JP2013032992A - Immunoassay device, immunoassay method and immunoassay program - Google Patents

Abstract

PROBLEM TO BE SOLVED: To solve the problem that a nozzle used for B/F separation is clogged by insoluble substances in an immunoassay technology including B/F separation processing.SOLUTION: An immunoassay device includes a B/F separation part for cleaning an antigen or an antibody which has reacted to an antibody binding carrier or an antigen binding carrier inside a reaction vessel and an unreacted free antigen or free antibody and separating the unreacted free antigen or free antibody by sucking it by a suction part (nozzle), and the immunoassay device includes: in a preceding stage of the B/F separation part, a photometric part having a light emitting part for irradiating the reaction vessel with light and a light receiving part for measuring transmitted light of the reaction vessel; and an insoluble substance determination part for determining presence/absence of insoluble substances to be a cause of clogging of the suction part on the basis of received light intensity in the light receiving part.

Description

  The present invention relates to an immunoassay technique including B / F separation, and more particularly to an insoluble matter determination technique of B / F separation.

  FIG. 1 is a diagram showing an example of an outline of the flow of a heterogeneous immunoassay method that requires B / F (Bound / Free) separation. As an immunoassay method such as an antigen or an antibody, a competitive method or a sandwich method is known. This is because an antigen-antibody reaction (immune reaction) is performed in a reaction vessel (cell or the like) 1 filled with the reaction solution 3, and is combined with the antigen / antibody solid-phased on the magnetic particles, polystyrene particles, plates, etc. In this method, an antigen / antibody is detected by binding an enzyme pre-labeled to a conjugate that forms a body. In this method, only the conjugate that has formed a complex with the antibody / antigen reacted with the immobilized antigen / antibody is left in the reaction container 1, and the conjugate that has not formed a complex with the unreacted antigen / antibody. Is removed from the reaction vessel 1.

  In the heterogeneous immunoassay, this removal operation is necessary, and generally called B / F separation. Usually, for this B / F separation, the antigen 103 or the antibody 101 for performing the antigen-antibody reaction is fixed in advance on a carrier in the reaction vessel 1 (FIG. 1A). ). For example, when magnetic particles 105 are used as a carrier, a solution containing a specific antigen 103 to be measured is added to the magnetic particles 105 on which the antibody 101 is fixed on the surface, and a primary immune reaction is performed in the reaction vessel 1. Later (FIG. 1B, the antigen 103 is bound to the antibody 101 fixed to the magnetic particle 105), and the magnet 5 is brought into contact with or brought close to the reaction vessel 1 so that the antibody 101 is placed on the inner wall in the reaction vessel 1. And the magnetic particles 105 bound to the antigen 103 are captured (FIG. 1C).

  In the state shown in FIG. 1C, non-magnetic components (impurities such as unreacted antigen 103a in the figure) containing impurities that have not been trapped on the inner wall of the reaction vessel 1 are removed by suction and the cleaning liquid is discharged. It discharges from the part 11. When the magnet 5 is sufficiently separated from the reaction vessel 1 and the washing solution is dispensed from the washing solution discharge section 11, the reaction product is separated from the inner wall of the reaction vessel 1 and the washing solution is spread over the entire magnetic particles 105 and is removed only by the above suction. Impurities (free antigen, antibody, etc.) that could not be removed can be removed. As shown in FIG. 1 (d), when an enzyme-labeled antibody 107 (an antibody 109 bound to an antibody 111) is added to cause a secondary immune reaction, an antigen 103 is immobilized on the antibody 101 immobilized on the bound magnetic particle 105. The enzyme-labeled antibody 107 binds to the complex that binds (complex X is formed). Here, in order to remove the unreacted enzyme-labeled antibody 107a, as shown in FIG. 1 (e), when the magnet 5 is brought into contact with or close to the reaction vessel 1 again, the reaction product ( The complex X) is captured, and the cleaning liquid containing the impurities and the like (including the unreacted enzyme-labeled antibody 107a) is discharged from the discharge section 11 by the suction nozzle 7 in the same manner as described above. .

  By repeating this operation, the washing effect of the reaction product is enhanced, and only the complex X consisting of the conjugate labeled with the enzyme in advance and the antigen-antibody can be left. Therefore, highly accurate analysis results (analysis results for determining the concentration, amount, etc. of the antigen 103) can be obtained. If the stirring operation is carried out after dispensing the cleaning liquid, further improvement of the cleaning effect can be expected. Next, as shown in FIG. 1F, the luminescent substrate 113 is added to the reaction solution. Then, the luminescent substrate 113 is consumed by the enzyme-labeled antibody 107, and a signal is emitted at that time. In this way, the signal of the complex X can be analyzed. For example, the greater the number of antigens 103, the greater the number of substrates consumed. Therefore, by measuring the signal in the state of FIG. 1 (f), the amount (concentration) of the antigen or antibody depending on the signal amount is measured. The concentration of the target substance in the sample can be known.

  Here, by reliably performing this B / F separation, the signal background in the immunoassay is reduced, and the sensitivity and accuracy of the measurement can be increased.

  Conventionally, increasing the number of washings to increase the reliability of B / F separation has a problem of hindering speeding up of measurement and high throughput. For this reason, various inventions for obtaining the maximum separation efficiency with the shortest operation have been made. As an example, improvement of the composition of the cleaning liquid used for B / F separation, reduction of the cleaning liquid remaining, and the like can be mentioned.

  The following Patent Document 1 describes a cleaning in performing an B / F separation in which an unbound molecule is removed by repeated discharge and discharge of a cleaning liquid in an antigen-antibody reaction in which a molecule fixed on a carrier surface and a molecule in a solution are bound. In the first time, discharge and subsequent discharge to become a predetermined concentration of cleaning liquid are performed, and in the second and subsequent times, discharge and subsequent discharge to become cleaning liquid that is not darker than the previous time are performed. The discharge is performed so that the cleaning liquid is thinner than the initial concentration, and the subsequent discharge is performed. Thereby, as a whole, the amount of the cleaning liquid can be reduced and the cleaning can be performed efficiently, and the influence of the remaining cleaning liquid can be reduced.

