Patent Application
20140178908
The present invention relates to a competitive immunoassay and an immunoassay apparatus.
Conventionally, the competitive immunoassay has been known as a procedure for measuring the concentration of small amounts of antigens and antibodies contained in a sample.
For example, a procedure described in Patent Document 1 is known as a procedure for extending a measurable concentration range in the competitive immunoassay and the immunoassay apparatus. In the procedure described in Japanese Patent Application Laid-Open (JP-A) No. 2011-80975, a plurality of calibration curves of different concentrations of a molecular recognition reagent are previously created, the calibration curve to be used for concentration conversion is selected depending on the absorbance of a measurement target specimen. Thus, in the procedure described in Patent Document 1, a measurable concentration range is extended by previously creating the plurality of calibration curves of different concentration ranges.
However, in the procedure of Patent Document 1, it is necessary to create the calibration curves of mutually different concentrations of the molecular recognition reagent. Accordingly, there are problems such that the usage of a calibrator necessary for creating calibration curves and the amount of the reagent are increased, and further the user's work rate necessary for creating calibration curves is increased. Furthermore, it is necessary to design a measurement device corresponding to the step of preparing a sample having a plurality of concentrations. Therefore, a problem such that the configuration of the measurement device is complicated is caused.
The scope of the present invention is defined solely by the appended claims, and is not affected to any degree by the statements within this summary.
A first aspect of the present invention is a method of competitive immunoassay. The method comprises: creating a first calibration curve created for a first reaction time period and a second calibration curve created for a second reaction time period longer than the first reaction time period; preparing a measurement sample containing a target substance in a specimen, a reactant, and a competitive substance; obtaining a reference information related to the concentration of the target substance after the first reaction time period since the preparation of the measurement sample; first outputting the concentration obtained based on the first calibration curve as a measurement result in the case where the reference information shows that the concentration of the target substance is less than a predetermined threshold; and second outputting the concentration obtained based on the second calibration curve as the measurement result in the case where the reference information obtained in the obtaining is information showing that the concentration of the target substance is more than or equal to the threshold.
A second aspect of the present invention is an apparatus for competitive immunoassay. The apparatus comprises: a sample preparation unit for preparing a measurement sample containing a target substance in a specimen, a reactant, and a competitive substance; a detection unit; and a control unit. The control unit: stores a first calibration curve created for a first reaction time period and a second calibration curve created for a second reaction time period longer than the first reaction time period; obtains predetermined reference information related to the concentration of the target substance after the first reaction time period since the preparation of the measurement sample based on the output of the detection unit; outputs the concentration obtained based on the first calibration curve as the measurement result in the case where the obtained reference information shows that the concentration of the target substance is less than a predetermined threshold; and outputs the concentration obtained based on the second calibration curve as the measurement result in the case where the reference information shows that the concentration of the target substance is more than or equal to the threshold.
A third aspect of the present invention is an apparatus for competitive immunoassay. The apparatus comprises: a sample preparation unit configured to prepare a measurement sample containing a target substance in a specimen, a reactant, and a competitive substance; a detection unit; and a control unit. The control unit: stores a first calibration curve created for a first reaction time period and a second calibration curve created for a second reaction time period longer than the first reaction time period; obtains the concentration of the target substance after the first reaction time period since the preparation of the measurement sample based on the output of the detection unit; outputs the concentration obtained based on the first calibration curve as the measurement result in the case where the obtained concentration shows that the concentration of a target substance is less than a predetermined threshold; and outputs the concentration obtained based on the second calibration curve as the measurement result in the case where the obtained concentration shows that the concentration of the target substance is more than or equal to the threshold.
A cover 1a and a display input unit 2 composed of a touch panel are disposed on the front surface of the biological sample analyzer 1. A control unit 3 for controlling each unit is disposed in a space at the right side in the biological sample analyzer 1, and a measurement unit 4 for detecting a signal from a measurement sample is disposed in a space at the lower left side in the biological sample analyzer 1. Further, a sample preparation unit 5 for preparing a measurement sample is disposed in the remaining space in the biological sample analyzer 1.
The sample preparation unit 5 includes a specimen setting unit 51, a standard sample setting unit 52, a reagent setting unit 53, a dispensing device 54, a reaction unit 55, and a liquid feeding device 56. A user opens the cover 1a to set various containers to the specimen setting unit 51, the standard sample setting unit 52, and the reagent setting unit 53.
