JPH03188356A - Liquid-sample analyzer and calibrating liquid - Google Patents

Abstract

PURPOSE:To correct the error caused by dilution by using two kinds of raw liquids of standard solutions having the compositions close to a sample, and preparing the calibrating liquids in multiple stages. CONSTITUTION:First raw liquid of standard solution 52 and second raw liquid of standard solution 54 are prepared for each item. Then, the number of the raw liquids of standard solutions is (item-number X 2) even if calibrating liquids for multiple stages are prepared for each item. The first raw liquid of standard solution 52 is commonly used for each item, and the second raw liquid of standard solution 54 is prepared for each item. Then, the number of the raw liquids of standard solutions to be prepared is (item-number + 1) even if the calibrating liquids in multiple stages are prepared. When the concentration of the calibrating liquid which is obtained by mixing the first and second raw liquids of standard solutions 52 and 54 is made to be an effective value, a matrix effect can be incorporated in the effective value.

Description

Detailed Description of the Invention (Field of Industrial Application) The present invention relates to a liquid sample analyzer that samples and measures a liquid sample and quantifies its components based on a calibration curve prepared in advance. The present invention relates to a liquid sample analyzer suitable for use in immunoassays such as enzyme immunoassays, and a calibration solution suitable for use in such a liquid sample analyzer.

(Prior Art) Since the calibration curve in EIA is curved, standard solutions for calibration at many concentration levels, usually 5 to 10 points, are required for one measurement item. In addition, recently there has been a strong demand for simultaneous measurement of multiple items in EIA, but if separate calibration liquids were prepared for each concentration of all measurement items, the number of calibration liquids alone would be several tens, which is not practical. It will no longer become.

Therefore, as a countermeasure to reduce the number of calibration solutions, there is a method to reduce the number of calibrations by stabilizing the calibration curve.

A known method is to assume that the curve of the calibration curve is the same each time and to measure only representative points on the calibration curve. but,
If the shape of the calibration curve varies, it is necessary to actually perform measurements at the required calibration points.

(Problem to be Solved by the Invention) Another method for reducing the number of calibration solutions is to use a highly concentrated standard solution as a stock solution, change the sample dispensing amount in several steps, and as a result change the dilution rate, and use a high concentration standard solution as the stock solution. Methods of preparing step-by-step calibration solutions are being considered. However, although this method is applicable to ordinary biochemical analysis where the specimen is significantly diluted with reagents, it is inconvenient for use in EIA, where the amounts of specimen and reagent are often about the same. In other words, this is because errors occur due to matrix effects due to differences in the properties of the standard solution and the sample.

SUMMARY OF THE INVENTION Therefore, an object of the present invention is to provide a liquid sample analyzer that is capable of automatically adjusting multi-stage calibration solutions and creating a calibration curve while reducing the number of standard solutions to be prepared. It is.

The present invention also includes: The object of the present invention is to provide a calibration solution convenient for creating a calibration curve with such a liquid sample analyzer.

(Means for Solving the Problems) In the present invention, two types of standard solution stock solutions having compositions close to those of the specimen are used to prepare a multi-stage calibration solution.

One type of standard solution stock solution is the first standard solution stock solution in which the component to be detected is relatively low concentration, and the other one type of standard solution stock solution is the first standard solution stock solution in which the component to be detected is much higher than the first standard solution stock solution. This is a second standard solution stock solution prepared for each item by concentration. If the specimen is serum, for example, the first and second standard solution stock solutions should all have a composition close to that of serum. The first standard solution stock solution is prepared for each item or commonly for each item, and the second standard solution stock solution is prepared for each item. A multi-stage calibration solution is created by mixing the first standard solution stock solution and the second standard solution stock solution at different ratios for each item.

A preferred calibration solution is one in which the calibration value of a multi-stage calibration solution, which is a mixture of the first standard solution stock solution and the second standard solution stock solution, is not a value calculated simply from the mixing ratio, but is prepared in advance using a large number of calibration solutions. Display it as the measured effective value.

FIG. 1 shows a liquid sample analyzer according to the present invention.

52 is the first standard solution stock solution, 54 is the second standard solution stock solution, 5
0 is a sampling means having a function of sampling the first standard solution stock solution 52 and the second standard solution stock solution 54 at a predetermined ratio into a reaction tube to create calibration points at multiple concentration levels.

Reference numeral 56 denotes a measurement section; 50 indicates a calibration curve creation means for creating a calibration curve from the measurement results from the measurement section 56 obtained by sampling; and 60 receives measurement data about the specimen from the measurement section 56, and the calibration curve creation means 58 This is a data processing means that performs data processing using the created calibration curve.