  In addition, in the chyle / hemolyzed specimen detection device described in Patent Document 2 below, based on the detected image information, an imaging unit that detects image information of the specimen before analysis processing for analyzing the specimen, the specimen And a chyle / hemolyzed sample detection unit for detecting the chyle state or hemolyzed state of the sample from the color of the sample. By detecting the chyle state or hemolysis state of the specimen from the color of the specimen using the image information of the specimen, the state of the specimen can be detected with high accuracy and efficiency. For example, the case where the sample color is yellow is set as a normal state, and the determination process is performed by teaching. For example, when the color of the specimen is red, pink, orange, or the like that is more reddish than the normal state, the hemolytic state is determined. On the other hand, when the color of the specimen is more turbid than the normal state, such as pink or milky white, it can be determined as a chyle state.

JP 2009-162733 A JP 2010-281604 A

  However, when the technique described in Patent Document 1 is used, there is a problem that the nozzle is clogged with insoluble matter in the suction step after the B / F separation. In the technique described in Patent Document 2, the degree of cloudiness is inspected based on the image information of the specimen, but there is a problem that the detection accuracy is not so good as in the case of visual observation.

  Further, as a problem in the B / F separation step, for example, in a measurement system such as HCV (hepatitis C virus) core antigen, an acid or alkali containing a surfactant is used to express the core antigen in the virus. The modification | denaturation pretreatment by is required. When this pretreatment is performed on a hyperproteinemia sample, there is a problem that irreversible protein denaturation occurs and various insoluble substances are generated.

  Denatured products, plasma (or serum) fibrin clots, insoluble matters such as impurities in urine and feces generated by such treatment clog the suction nozzle 7 used for B / F separation. There is a problem that a failure / trouble of the apparatus including the separation processing unit occurs.

  In addition, since the B / F separation process is incomplete, the measurement result becomes false positive or false negative, thereby causing a medical malpractice.

  An object of the present invention is to solve the problem that a nozzle used for B / F separation is clogged with insoluble matter in an immunoassay technique including B / F separation treatment. Moreover, it aims at improving the detection accuracy of an insoluble matter.

  In order to achieve the above object, in the immunoassay apparatus including B / F separation of the present invention, the presence or absence of insoluble matter is determined based on the insoluble matter detection portion and the photometric result before the B / F separation portion. An insoluble matter determination unit, and in the insoluble matter determination unit, a non-magnetic component (insoluble matter) containing impurities not captured by the inner wall of the reaction vessel in the B / F separation unit Whether or not nozzle clogging can occur is determined in advance based on the light transmittance. If the transmittance is not high enough to prevent nozzle clogging, the sample can clog the nozzle with insoluble matter. It is characterized by determining that the property is high.

  By determining the presence or absence of insoluble matter before nozzle suction by the B / F separation unit, nozzle clogging caused by insoluble matter is prevented, and B / F separation processing caused by insoluble matter is incomplete. This can be suppressed, and the cause of medical errors due to false positive or false negative measurement results such as antigen concentration can be reduced.

  For example, in order to avoid trouble of an immunoassay device in B / F separation or medical error, a light receiving element (photodiode or the like) disposed on the opposite side is irradiated with light or the like before the B / F separation step. ) To determine the presence or absence of insoluble matter in the reaction vessel based on the change in absorbance (or the change in photodiode voltage). For determination of this insoluble matter, an integrated value or peak value of absorbance (or voltage) can be used. If it is determined that there is insoluble matter, the sample is discharged or discarded before entering the B / F separation step. In addition, when using LED as a light source, all or any of red (wavelength 635nm vicinity), green (wavelength 525nm vicinity), and blue (wavelength 470nm vicinity) can be used. As the light source, light having a wavelength band including a visible light region can be used.

  According to one aspect of the present invention, the unbound free antigen or free antibody is washed with the antigen or antibody reacted with the antibody-bound magnetic particle or antigen-bound magnetic particle in the reaction vessel, and the unreacted free antigen or free antibody An immunoassay device equipped with a B / F separation part that separates by sucking with a suction part (nozzle), a light emitting part that irradiates the reaction container with light before the B / F separation part, A photometric unit including a light receiving unit that measures the transmitted light of the reaction vessel, and an insoluble matter determining unit that determines the presence or absence of an insoluble matter that causes clogging of the suction unit based on the light reception intensity in the light receiving unit. And an immunoassay device characterized by comprising:

  Further, a threshold storage unit for storing a threshold for determining the presence or absence of an insoluble material is provided, and the insoluble material determination unit is based on the magnitude relationship between the light receiving intensity of the light receiving unit and the threshold Preferably, the presence or absence of insoluble matter is determined. As the threshold value, an integrated value of absorbance (or voltage) is preferably used. An LED is preferably used as the light source of the light emitting unit. A red LED is preferably used as the LED. Even if a coexisting substance is mixed, if it is a red LED, a sample that does not exceed the threshold value is not determined as a sample containing insoluble matter.

  The light receiving unit is a first light receiving element including red in a detection wavelength band and a second light receiving element including red in a detection wavelength band, and a filter unit for filtering other than red is provided on the light receiving surface side. It is preferable that the insoluble matter determination unit performs insoluble matter determination based on a difference in received light intensity from the light receiving element. Even if it is insoluble in the first light receiving element, if it is red in the second light receiving element, it is not determined as an insoluble substance as a coexisting substance of hemoglobin.

  The threshold values are the first received light intensity in a reaction vessel containing a precipitate having a particle size approximate to the opening diameter of the suction portion as a model sample, and the second received light in a reaction vessel not containing a precipitate. It is preferably determined as a value between the received light intensity.

  Preferably, the insoluble matter determination unit that determines the presence or absence of an insoluble matter that causes clogging of the suction unit determines the presence or absence of the insoluble matter based on an integrated value of parameters that depend on the light reception intensity in the light receiving unit.

  It is preferable that the photometric unit has an operation unit that is disposed at a position where the light emitting unit and the light receiving unit face each other and scans the reaction container in the vertical direction. Thereby, the integrated value can be obtained.

  It is preferable that a disposal unit that discharges or discards the reaction container determined by the insoluble matter determination unit to be insoluble is provided in the vicinity of the photometry unit.

  According to another aspect of the present invention, the antigen in the reaction vessel or the antigen reacted with the antigen-binding magnetic particles or the antibody and the unreacted free antigen or antibody are washed and aspirated (nozzle) for separation. / F separation method including an F separation process, comprising: a light emitting unit for irradiating the reaction vessel with light and a light receiving unit for measuring the transmitted light of the reaction vessel as a pre-step of the B / F separation step And an insoluble matter determination step for determining the presence or absence of an insoluble matter causing the clogging of the suction portion based on the light reception intensity in the light receiving portion. The

  Further, the present invention may be a program for causing a computer to execute the above-described immunoassay and a computer-readable recording medium for recording the program.

