did not exhibit the advantages of scattered light measurement as in the case of the single particle system (Comparative Examples 1 to 9).

When the small particle size of the latex particles is 100 nm and the particle concentration is 0.35 to 0.15 w / v% (Comparative Examples 16 to 17, Example 12), the absorbance measurement value on the short wavelength 570 nm side is the upper limit of measurement. The absorbance of 3.0 was exceeded, and it became impossible to measure (the variation coefficient of absorbance is expressed as "ERROR"). Therefore, the detection coefficient of variation ratio in the same range cannot be calculated. However, the absorbance has an inversely proportional relationship with the measurement wavelength, and by measuring the measurement wavelength at a wavelength longer than 570 nm, it becomes 3.0 or less, which is the upper limit of measurement, and it is possible to calculate the detection coefficient of variation ratio. There is sex. The coefficient of variation of the scattered light measurement of Example 12 in which the particle concentration of the small particle size is 0.15 w / v% is a sufficiently low value of 4.2%, and the detection coefficient of variation ratio is 1 or less depending on the wavelength selection of the absorbance. It can be considered that there is a sufficient possibility of becoming. Further, when the particle concentration of the small particle diameter (60, 70, 100 nm) is 0.074 w / v% (Examples 8 to 11), the detection coefficient of variation ratio is 1 or less, and the advantage of the scattered light detection system is exhibited. It was suggested that it was a reagent design.

(Example 13, Comparative Example 18)
Similar evaluations were performed with different reagent items for the purpose of verifying whether the results obtained in Examples 8 to 12 and Comparative Examples 10 to 17 were unique to the CRP measurement reagent.

(Example 13)
A second reagent (R2) was prepared and measured in the same manner as in Example 9, except that the binding partner supported on the latex was changed to an anti-ferritin (FER) polyclonal antibody and changed as shown in Table 7. rice field. The results obtained are shown in Table 8. The detection coefficient of variation ratio is 1 or less, and both the particle size and the particle concentration are equivalent to the ranges obtained in the evaluations in the CRP measuring reagent (Examples 8 to 12 and Comparative Examples 10 to 17), and are consistent with each other. It was confirmed.

(Comparative Example 18)
A second reagent (R2) was prepared in the same manner as in Example 9 except that the binding partner supported on the latex was changed to an anti-β2-microglobulin (BMG) polyclonal antibody and changed as shown in Table 7. Measurements were made. The results obtained are shown in Table 8. The detection coefficient of variation ratio is 1 or more, and both the particle size and the particle concentration are equivalent to the ranges obtained in the evaluations in the CRP measuring reagent (Examples 8 to 12 and Comparative Examples 10 to 17), and are consistent with each other. It was confirmed.

(Comparative Example 19, Example 14)
A second reagent (R2) was prepared and measured in the same manner, except that the sample concentration was changed as shown in Table 9, based on the reagent of Example 9. However, in Comparative Example 19, the scattered light measurement was not performed, and only the absorbance measurement was performed. In this evaluation, the following were added as additional evaluation items. The results obtained are shown in Table 10.

[High concentration range measurement upper limit]
The upper limit of measurement in the high concentration range was the maximum concentration of the target substance to be measured in order to obtain a calibration curve.

As a result, in Example 14, even if the sample amount was reduced to 0.680% from Comparative Example 19, the detection coefficient of variation ratio was 1 or less, and the upper limit of measurement in the high concentration range was Comparative Example 19 (32 mg / dL). ), A good result was obtained as a clinical reagent that increased by 1.5 times to 47 mg / dL.

(Comparative Example 20, Example 15)
For the purpose of verifying whether the result obtained in Example 14 is a result peculiar to the CRP measuring reagent, the same evaluation was performed with the FER measuring reagent as a different reagent item. A second reagent (R2) was prepared and measured in the same manner, except that the sample amount was changed as shown in Table 9 based on the reagent of Example 13. However, in Comparative Example 20, only the absorbance was measured without measuring the scattered light. The results obtained are shown in Table 10.

As a result, even if the sample concentration was reduced to 0.290% from Comparative Example 20, the detection coefficient of variation ratio was 1 or less, and the upper limit of measurement in the high concentration range was 3 as compared with Comparative Example 20 (1000 ng / mL). Good results were obtained as a clinical reagent that increased by a factor of 3.5 to 3500 ng / mL.

From the results in Tables 1 to 10, it was found that the latex particle-enhanced immunoaggregation measuring method and its reagent of the present invention are excellent in sensitivity (detection sensitivity ratio) and dynamic range (high concentration range measurement upper limit).

2 Specimen 3 Reaction solution 4 Reagent 10 Specimen disk 11 Disc body 12 Drive unit 15 Specimen cup 20 Reaction disk 21 Disc body 22 Drive unit 25 Reaction vessel 28 Constant temperature bath 30 Reagent disk 31 Disc body 32 Drive unit 35 Reagent bottle 38 Reagent cold storage 41 Specimen dispensing mechanism 42 Reagent dispensing mechanism 43 Stirring unit 44 Absorption photometer 45 Scattered photometer 46 Cleaning unit (tank)
50 Analysis control unit 70 Storage unit 71 Output unit 72 Input unit 100 Computer 101 Interface circuit

Claims

A method for measuring a target substance by a latex agglomeration method, which comprises reacting a suspension of sensitized latex particles with a target substance, and then measuring the aggregation of the sensitized latex particles from an amount of optical change. The volume average particle diameter of the sensitized latex particles before sensitization was 80 nm to 335 nm, and the final concentration of the sensitized latex particles in the reaction system was 0.005 to 0.170 w / v%. The final concentration of the latex particles with a particle size of 80 nm or less before sensitization in the reaction system is 0.09 w / v% or less, and the optical changes are the absorbance change and the scattered light change. Method.
The method according to claim 1, wherein the amount of change in absorbance and the amount of change in scattered light are measured using light having a wavelength in the range of 1 to 10 times the volume average particle diameter of the sensitized latex particles before sensitization. ..
As the measurement wavelength of the amount of change in absorbance, two wavelengths selected in the range of 500 to 900 nm are used, and the two further selected measurement wavelengths use a main wavelength and a sub-wavelength longer than the main wavelength, and further. The method according to claim 1 or 2, wherein the amount of change in scattered light in the first half uses one wavelength selected in the range of 500 to 900 nm.
When the lower and upper limits of the measured values to be achieved are specified for the target substance, 0.7 times or less of the minimum sample volume concentration required to achieve the lower limit only by absorbance measurement. The method according to any one of claims 1 to 3, which is carried out at the sample amount concentration of.
The method according to any one of claims 1 to 4, wherein at least one type of scattering is measured within a range of a scattering angle of 10 ° to 30 ° in the measurement of the amount of change in scattered light.
Measurement of the target substance by the latex agglomeration method, which comprises reacting the suspended liquid of the sensitized latex particles with the target substance, and then measuring the aggregation of the sensitized latex particles from the amount of change in absorbance and the amount of change in scattered light. As a reagent, the volume average particle diameter of the sensitized latex particles before sensitization is 80 nm to 335 nm, and the final concentration of the sensitized latex particles in the reaction system is 0.005 to 0.10 w / v%. A reagent having a final concentration of 0.09 w / v% or less in a reaction system of particles having a particle size of 80 nm or less before sensitization of the sensitized latex particles.
The reagent according to claim 6, wherein the volume average particle diameter of the sensitized latex particles before sensitization is in the range of 1 to 1/10 of the measurement wavelengths of the amount of change in absorbance and the amount of change in scattered light.