A preferred calibration solution of the present invention is composed of a first standard solution stock solution and a second standard solution stock solution, and displays the predicted effective value when both standard solution stock solutions are mixed at a specified mixing ratio. It is something that exists.

In a preferred embodiment, the first standard solution stock solution is commonly used for each item.

(Function) If you prepare the first standard solution stock solution and the second standard solution stock solution for each item, even if you create multi-stage calibration solutions for each item,
The number of standard solution stock solutions to be prepared is (number of items x 2).

The first standard solution stock solution is commonly used for each item, and the second
Prepare stock solutions of standard solutions for each item.

Even if multi-stage calibration solutions are prepared for each item, the number of standard solution stock solutions to be prepared is (number of items + 1).

If the concentration of the calibration solution obtained by mixing the first standard solution stock solution and the second standard solution stock solution is an effective value rather than a calculated value, the matrix effect can be included in the effective value. This makes the calibration curve more accurate, for example when the sample is serum.

Instead of serum, the first standard solution stock solution can be replaced with a substance with a simpler composition, such as a buffer solution or distilled water.

(Example) An example of creating seven levels of calibration solutions for a certain measurement item will be explained. Both the first standard solution stock solution and the second standard solution stock solution are composed of the same components as the serum, which is the liquid to be measured. The first standard solution stock solution contains 2 ng/mQ of serum components, and this is a serum that contains almost no components (analytes) to be detected, and this component can be considered to be a so-called negative serum. can. Serum that is negative for all items can be used as a common first standard solution stock solution for each item. Note that the concentration of each component in the first standard solution stock solution does not need to be O, it is sufficient that the concentration of each component is known and is sufficiently low.The second standard solution stock solution has a serum concentration of 400 ng/m12. It is a stock solution containing the ingredients.

The calibration solution, which is a mixture of both standard solution stock solutions, also has a component similar to serum, and matrix errors caused by differences in the components of the calibration solution, such as when diluting with distilled water, can be suppressed. Multi-stage, for example, seven-stage calibration solutions are automatically created from these two standard solution stock solutions. The mixing ratio is shown in Table 1. *
The total amount of calibration solution after a is 100 μQ.

Even if we prepared the first standard solution stock solution and the second standard solution stock solution for each item in Table 1, if we were to conduct simultaneous analysis of 6 items, we would only need to prepare 12 types of standard solution stock solutions. Become.

If standard solutions were prepared for each calibration point as in the past, 42 standard solutions would be required.

To further reduce the number of standard solutions, if the first standard solution stock solution is used as the standard solution stock solution common to all items, it is possible to
In the example of the items, if seven calibration points are created for each item, the types of standard solution stock solutions can be reduced to seven types in total.

FIG. 2 shows a flowchart of the operation for creating a calibration curve for one item.

The sampling amount of each of the first standard solution stock solution and the second standard solution stock solution and the sampling amount of the reagent are stored. For each calibration point, the first standard solution stock solution and the second standard solution stock solution are sampled and reagents are added. Absorbance is measured for each calibration point and a calibration curve is created.

As a result, a calibration curve as shown in FIG. 3 is obtained.

Calibration curves are created in the same manner for two or more measurement items.

Table 2 shows an example of using an effective value instead of a calculated value as the concentration of a mixed solution.The adjusted concentration is a calculated value based on the mixing ratio, and the effective value is an actual value measured using a reliable device. . In this case, the matrix effect can be included in the effective value.

An example of a liquid sample analyzer to which the present invention is applied is shown in FIGS. 4 to 8.

2 is a reaction line consisting of a snake chain, 24
0 reaction tube holders are connected in series. Each reaction tube holder can hold one reaction tube 4.
The reaction tube 4 is a disposable one made of plastic, and its size is similar to that of a hole in a microplate, with an inner diameter of 8 mm and a depth of 12 mm. The reaction line 2 is assumed to move one step in the direction of the arrow every 15 seconds, and completes one cycle in 60 minutes.

Reference numeral 6 denotes a reaction tube supply device, which sequentially supplies reaction tubes 4 of selected measurement items to the reaction line 2 one by one. The starting point is the position where the reaction tube 4 is supplied. The sample dispensing section 8 is placed at a position 0.75 minutes from the start point along the direction of movement of the reaction line 2, and the reagent dispensing section 1 is placed at a position 1.5 minutes later.
0 is placed, and the cleaning mechanism 12 is placed at a position 44.5 minutes later.