  ADVANTAGE OF THE INVENTION According to this invention, the nozzle clogging resulting from the insoluble matter in a B / F separation process can be prevented in the immunoassay apparatus containing B / F separation.

  In addition, the B / F separation process due to insoluble matter is suppressed from becoming incomplete, and the measurement result such as the concentration of the target substance becomes false positive or false negative. Factors can be reduced.

It is a schematic diagram which shows the flow of the immunoassay including B / F separation by one embodiment of this invention. It is a perspective view which shows schematic structure of the immunoassay apparatus by one embodiment of this invention. It is a disassembled perspective view which shows schematic structure of the immunoassay apparatus by one embodiment of this invention. It is a top view which shows schematic structure of the immunoassay apparatus by one embodiment of this invention. It is a figure which shows schematic structure of the principal part of the immunoassay apparatus by one embodiment of this invention. It is a figure which shows schematic structure of an insoluble matter measurement part among the immunoassay apparatuses by one embodiment of this invention. It is a functional block diagram which shows the schematic structural example of the immunoassay apparatus by one embodiment of this invention. It is a figure which shows the photometry result using the standard model sample of Example 1, (a) is red LED, (b) is green LED, (c) is a light-receiving part (photograph) when using blue LED. It is a figure which shows the relationship between the voltage depending on the light reception intensity | strength of a diode), and the scanning timing to the z direction of a reaction container. It is a figure which shows the photometry result using the standard model sample of Example 1, (a) is red LED, (b) is green LED, (c) is a light-receiving part (photograph) when using blue LED. It is a figure which shows the relationship between the light absorbency which depends on the light reception intensity | strength of a diode), and the scanning timing to the z direction of a reaction container. 6 is a diagram showing an example of a reaction container group used for immunoassay using a model sample of Example 2. FIG. It is a figure which shows the photometry result using the model sample of Example 2, (a) is red LED, (b) is green LED, (c) is a light-receiving part (photodiode in the case of using blue LED) Is a diagram showing the relationship between the voltage depending on the received light intensity and the scan timing of the reaction vessel in the z direction. It is a figure which shows the photometry result using the model sample of Example 2, (a) is red LED, (b) is green LED, (c) is a light-receiving part (photodiode in the case of using blue LED) It is a figure which shows the relationship between the light absorbency which depends on the light reception intensity | strength of ()), and the scanning timing to the z direction of a reaction container. 6 is a diagram showing an example of a reaction container group used for immunoassay using an actual sample of Example 3. FIG. It is a figure which shows the photometry result using the real sample of Example 3, (a) is red LED, (b) is green LED, (c) is a light-receiving part (photodiode in the case of using blue LED) Is a diagram showing the relationship between the voltage depending on the received light intensity and the scan timing of the reaction vessel in the z direction. It is a figure which shows the photometry result using the real sample of Example 3, (a) is red LED, (b) is green LED, (c) is a light-receiving part (photodiode in the case of using blue LED) It is a figure which shows the relationship between the light absorbency which depends on the light reception intensity | strength of ()), and the scanning timing to the z direction of a reaction container. It is a figure which shows the photometry result of the test substance containing the coexisting substance of Example 4, (a) is red LED, (b) is green LED, (c) is a light-receiving part (photograph) when using blue LED. It is a figure which shows the relationship between the voltage depending on the light reception intensity | strength of a diode), and the scanning timing to the z direction of a reaction container. It is a figure which shows the photometry result using the photometry result of the test substance containing the coexistence substance of Example 4, (a) is red LED, (b) is green LED, (c) is a case where blue LED is used. It is a figure which shows the relationship between a light absorbency and the scan timing to the z direction of a reaction container. It is a figure which shows the photometry result at the time of using the immunoassay apparatus of Example 5, using red LED, (a) is BGG dependence, (b) is the amount dependence of a deposit, (c) is specimen dependence. It is a figure which shows the relationship between the voltage which depends on the light reception intensity | strength of a light reception part (photodiode), and the scan timing to the z direction of a reaction container. It is a figure which shows the photometry result at the time of using the immunoassay apparatus of Example 5, using red LED, (a) is BGG dependence, (b) is the amount dependence of a deposit, (c) is specimen dependence. It is a figure which shows the relationship between the light absorbency and the scan timing to the z direction of a reaction container. It is a figure which shows the photometry result at the time of using the real sample of plasma / serum as a model sample of the coexisting substance using the immunoassay apparatus of Example 6, red LED is used, (a) is a model sample of a coexisting substance (B) is a figure which shows the relationship between the voltage which depends on the light reception intensity | strength of a light-receiving part (photodiode), and the scanning timing to the z direction of a reaction container of chyle plasma and (c) Hb plasma. It is a figure which shows the photometry result at the time of using the real sample of plasma / serum as a model sample of the coexisting substance using the immunoassay apparatus of Example 6, red LED is used, (a) is a model sample of a coexisting substance (B) is a figure which shows the relationship between the light absorbency of a light-receiving part (photodiode) and the scanning timing to the z direction of a reaction container of chyle plasma, (c) is Hb plasma. It is a flowchart figure which shows the flow of the immunoassay method by a 1 step method. It is a flowchart figure which shows the flow of the immunoassay method by a delayed 1 step method. It is a flowchart figure which shows the flow of the immunoassay method by a 2 step method (1). It is a flowchart figure which shows the flow of the immunoassay method by a 2 step method (2). It is a flowchart figure which shows the flow of the immunoassay method by the two-step method with pre-processing. It is a flowchart figure which shows the flow of the immunoassay method by the two-step method with predilution.

Hereinafter, an insoluble matter determination technique according to an embodiment of the present invention will be described with reference to the drawings.
In a heterogeneous assay system that requires B / F separation, B / F separation is a process in which an antigen (or antibody) reacted with antibody (or antigen) -bound magnetic particles and an unreacted free antigen (or antibody) are washed and separated. Is the method.