A container for housing a specimen containing a target substance is set in the specimen setting unit 51. A container for housing a standard sample (calibrator) which contains a target substance and has a known concentration is set in the standard sample setting unit 52. In this embodiment, ten standard samples (1) to (10) having different concentrations of the target substance are used. 10 containers for housing the standard samples are set in the standard sample setting unit 52. A container for housing a reaction buffer solution containing a reactant and a container for housing a reagent containing a competitive substance (hereinafter, referred to as “competitive reagent”) are set in the reagent setting unit 53. An empty cuvette is set in the reaction unit 55.
In this embodiment, the target substance is an antigen. Specifically, it is thyroxine (T4) which is a type of thyroid hormone. The standard samples (1) to (10) are prepared by adding thyroxine (manufactured by Nacalai Tesque, Inc.) to normal human serum from which triiodothyronine (T3) and thyroxine (T4) are removed (manufactured by CC Biotech Corporation). The concentration of thyroxine in the standard samples (1) to (10) is set as shown in Table 1 below.
Further, the reactant is an antibody. Specifically, it is an anti-thyroxine antibody. The reaction buffer solution is prepared by adjusting the pH of 16 mg/mL of 3,3-dimethyl glutaric acid, 1 mg/mL of 2-amino-2-methyl-1,3-propanediol, 12 mg/mL of tris, 1.5% (w/v) BSA, 1.5% (w/v) PVA105 (manufactured by Kuraray Co., Ltd.), 0.58% (w/v) NaCl, 0.016% (w/v) NaN3, 0.2 mg/mL of 8-anilino-1-naphthalenesulfonic acid ammonium, and 0.5 μg/mL of anti-thyroxine antibody (manufactured by Medix Biochemica) to 7.1.
Further, the competitive substance is a labeled antigen. Specifically, it is a triiodothyronine-sensitized latex produced by sensitizing triiodothyronine (T3) to polystyrene latex particles. The competitive reagent is prepared by sensitizing triiodothyronine (BSA-X-T3; manufactured by Michigan Diagnostics) to polystyrene latex particles having a diameter of 0.8 μm to produce a triiodothyronine-sensitized latex, adding 25 mM of an MOPSO buffer (MOPSO (manufactured by Dojindo Laboratories), 30 mM of NaCl, 0.1% (w/v) NaN3, 6% (w/v) sucrose, and 2% (w/v) BSA, pH 7.1) to the produced triiodothyronine-sensitized latex so as to be 1% (w/v).
Subsequently, the dispensing device 54 aspirates and discharges a predetermined amount of liquid from the tip thereof and is configured to be able to move in side-to-side, up-and-down, and back-and-forth directions by a driving device (not shown). The dispensing device 54 appropriately dispenses a specimen, a standard sample, a reaction buffer solution, and a competitive reagent into the cuvette of the reaction unit 55.
The reaction unit 55 includes a temperature regulating mechanism (not shown) for maintaining a constant temperature of the solution in the cuvette and a stirring mechanism (not shown) for stirring the solution in the cuvette. The specimen, the reaction buffer solution, and the competitive reagent are mixed in the cuvette set in the reaction unit 55 to prepare a measurement sample. Before the specimen measurement, the standard sample, the reaction buffer solution, and the competitive reagent are mixed in the cuvette set in the reaction unit 55 to prepare a measurement sample for creating calibration curves.
If the reaction buffer solution containing the reactant (antibody) is mixed with the competitive reagent containing the competitive substance (labeled antigen), the reactant and the competitive substance are aggregated by the antigen-antibody reaction. If the specimen or the standard sample, the reaction buffer solution and the competitive reagent are mixed, the target substance (antigen) in the specimen or the standard sample and the competitive substance (labeled antigen) in the competitive reagent are competitively reacted with the reactant (antibody) in the reaction buffer solution. That is, the biological sample analyzer 1 allows the antigen-antibody reaction to be competitively caused by the competitive assay based on the latex agglutination. The measurement unit 4 detects aggregates composed of the reactant and the competitive substance.
The liquid feeding device 56 is composed of an aspiration tube 56a for aspirating a measurement sample, a liquid feeding tube 56b which feeds the measurement sample aspirated from the aspiration tube 56a to the measurement unit 4, and a pump 56c which aspirates the measurement sample and feeds it to the measurement unit 4. The liquid feeding device 56 is configured to be able to move in side-to-side, up-and-down, and back-and-forth directions by a driving device (not shown). A predetermined amount of the measurement sample in the cuvette set in the reaction unit 55 is fed to the measurement unit 4 by the liquid feeding device 56.