  First, an immunoassay apparatus having a B / F separation unit and a photometry unit according to an embodiment of the present invention will be described. FIG. 1 is a schematic diagram showing a flow of immunoassay including B / F separation according to an embodiment of the present invention. Since FIG. 1 was mentioned above, description is abbreviate | omitted.

  FIG. 2A is a perspective view showing a schematic configuration of an immunoassay device according to one embodiment of the present invention. FIG. 2B is an exploded perspective view showing a schematic configuration of the immunoassay device according to one embodiment of the present invention.

  In addition, the immunoassay apparatus A according to the present embodiment includes an auto cell feeder 37 that automatically supplies reaction containers 1 to the apparatus one by one, an incubation table 39 for performing incubation, and a B / F separation process. A B / F separation mechanism 41 for performing the detection and a detection unit (insoluble matter detection unit) 45 for detecting the insoluble matter are included.

  FIG. 3 is a plan view showing a schematic configuration example of the immunoassay device according to one embodiment of the present invention. As shown in FIG. 3, when the immunoassay apparatus A according to an embodiment of the present invention is viewed from the top, for example, a sample dispensing unit 20 that dispenses a specimen into a reaction container 1 and a reagent dispenser that dispenses a reagent. Injection part (1) 50 or (2) 23, reaction container agitation part 47 or 55a for agitating the primary or secondary immune reaction solution in reaction container 1 into which the specimen / reagent has been dispensed, and primary or secondary Incubation tables (1) 43 and (2) 44 for performing an immune reaction, a reagent cooler 51 for cooling the reagent, a reaction container transporter 57 for transporting the reaction container, a reagent nozzle washing tank 61, and detection of insoluble matter An insoluble matter detection unit (photometric unit) 63, a photometric unit 63 detects insoluble matter, a discarding unit 64 that discharges or discards a specimen to be discharged or discarded, and a photometric unit 63 detects insoluble matter. Do not discharge or dispose of B / F separation unit 65 that performs B / F separation processing of a sample to be subjected to / F separation, and B / F dispensing units (1) 67 and (2) that discharge cleaning liquids 1 and 2 in the B / F separation processing 69, a B / F cleaning unit 65a, a reagent nozzle cleaning tank 70 for cleaning the reagent nozzle, a detection unit 97 for detecting a signal, and a discarding unit 64 for discarding each reaction container 1 after signal detection. doing. It should be noted that any one of the above configurations can be replaced with another configuration, or a part of the configuration can be provided to another device.

The operation of the apparatus will be described.
FIG. 4 is a diagram showing a schematic configuration of a main part of the immunoassay device according to one embodiment of the present invention. As shown in FIG. 4A, the reaction vessel 1 is inserted into the photometry unit 63 by the reaction vessel transport unit 57, and the transmitted light of the LED emission is measured by the light receiving element while moving in the Z-axis direction. The voltage or absorbance of the light receiving element depending on the quantity is obtained. Based on the obtained voltage or absorbance, a reaction container that exceeds a certain threshold value can be used as a specimen as transparent and free of insoluble matter. A reaction container that does not exceed a certain threshold value is judged to be insoluble, and cannot be used as a specimen.

  FIG. 5 is a diagram illustrating a detailed configuration example of the insoluble matter detection unit (photometry unit) 63 in the immunoassay device according to the embodiment of the present invention. The reaction container transport unit 57 can hold the reaction container 1 and move it in the vertical (Z-axis) direction. The insoluble matter detection unit 63 has a cavity 63a, and a light emitting unit (LED) 81, a light receiving unit (photodiode, PD) 85, and a position opposite to each other with the reaction vessel 1 on the inner wall of the cavity 63a interposed therebetween, Is arranged. Light emitted from the LED (light emitting unit) 81 is transmitted through the central portion of the reaction vessel 1 and irradiated to the PD (light receiving unit) 85. The irradiated light is converted into a voltage depending on the amount of light received by the PD 85, and the absorbance can be obtained. The higher the voltage, the less insoluble matter in the specimen, that is, the lower the absorbance. Therefore, the amount of insoluble matter in the sample can be estimated from the voltage or absorbance of the light receiving element.

  FIG. 6 is a functional block diagram showing a schematic configuration example of the immunoassay device according to one embodiment of the present invention. First, the sample in the reaction container 1 is pre-processed in the pre-processing unit 91, and then photometrically measured by the insoluble matter detection unit 63 after the primary immune reaction. Next, based on the photometric result in the insoluble matter detection unit 63, the insoluble matter judgment unit 95 determines the presence or absence of an insoluble matter that causes clogging of the nozzle. If there is no such insoluble matter, the B / F separation is performed. The unit 65 performs B / F (1) separation. Subsequently, after the secondary immune reaction, B / F (2) separation is performed by the B / F separation unit 65, the signal amount is analyzed by the detection unit 97, and the antigen and antibody are quantified and qualitatively analyzed. Here, the insoluble matter determination unit 95 is capable of nozzle clogging based on a comparison result with a threshold value stored in the threshold value storage unit 96 in advance or a threshold value input from the threshold value input unit 98 or the like. Determine sex.

Next, the specific processing content of insoluble matter determination is demonstrated.
Since the nozzle diameter is 0.6 to 0.8 mm, a standard model specimen containing a precipitate having a slightly larger diameter can be used as a standard sample of insoluble matter that clogs the nozzle.

[Example 1]
FIG. 7 shows the relationship between the scan timing along the z-axis of the reaction vessel 1 and the output voltage of the PD (corresponding to the transmittance of the LED light) in each of the red LED, green LED, and blue LED. It is the figure which measured about a lot of each. From FIG. 7, it is possible to obtain the sum of voltages (voltage integrated value) during the scanning period in the degree of precipitate. Similarly, for LED (green) and LED (blue), the sum of voltages (voltage integrated value) during the scanning period is obtained as shown in Table 1. For example, 288 points are measured at 0.5 ms. When switching LEDs with actual device data, 288 points are divided by the number of LEDs to be metered, that is, if there are two red and green, 144 points each. With a simple device having only a photometric unit, it is possible to measure 288 points at each wavelength.

  In Table 1, it can be seen that the sum (voltage integrated value) of the voltage during the scan decreases with the amount of precipitate in any of the case of LED (red), LED (green), and LED (blue). Here, since there is a threshold value of the integrated voltage value related to nozzle clogging between no precipitate and a small amount, as shown in Table 2, the threshold value of the integrated voltage value related to nozzle clogging in each color LED is: They can be determined as 35000, 25000, and 20000, respectively. If it is below these threshold values, nozzle clogging will occur.

  This threshold value serves as a reference value for determining whether or not to clog a nozzle in a similar specimen described below. This reference value is stored in the threshold storage unit 96. Alternatively, it is input from the threshold value input unit 98.

  FIG. 8 is a diagram showing a photometric result using a standard model sample, and corresponds to FIG. FIG. 8A shows the red LED, FIG. 8B shows the green LED, and FIG. 8C shows the absorbance depending on the light receiving intensity of the light receiving unit (photodiode) and the z direction of the reaction vessel. It is a figure which shows the relationship with the scan timing to. From FIG. 8, the integrated value of the absorbance during the scan in the amount of the precipitate can be obtained. Similarly, for LED (green) and LED (blue), the sum of absorbance (integrated value) during scanning is as shown in Table 3. The threshold value of the integrated value in the presence or absence of sediment is obtained. Similarly, the LED (green) and LED (blue) are obtained as follows.

  When the absorbance is used as a reference, the threshold values of LED (red), LED (green), and LED (blue) are 150,000, 160000, and 350,000 as shown in Table 4. If it is above these threshold values, nozzle clogging will occur.

  This threshold value also serves as a reference value for determining whether or not to clog a nozzle in a similar specimen described below. This reference value is stored in the threshold storage unit 96. Alternatively, it is input from the threshold value input unit 98.

[Example 2]
FIG. 9 is a diagram showing an example of a reaction container group used for an immune reaction using a model specimen.

  As model sample 1, Bovine γG (BGG) was added to human normal serum (NHS) to produce a pseudo high protein serum and pretreated. In order from the reaction container on the left side of FIG. It is a figure which shows a mode, and it turns out that the cloudiness of the insoluble matter has generate | occur | produced in a certain reaction container. Insoluble matter is detected by scanning and irradiating LED light from the bottom to the top of the reaction container and receiving the light by the light receiving element, and integrating the voltage value of the light receiving element depending on the amount of light transmitted through the reaction container.

  Table 5 shows NHS, + 0.5% BGG, + 1% BGG, + 2% BGG, + 4% BGG, + 6% BGG, + 8% BGG, + 10% for LED (red), LED (green), and LED (blue). It is a table | surface which shows the sum (voltage integration value) of the voltage during the scan of BGG. From Table 6, 35000, 25000, and 20000 are stored in the threshold value storage unit 96 as threshold values for LED (red), LED (green), and LED (blue) (the same values as in Table 2). Referring to this threshold value, from the values actually obtained in FIG. 10, the integrated voltage values exceeding the threshold value are + 1% BGG for LED (red) and + 0.5% BGG for LED (green), respectively. , LED (blue) is also + 0.5% BGG, and from these results, it can be determined that a specimen from + 1% BGG to + 0.5% BGG is a specimen that is highly likely not to clog the nozzle. From this photometric result, up to + 0.5% BGG sample is used for the B / F separation process if safety is expected, and BGG samples with higher concentrations than that are discharged or discarded.

  FIG. 11 is a diagram showing a photometric result using a model specimen, where (a) shows a red LED, (b) shows a green LED, and (c) shows a absorbance and a reaction container when a blue LED is used. It is a figure which shows the relationship with the scan timing to az direction.

  Table 7 shows NHS, + 0.5% BGG, + 1% BGG, + 2% BGG, + 4% BGG, + 6% BGG, + 8% BGG, + 10% for LED (red), LED (green), and LED (blue). It is a table | surface which shows the light absorbency integrated value during the scan of BGG. From Table 8, 150000, 160000, and 350,000 are stored in the threshold value storage unit 96 as the threshold values for LED (red), LED (green), and LED (blue) (same values as in Table 4). Referring to this threshold value, from the values actually obtained in FIG. 11, the integrated absorbance values below the threshold value are + 1% BGG for LED (red) and + 0.5% BGG for LED (green), respectively. , LED (blue) is also + 0.5% BGG, and from these results, it can be determined that a specimen from + 1% BGG to + 0.5% BGG is a specimen that is highly likely not to clog the nozzle. From this photometric result, up to + 0.5% BGG sample is used for the B / F separation process if safety is expected, and a BGG sample with a higher concentration than that is discharged or discarded. Similar results are obtained for the integrated voltage value and the integrated absorbance value.

  In this way, it can be seen that each sample of NHS and + 0.5% BGG can be processed without actually clogging the nozzle by B / F separation. That is, these samples do not cause nozzle clogging, and can be said to be samples that may be subjected to B / F separation.

[Example 3]
FIG. 12 is a diagram showing an example of a reaction container group used for an immune reaction using a real sample.

  A high protein plasma sample is used as the actual sample. In order from the reaction container on the left side of FIG. 12, human normal serum and the remaining six reaction containers show various insolubles generated after acid / alkali pretreatment (from the left, # 4, # 6, # 39, # 43, # 72, and # 82), it can be seen that the insoluble white turbidity is generated. Insoluble matter is detected by scanning and irradiating the reaction vessel with LED light and integrating the voltage value of the light receiving element depending on the amount of light.

  FIG. 13 is a diagram showing a photometric result using an actual specimen, where (a) shows a red LED, (b) shows a green LED, and (c) shows a light receiving unit (photodiode) when using a blue LED. Is a diagram showing the relationship between the voltage depending on the received light intensity and the scan timing of the reaction vessel in the z direction.

  From FIG. 13, the sum of voltages (voltage integrated value) during scanning in various specimens can be obtained (Table 9).

  Table 10 is a table corresponding to Table 2. From Tables 9 and 10, this sample has a voltage integration below the threshold value for samples other than NHS, and these samples contain insoluble matter and may clog the nozzle. I understand.

  FIG. 14 is a diagram showing a photometric result using an actual sample, where (a) is a red LED, (b) is a green LED, and (c) is a absorbance and a reaction container when a blue LED is used. It is a figure which shows the relationship with the scan timing to az direction. From FIG. 14, it is possible to obtain integrated values of absorbance during scanning in various specimens (Table 11).

  Table 12 is a table corresponding to Table 4. According to Tables 11 and 12, in this sample, samples other than NHS have absorbance integrated values that are equal to or higher than the threshold value, and these samples contain insoluble matter and may clog the nozzle. I understand that. This result is the same as the result of FIG.