The measurement unit 4 includes a flow cell 41, a laser light source 42, a condenser lens 43, a light collecting lens 44, a pinhole 45, and a photo diode 46. The flow cell 41 passes the measurement sample prepared by the sample preparation unit 5 in a state of being enclosed in a sheath liquid, and as shown in
Laser light emitted from the laser light source 42 passes through the condenser lens 43 and is delivered to the pore portion 41d of the flow cell 41. Thus, the measurement sample passing through the inside of the pore portion 41d is irradiated with the laser light. The light collecting lens 44 focuses forward scattered light from each of the particles in the measurement sample irradiated with the laser light. The photo diode 46 receives the forward scattered light passed through the pinhole 45, performs photoelectric conversion of the received forward scattered light, and produces a forward scattered light signal. The produced forward scattered light signal is sent to the control unit 3.
Here, if aggregates (agglomerated particles) produced by the antigen-antibody reaction of the reactant (antibody) and the competitive substance (labeled antigen) are compared with other particles not aggregated (single particles), the agglomerated particles are larger than the single particles. Thus, the control unit 3 can determine whether particles passed through the pore portion 41d of the flow cell 41 are agglomerated or single particles based on the magnitude of the forward scattered light signal. The control unit 3 can separately calculate single particles and agglomerated particles based on the received forward scattered light signal so that the aggregation degree can be determined. As the aggregation degree in this embodiment, a value of P/T calculated based on the number of single particles (M), the number of agglomerated particles (P), and the total particle number (T) (the total of M and P) is used.
The control unit 3 includes a microcomputer having a storage device such as CPU, ROM or RAM and a circuit which processes various signals. Thus, the control unit 3 has functions of a memory unit 31, an analysis unit 32, and an operation control unit 33.
The memory unit 31 stores an analysis program which analyzes the forward scattered light signal and a control program which controls the operation of each of the devices. The memory unit 31 stores data of the received forward scattered light signal, the step results by the analysis program, data of calibration curves, and various set contents. The analysis unit 32 analyzes the forward scattered light signal based on the analysis program and calculates the concentration of the target substance contained in the measurement sample. The calculation results by the analysis unit 32 are output to the display input unit 2. The operation control unit 33 controls the operation of the units of the devices based on the control program stored in the memory unit 31.
With reference to
With reference to
With reference to
Thus, the aggregation degree of the antibody s2 and the competitive substance s3 changes depending on the concentration of the antigen s1 in the sample. The aggregation of the antibody s2 and the competitive substance s3 progresses according to the reaction time.
In the specimen measurement, a calibration curve is first created using the standard samples (1) to (10). Here, the reaction buffer solution is mixed with each of the standard samples having a predetermined concentration. Further, the competitive reagent is mixed. The sample thus prepared is measured by the measurement unit 4 at the timing after a predetermined reaction time.
As described with reference to
In the specimen measurement, the reaction buffer solution is mixed with the specimen, and further the competitive reagent is mixed. When the predetermined reaction time is passed, the concentration of the antigen s1 contained in the specimen is obtained on the basis of the aggregation degree calculated based on the forward scattered light from the specimen and the previously created calibration curve corresponding to the predetermined reaction time. For example, in
However, the slope (gradient) of the created calibration curve is generally large near the center, but it is small near both ends as shown in
Hereinafter, a procedure for creating two calibration curves, using the calibration curves properly, and obtaining the concentration of the target substance with high accuracy will be described.
The control unit 3 first dispenses a predetermined amount (10 μL in this embodiment) of one of the standard samples (1) to (10) into an empty cuvette set in the reaction unit 55 (S101). Subsequently, the control unit 3 dispenses a predetermined amount (80 μL in this embodiment) of the reaction buffer solution into the cuvette (S102), and starts to count the reaction time after dispensing the reaction buffer solution (S103). Subsequently, the control unit 3 feeds a predetermined amount (10 μL in this embodiment) of the competitive reagent to the cuvette when the reaction time reaches a predetermined time (50 seconds in this embodiment) (S104). Then, the control unit 3 heats the cuvette to a predetermined temperature (45° C. in this embodiment) by the reaction unit 55 and waits the step until the reaction time reaches t1 (320 seconds in this embodiment) (S105).
When the reaction time reaches t1 (S105: YES), the control unit 3 feeds only a predetermined amount of the measurement sample in the cuvette to the measurement unit 4, executes the measurement by the measurement unit 4, and calculates an aggregation degree v1 of the measurement sample 5 times (S106). Subsequently, the control unit 3 waits the step until the reaction time reaches t2 (1370 seconds in this embodiment) (S107). When the reaction time reaches t2 (S107: YES), the control unit 3 feeds only a predetermined amount of the measurement sample in the cuvette to the measurement unit 4, executes the measurement by the measurement unit 4, and calculates an aggregation degree v2 of the measurement sample 5 times (S108).