  As described above, the threshold value obtained based on the integrated voltage or the absorbance integrated value for the precipitate having a diameter slightly larger than the nozzle diameter is stored, and the actual value is calculated based on this threshold value. In the photometric process before the B / F separation process of the sample, the photometric unit determines whether or not the sample clogs the nozzle based on whether or not the integrated value of the sample is larger than the threshold value. can do.

  Therefore, before the B / F separation process, the presence or absence of insoluble matter in the specimen can be examined nondestructively, and nozzle clogging in the B / F separation process can be prevented.

  Thus, in an immunoassay apparatus including B / F separation, nozzle clogging caused by insoluble matter in B / F separation treatment is prevented, and B / F separation treatment caused by insoluble matter is incomplete. It is possible to reduce the cause of medical error due to false positive or false negative measurement results such as the concentration of the target substance.

  Next, the influence of coexisting substances will be described. Samples often contain coexisting substances such as direct bilirubin (Bil-F), conjugated bilirubin (Bil-C), chyle, and hemolysis (Hb), which affect the insoluble matter detection process. There is a possibility to give. Hereinafter, an example of an immunoassay technique considering the influence of coexisting substances will be described.

[Example 4]
FIG. 15 is a diagram showing the photometric results of a specimen containing a coexisting substance, where (a) shows a red LED, (b) shows a green LED, and (c) shows a light receiving unit (photo) when a blue LED is used. It is a figure which shows the relationship between the voltage depending on the light reception intensity | strength of a diode), and the scanning timing to the z direction of a reaction container. The coexisting substances include NHS, Bil-F · L (about 4 mg / dL), Bil-F · H (about 20 mg / dL), Bil-C · L (about 4 mg / dL), Bil-C · H (about 20 mg / dL), chyle · L (about 280 turbidity), chyle · H (about 1400 turbidity), Hb · L (about 95 mg / dL), Hb · H (about 480 mg / dL) doing.

Table 13 shows the measurement results, and Table 14 corresponds to Table 2 and shows threshold values in the case of voltage conversion.
Table 13 shows the result of voltage integration. As can be seen from Table 13, there are many LEDs (green) and LEDs (blue) that do not exceed the threshold value. In fact, coexisting substances are contained in the sample and B / F separation processing is performed. In spite of the fact that the coexisting substances are included, there is a problem that the integrated value is equal to or lower than the threshold value and is to be discharged or discarded.

  Therefore, it is preferable to use LED (red) as the LED of the light source. Further, even if the LED (red) is used, it is impossible to determine whether Hb · L and Hb · H are coexisting substances with threshold values. Therefore, in addition to the first PD 85 (for visible light) of the photometry unit 63, a similar second PD is attached with a filter that transmits only red (it is detected visually). When it is detected from the measurement results of the two first and second PDs that the first PD is below the threshold value but the second PD is red, this is expressed as Hb · L, Hb · It is better to determine that it is a coexisting substance of H. By doing so, it is possible to prevent the specimen containing the coexisting substance from being removed as an insoluble matter. It can also be determined visually.

  FIG. 16 is a diagram showing the photometric results of a specimen containing a coexisting substance, where (a) is a red LED, (b) is a green LED, and (c) is a absorbance and reaction container when using a blue LED. It is a figure which shows the relationship with the scan timing to the z direction.

Table 15 shows the measurement results, and Table 16 corresponds to Table 4 and shows threshold values in the case of absorbance.
In many cases, LED (green) and LED (blue) exceed the threshold value, and the coexisting substance should actually be included in the specimen and the B / F separation process should be performed. Is included, the absorbance integrated value is equal to or higher than the threshold value, and is subject to discharge or disposal. Therefore, as in the case described above, it is preferable to use an LED (red) as the LED. Further, even if the LED (red) is used, it is impossible to determine whether Hb · L and Hb · H are coexisting substances with threshold values. Therefore, in addition to the first PD 85 (for visible light) of the photometry unit 63, a similar second PD is attached with a filter that transmits only red (it is detected visually). When it is detected from the measurement results of the two first and second PDs that the first PD is below the threshold value but the second PD is red, this is expressed as Hb · L, Hb · You may make it determine with the coexisting substance of H. In this way, even in the case of absorbance measurement, it is possible to prevent the specimen containing the coexisting substance from being removed as an insoluble matter.

[Example 5]
FIG. 17 is a diagram of photometric results using an immunoassay device (actual device), where (a) shows the case of using a red LED, NHS, + 0.5% BGG, + 1% BGG, + 2% BGG, +4. It is a table | surface which shows the sum (voltage integrated value) of the voltage during the scan of% BGG, + 6% BGG, + 8% BGG, + 10% BGG. (B) is a figure which shows the measurement result in the case of no precipitate and few to many, (c) is a figure which shows the measurement result of NHS and various specimens.

  Table 18 sets a threshold value of 20000 between precipitate 0 (NHS) and small cases. From the measurement results shown in Table 17, it can be determined that the case of + 1% BGG is the limit at which the nozzles are not clogged. Further, it can be seen that all the displayed samples from # 4 to # 82 do not exceed the threshold value and are samples that may cause nozzle clogging due to insoluble matter.

  FIG. 18 is a diagram of photometric results using an immunoassay device, where (a) is NHS, + 0.5% BGG, + 1% BGG, + 2% BGG, + 4% BGG, + 6% BGG, + 8% BGG, +10 It is a table | surface which shows the sum (absorbance integrated value) of the light absorbency during the scan of% BGG. (B) is a figure which shows the measurement result in the case of no precipitate and few to many, (c) is a figure which shows the measurement result of NHS and various specimens.

  Similarly, 400,000 is determined as the absorbance threshold between precipitates 0 and low (Table 20). Based on this value, as shown in Table 19, it can be determined that the case of + 1% BGG is the limit at which the nozzles are not clogged. Furthermore, all the displayed samples from # 4 to # 82 exceed the threshold value, and it can be seen that the samples cause nozzle clogging due to insoluble matter.

[Example 6]
FIG. 19 is a diagram showing a photometric result when a plasma / serum real sample is used as a model sample of a coexisting substance using an immunoassay device, (a) a model sample of the coexisting substance, and (b) a chyle. (C) is a diagram showing the relationship between the voltage of the light receiving unit (photodiode) and the scanning timing of the reaction vessel in the z direction for Hb plasma.

  If the threshold value is 20000 between the precipitate 0 (NHS) and one case in Table 18 obtained above, it is determined that all of Table 21 except Hb and H are not insoluble. Hb / H is originally a coexisting substance, but as described above, even if an LED (red) is used, Hb · L and Hb · H are threshold values and whether or not they are coexisting substances. Cannot be determined. Therefore, in addition to the first PD 85 (for visible light) of the photometry unit 63, a similar second PD is attached with a filter that transmits only red (it is detected visually). When it is detected from the measurement results of the two first and second PDs that the first PD is below the threshold value but the second PD is red, this is expressed as Hb · L, Hb · You may make it determine with the coexisting substance of H.

  FIG. 20 is a diagram showing a photometric result when a plasma / serum real sample is used as a model sample of a coexisting substance using an immunoassay device, and (a) a model sample of the coexisting substance using a red LED. (B) is a figure which shows the relationship between the light absorbency of a light-receiving part (photodiode) and the scanning timing to the z direction of a reaction container of chyle plasma, (c) is Hb plasma.

  In this case, using the threshold value of 400000 shown in Table 24 (corresponding to Table 20), all coexisting substances such as serum and plasma can be determined as substances without nozzle clogging.

[Example 7]
Next, some examples of the flow of processing for measuring a specimen using an actual apparatus will be described more specifically.

FIG. 21 is a flowchart showing the flow of the immunoassay method by the one-step method.
In this one-step method, an enzyme label, a sample (analyte), and magnetic particles are dispensed (step S1), stirring / incubation (37 ° C.) is performed (step S2), and then insoluble matter photometry (LED) is performed. -Select "No" (step S3). If insoluble matter photometry is performed and the photometric result is no insoluble matter, B / F separation cleaning operation (magnetization, reaction solution suction / washing solution dispensing, stirring) is performed. (Step S4). Next, the luminescent reagent 1 is added and stirred (step S5), the luminescent reagent 2 (substrate) is added (step S6), and the amount of luminescence is measured (step S7). Thereby, the possibility of nozzle clogging can be determined before the B / F separation process, and nozzle clogging can be prevented in advance.

[Example 8]
FIG. 22 is a flowchart showing the flow of an immunoassay method using the delayed one-step method. That is, an immune reaction solution, a sample (specimen), and magnetic particles are dispensed (step S11). Next, stirring / incubation (37 ° C.) is performed (step S12), an enzyme label is added (step S13), and stirring / incubation (37 ° C.) is performed (step S14). Next, it is selected whether or not to perform photometric processing (LED) of insoluble matter (step S15). If insoluble matter photometry is performed and the photometric result is insoluble, there is no B / F separation cleaning operation (magnetization, Reaction solution suction / washing solution dispensing and stirring are performed (step S16). Further, the luminescent reagent 1 is added and stirred (step S17), the luminescent reagent 2 (substrate) is added (step S18), and the amount of luminescence is measured (step S19). Thereby, the possibility of nozzle clogging can be determined before the B / F separation process, and nozzle clogging can be prevented in advance.

[Example 9]
FIG. 23 is a flowchart showing the flow of the immunoassay method by the two-step method (1). An immune reaction solution, a sample (specimen), and magnetic particles are dispensed (step S21), and stirring / incubation (37 ° C.) is performed (step S22). Next, it is selected whether or not to perform photometric processing (LED) of insoluble matter (step S23). If insoluble matter photometry is performed and the photometric result is insoluble, there is no B / F separation cleaning operation (magnetization, Reaction solution suction / washing solution dispensing and stirring are performed (step S24). An enzyme label diluent and an enzyme label are added (step S25). Further, stirring / incubation (37 ° C.) is performed (step S26), and B / F separation and cleaning operations (magnetization, reaction solution suction / washing solution dispensing, stirring) are performed (step S27). Next, the luminescent reagent 1 is added and stirred (step S28), and the luminescent reagent 2 (substrate) is used (step S29) to measure the amount of luminescence (step S30). Thereby, the possibility of nozzle clogging can be determined before the B / F separation process, and nozzle clogging can be prevented in advance.

[Example 10]
FIG. 24 is a flowchart showing the flow of the immunoassay method by the two-step method (2). First, the immune reaction solution 1 and the immune reaction solution 2 are dispensed (step S31), and after stirring, the sample (specimen) and magnetic particles are mixed (step S32). Next, stirring / incubation (37 ° C.) is performed (step S33). Next, it is selected whether or not to perform insoluble matter photometry (LED) (step S34). If insoluble matter photometry is performed and the photometric result is insoluble, there is no B / F separation cleaning operation (magnetization, Reaction solution suction / washing solution dispensing and stirring are performed (step S35). Next, the enzyme label diluent and the enzyme label are mixed (step S36), and stirring / incubation (37 ° C.) is performed (step S37). Next, a B / F separation and cleaning operation (magnetization, reaction liquid suction / washing liquid dispensing, stirring) is performed (step S38), and the luminescent reagent 1 is added and stirred (step S39). Then, using the luminescent reagent 2 (substrate) (step S40), the amount of luminescence is measured (step S41). Thereby, the possibility of nozzle clogging can be determined before the B / F separation process, and nozzle clogging can be prevented in advance.

[Example 11]
FIG. 25 is a flowchart showing the flow of the immunoassay method by the two-step method with preprocessing. This method is a two-step method including pre-processing automation.

  First, the pretreatment liquid and the sample (specimen) are used (step S51), and stirring / incubation (37 ° C.) is performed (step S52). Next, the immune reaction solution is mixed (step S53), stirred, magnetic particles are mixed (step S54), and stirring / incubation (37 ° C.) is performed (step S55). Next, it is selected whether or not to perform insoluble matter photometry processing (LED) (step S56). If insoluble matter photometry is performed and the photometric result is insoluble, there is no B / F separation cleaning operation (magnetization, Reaction solution suction / washing solution dispensing and stirring are performed (step S57). Next, stirring / incubation (37 ° C.) is performed using the enzyme-labeled diluted solution and the enzyme-labeled product (step S58) (step S59). Next, a B / F separation and cleaning operation (magnetization, reaction solution suction / washing solution dispensing, stirring) is performed (step S60). Further, the luminescent reagent 1 is mixed (step S61) and stirred, and the luminescent reagent 2 (substrate) is used (step S62) to measure the luminescence amount (step S63). Thereby, the possibility of nozzle clogging can be determined before the B / F separation process, and nozzle clogging can be prevented in advance.