Subsequently, the control unit 3 averages the five aggregation degrees v1 of the standard sample to calculate an aggregation degree a1 as shown in
When the calculation of the aggregation degrees of all the standard samples is completed (S110: YES), the control unit 3 creates a calibration curve T1 as shown in
Subsequently, the control unit 3 calculates a threshold As related to the aggregation degree for determining which the calibration curves T1 and T2 are used in order to obtain the concentration of a specimen in the step of measuring a specimen to be described below (S113). The procedure for calculating the threshold As will be described later with reference to
The control unit 3 first dispenses a predetermined amount (10 μL in this embodiment) of the specimen into an empty cuvette set in the reaction unit 55 (S201). Subsequently, the control unit 3 dispenses a predetermined amount (80 μL in this embodiment) of the reaction buffer solution into the cuvette (S202), and starts to count the reaction time after dispensing the reaction buffer solution (S203). Subsequently, the control unit 3 feeds a predetermined amount (10 μL in this embodiment) of the competitive reagent to the cuvette when the reaction time reaches a predetermined time (50 seconds in this embodiment) (S204). Then, the control unit 3 heats the cuvette to a predetermined temperature (45° C. in this embodiment) by the reaction unit 55 and waits the step until the reaction time reaches t1 (S205). When the reaction time reaches t1 (S205: YES), the control unit 3 feeds only a predetermined amount of the measurement sample in the cuvette to the measurement unit 4, executes the measurement by the measurement unit 4, and calculates an aggregation degree A1 of the measurement sample (S206).
Subsequently, the control unit 3 determines whether the calibration curve T1 created previously is used (S207). Specifically, as shown in
When the aggregation degree A1 is larger than the threshold As (S207: YES), as shown in
On the other hand, when the aggregation degree A1 is less than or equal to the threshold As (S207: NO), the control unit 3 determines whether the biological sample analyzer 1 continues the measurement based on the set contents stored in the memory unit 31 (S211). The set contents are previously set by a user via a setting screen 21. The step of storing the set contents and the setting screen 21 will be described later with reference to
When it is set so that the biological sample analyzer 1 does not continue the measurement (S211: NO), steps S208 to S210 described above are performed. On the other hand, when it is set so that the biological sample analyzer 1 continues the measurement (S211: YES), the control unit 3 waits the step until the reaction time reaches t2 (S212). When the reaction time reaches t2 (S212: YES), the control unit 3 feeds only a predetermined amount of the measurement sample in the cuvette to the measurement unit 4, executes the measurement by the measurement unit 4, and calculates the aggregation degree A2 of the measurement sample as shown in
Subsequently, the control unit 3 obtains the concentration C2 from the aggregation degree A2 based on the calibration curve T2 as shown in
Thus, the slope (gradient) of the calibration curve T2 corresponding to the aggregation degree A2 is larger than the slope (gradient) of the calibration curve T1 corresponding to the aggregation degree A1. Thus, as shown in
With reference to
When the user pushes the OK button 213 (S303: YES), the control unit 3 stores the set contents in the memory unit 31 depending on the radio button selected from the radio buttons 211 and 212 (S305). That is, when the OK button 213 is pushed in a state where the radio button 211 (continue) is selected, it is set that the biological sample analyzer 1 performs the measurement not using the calibration curve T1 but using the calibration curve T2 (executes S212 to S216) in the case where the aggregation degree A1 calculated in S206 of
When the user pushes the cancel button 214 (S304: YES), the control unit 3 discards the state of the radio buttons 211 and 212 and closes the setting screen 21 (S306). The set contents set by the user via the setting screen 21 are read from the memory unit 31 by the control unit 3 in S211 of
Subsequently, in the case where a plurality of calibration curves are created, the range of the aggregation degree suitable for using the calibration curves (the range of concentration) is examined. The result will be described.
In
Table 2 below shows five coefficients of variation (CV) corresponding to five aggregation degrees calculated when determining points on the calibration curves T11 to T15 of
In this case, the coefficient of variation (CV) means variations in five concentrations c taken from five aggregation degrees v obtained at the time of creating of the calibration curve (for example, v1 and v2 obtained in S106 and S108 of
According to Table 2 above, the case where the coefficient of variation (CV) becomes 10% or less is as follows: In the case where the reaction time is 170 seconds (the calibration curve T11), the concentration is from 1 to 10 μg/dL. In the case where the reaction time is 320 seconds (the calibration curve T12), the concentration is from 2.5 to 10 μg/dL. In the case where the reaction time is 620 seconds (the calibration curve T13), the concentration is from 2.5 to 25 μg/dL. In the case where the reaction time is 870 seconds (the calibration curve T14), the concentration is from 2.5 to 25 μg/dL. In the case where the reaction time is 1370 seconds (the calibration curve T15), the concentration is from 5 to 50 μg/dL. That is, the reaction time capable of calculating the concentration when the coefficient of variation (CV) is 10% or less is shorter as the concentration is low, and is longer as the concentration is high.
In this embodiment, in the case where a plurality of calibration curves are created, the range of the aggregation degree suitable for using the calibration curves (the range of concentration) is set based on the range in which the coefficient of variation (CV) is 10% or less. For example, as shown in
Then, for example, the threshold Cs related to the concentration is set within a range R3 in which the ranges R1 and R2 are overlapped. If the concentration obtained based on the calibration curve T12 is less than or equal to Cs, the concentration is obtained based on the calibration curve T12. If the concentration obtained based on the calibration curve T12 is larger than Cs, the concentration is obtained using not the calibration curve T12, but the calibration curve T15. In S113 of
As described above, according to this embodiment, the calibration curves T1 and T2 can be created by changing the reaction time from the same standard samples (1) to (10). Accordingly, the amounts of the standard sample, the reaction buffer solution, and the competitive reagent, which are necessary for creating the calibration curves, can be reduced, and the operation of the user which is necessary for creating the calibration curves, can be reduced.
According to this embodiment, the aggregation degree A1 obtained when the reaction time is t1 in the specimen measurement is larger than the threshold As, the concentration C1 is obtained using the calibration curve T1. On the other hand, if the aggregation degree A1 is less than or equal to the threshold As, the concentration C2 is obtained using the calibration curve T2 in the case of continuing the measurement. Thus, the calibration curves T1 and T2 created previously can be used properly so that the accuracy of the concentration to be obtained becomes high.
According to this embodiment, the user can set whether the biological sample analyzer 1 continues the measurement in the case of low aggregation degree (high concentration) via the setting screen 21 shown in
The embodiments of the present invention have been described above. However, the embodiments of the present invention are not limited thereto.
For example, in the above embodiments, the target substance has been described as thyroxine (T4) which is a type of thyroid hormone. However, it is not limited thereto, and it may be other antigens. Further, the target substance may be not only the antigens but also antibodies.
In the above embodiments, polystyrene latex particles are used when preparing the competitive reagent. However, the particles are not particularly limited as long as they are particles used for immunoassay. Examples thereof include magnetic particles, polymer particles, metal oxide particles, glass particles, erythrocytes, and gelatin particles. As the polymer particles, latex particles are preferred.
Although the latex agglutination is used in the above embodiment, the ELISA method may be used.
In the above embodiments, the aggregation degree is calculated based on the forward scattered light signal obtained by the measurement unit 4 based on flow cytometry, and the concentration of the target substance is obtained based on the aggregation degree. However, the present invention is not limited thereto. The concentration of the target substance may be obtained based on the turbidity of the measurement sample obtained by a light source and a photodetector in the measurement unit 4. Further, other information may be used as long as the aggregation degree and the concentration of the target substance can be found.
In the above embodiments, two calibration curves T1 and T2 are created in the step of creating calibration curves of
In the above embodiments, two calibration curves T1 and T2 are created and then the step of measuring a specimen of
Further, it is not necessary to always perform the step of creating calibration curves of
In the above embodiments, when the aggregation degree A1 is less than or equal to the threshold As in the step of measuring a specimen of
In the above embodiments, it is determined which the calibration curves T1 and T2 are used in order to obtain the concentration based on the information about whether the aggregation degree A1 obtained in S206 of
When the aggregation degree A1 is calculated with reference to
In the above embodiments, when it is not set so that the biological sample analyzer 1 continues the measurement in S211 of
With reference to
In the case where the biological sample analyzer 1 is not set to continue the measurement (S211: NO), it suffices that the control unit 3 obtains the concentration C1 from the aggregation degree A1 based on the calibration curve T1, terminates the measurement (S221, S222) as shown in
In addition, the embodiments of the present invention can be appropriately modified in various ways within the scope of the technical spirit shown in the claims.
| Number | Date | Country | Kind |
|---|---|---|---|
| 2012-279949 | Dec 2012 | JP | national |