[Example 12]
FIG. 26 is a flowchart showing the flow of the immunoassay method by the two-step method with pre-dilution.

  First, the sample diluent and the sample (sample) are dispensed (step S71) and stirred, and then the immune reaction solution and the diluted sample are mixed (step S72) and stirred. Next, magnetic particles are added (step S73), and stirring / incubation (37 ° C.) is performed (step S74). Next, it is selected whether or not to perform insoluble matter photometry (LED) (step S75). If insoluble matter photometry is performed and the photometric result is insoluble, there is no B / F separation cleaning operation (magnetization, Reaction solution suction / washing solution dispensing and stirring are performed (step S76). Next, the enzyme label diluent and the enzyme label are mixed (step S77), and stirring / incubation (37 ° C.) is performed (step S78). Further, a B / F separation and washing operation (magnetization, reaction liquid suction / washing liquid dispensing, stirring) is performed (step S79), and the luminescent reagent 1 is mixed and stirred (step S80). Using the luminescent reagent 2 (substrate) (step S81), the amount of luminescence is measured (step S82). Thereby, the possibility of nozzle clogging can be determined before the B / F separation process, and nozzle clogging can be prevented in advance.

  As described above, according to the immunoassay device according to the present embodiment, insoluble materials can be detected non-destructively in various methods without depending on the presence or absence of pretreatment, the number of reagent items, and the like. In order to perform the B / F process after performing, in the immunoassay apparatus including the B / F separation, the nozzle clogging caused by the insoluble matter in the B / F separation treatment is prevented, and the B / F caused by the insoluble matter is prevented. The incomplete separation process can be suppressed, and the cause of medical errors due to false positive or false negative measurement results such as the concentration of the target substance can be reduced.

  In the above-described embodiment, the configuration and the like illustrated in the accompanying drawings are not limited to these, and can be appropriately changed within a range in which the effect of the present invention is exhibited. In addition, various modifications can be made without departing from the scope of the object of the present invention.

  The present invention can be used for immunoassay techniques.

DESCRIPTION OF SYMBOLS 1 ... Reaction container, 3 ... Reaction liquid, 5 ... Magnet, 7 ... Suction nozzle, 11 ... Discharge part, 21 ... Item reagent cold storage, 23 ... Sample dispensing mechanism, 25 ... Sample conveyance part, 27 ... Alkaline B / F cleaning solution,
31 ... Substrate cooler, 33 ... Waste container, 35 ... Main door, 37 ... Auto cell feeder, 39 ... Incubation table, 41 ... B / F separation mechanism, 45 ... Detection unit, 57 ... Reaction container transport unit, 61 ... Reagent Nozzle cleaning tank, 63 ... Insoluble matter detection part (photometry part), 63a ... Cavity, 64 ... Disposal part, 65 ... B / F separation part, 81 ... LED, 85 ... PD, 91 ... Pretreatment part, 95 ... Insoluble matter Determination unit 96... Threshold value storage unit 97 97 detection unit 98.

Claims (12)

The antibody-bound carrier in the reaction solution or the antigen or antibody reacted with the antigen-bound carrier and the unreacted free antigen or free antibody are washed and separated by sucking the unreacted free antigen or free antibody with a suction part (nozzle). An immunoassay device comprising a B / F separation unit
A photometric unit comprising a light emitting unit for irradiating the reaction vessel with light and a light receiving unit for measuring the transmitted light of the reaction vessel, upstream of the B / F separation unit;
Based on the received light intensity in the light receiving unit, an insoluble matter determination unit that determines the presence or absence of insoluble matter that causes clogging of the suction unit;
An immunoassay device comprising: Furthermore, a threshold storage unit for storing a threshold for determining the presence or absence of insoluble matter is provided,
The immunoassay apparatus according to claim 1, wherein the insoluble matter determination unit determines the presence or absence of an insoluble material based on a magnitude relationship between a light reception intensity of the light receiving unit and the threshold value.   The immunoassay device according to claim 1, wherein the carrier is a magnetic particle.   The immunoassay device according to any one of claims 1 to 3, wherein an LED is used as a light source of the light emitting unit.   The immunoassay device according to claim 4, wherein a red LED is used as the LED.   The light receiving unit is a first light receiving element including red in a detection wavelength band and a second light receiving element including red in a detection wavelength band, and a filter unit for filtering other than red is provided on the light receiving surface side. The immunoassay device according to claim 5, wherein the insoluble matter determination unit determines an insoluble matter based on a difference in received light intensity with respect to the light receiving element.   The threshold value includes a first light-receiving intensity in a reaction vessel containing a model sample having a precipitate approximate to the opening diameter of the suction part as a model sample, and a second light intensity in a reaction vessel not containing a model sample. The immunoassay device according to any one of claims 1 to 6, wherein the immunoassay device is determined as a value between the received light intensity.   The insoluble matter determination unit that determines the presence or absence of an insoluble matter that causes clogging of the suction unit determines the presence or absence of an insoluble matter based on an integrated value of parameters that depend on the light receiving intensity in the light receiving unit. The immunoassay device according to any one of claims 1 to 7.   The said photometry part is arrange | positioned in the position where the said light emission part and the said light-receiving part oppose, and has an operation part which scans the said reaction container to a perpendicular direction, Any one of Claim 1-8 characterized by the above-mentioned. The immunoassay device according to claim 1.   The immunoassay device according to claim 9, further comprising a discarding unit that discharges or discards the reaction container determined by the insoluble matter determination unit to be insoluble in the vicinity of the photometry unit. This is an immunoassay method including a B / F separation process in which an antigen or an antibody reacted with an antibody or an antigen-binding carrier in a reaction container or an unreacted free antigen or an antibody is washed and separated by aspiration (nozzle). And
As a pre-step of the B / F separation step, a photometric step comprising a light emitting unit for irradiating the reaction vessel with light and a light receiving unit for measuring the transmitted light of the reaction vessel;
Insoluble matter determination step for determining the presence or absence of insoluble matter that causes clogging of the suction portion based on the light receiving intensity in the light receiving portion;
An immunoassay characterized by comprising:   A program for causing a computer to execute the immunoassay method according to claim 11.

Images