44. The substrate liquid dispensing unit 14 is placed at a position after 75 minutes,
The absorbance measurement unit 16 is placed at a position after 58.75 minutes,
A discharge/cleaning section 18 for discharging or cleaning residual liquid is disposed at a position after 59.25 minutes, and a reaction tube removal mechanism 20 is disposed at a position after 59.75 minutes.

Reaction tube supply bag! ! The processing operation parts other than 6 use mechanisms already included in commercially available EIA automatic analyzers and biochemical analyzers. As the sample dispensing unit 8, a turntable type one is shown in the embodiment, and a sample cup 8b is arranged along the circumference of the turntable 8a, and the sample is distributed into the reaction tubes 4 by a pipetter 8c. be noted.

9 is a first standard solution stock solution, which is dispensed into the reaction tube 4 by a pipetter 8c. Since the first standard solution stock solution 9 is commonly used for all items, it requires a size of about 5 mm, and a particularly large cup is used as the cup. A second standard solution stock solution is prepared for each item and has a value of about 0.5 to 1 mβ. The second standard solution stock solution is placed at the position indicated by sample cup a"f on the turntable 8a of the sample dispensing section. One type of first standard solution stock solution 9 and six types of second standard solution stock solution a. If we create 7 calibration points for each item from = f, we can create 6×7=42 calibration points.

The reagent dispensing unit 10 is also exemplified as a turntable type, and reagents in reagent bottles 10b arranged along the circumference of the turntable 10a are dispensed into the reaction tube 4 by a dispenser 10c.

However, the sample dispensing section 8 and the reagent dispensing section 1o may be of other types, such as a rack type.

A specific example of the reaction tube supply device 6 will be explained with reference to FIGS. 5 to 9.

A reaction tube rack 24 is attached to the frame 22 of the reaction tube supply device 6 by a guide 26 so as to be able to slide in the horizontal direction in the figure.A screw thread 30 driven by a stepping motor 28 passes through the reaction tube rack 24. and threaded rod 30
The reaction tube rack 24 is slid and moved along the guide 26 by the rotation of the frame 22.The reaction tube rack 24 is arranged at an angle with the frame 22 with one end facing down.

The reaction tube rack 24 has six grooves 32 extending vertically in the figure.
is provided, and one end of the lower side of each groove 32 faces the wall surface of the frame 22. In each groove 32, twelve reaction tubes 4a each having twelve reaction tubes connected in a straight line are arranged. The six grooves 32 correspond to different measurement items from A to F.
In each groove 32, a connecting reaction tube 4a in which an antigen or antibody for each measurement item is immobilized is arranged.

The connecting reaction tube 4a has the shape shown in FIG. 6 and is made of plastic. The plurality of reaction tubes 4 are connected in a straight line by a connecting part 4b, and the connecting part 4b is shaped thin and can be easily broken.The reaction tube 4 corresponds to one hole of a 966 microplate. It is the size,
Antigens or antibodies for the same measurement item are immobilized in a series of reaction tubes 4.

Returning to FIG. 5, three connecting reaction tubes 4a can be arranged in each groove 32 of the reaction tube rack 24, and therefore 36 reaction tubes are arranged in each groove 32.

A reaction tube take-out mechanism 34 is provided at the lower end of the frame 22.The reaction tube take-out mechanism 34 has one reaction tube at the tip of the connected reaction tube 4a for the measurement item selected by the stepping motor 28. An air cylinder 36 for taking out the tube 4 and a reaction line 2 for the taken out and cut reaction tube.
A compressed air inlet 38 is provided for sending out compressed air.

FIG. 7 shows the internal structure of the reaction tube take-out mechanism 34.

A partition plate 40 that is moved vertically by an air cylinder 36 is provided within the reaction tube take-out mechanism 34, and a notch 42 is provided in the partition plate 40. The partition plate 40 is in contact with the lower end surface of the reaction tube rack 24, and when the partition plate 40 is lowered, the position of the tip of the reaction tube 4a is changed when the reaction tube rack 24 is moved laterally by the stepping motor 28. is regulated by a partition plate 4o. A reaction tube housing section 44 is provided outside the cutout section 42 . As shown in FIG. 8, a compressed air inlet 38 is led to the reaction tube housing 44, and the reaction tubes dropped into the housing 44 are guided by compressed air to the reaction line via a tube 46.

When the partition plate 4o is moved upward by the air cylinder 36, the notch 42 is removed from the notch 42.
One reaction tube 4 at the tip of the connecting reaction tube 4a in the groove located at position 2 comes out from the notch 42 and enters the reaction tube storage section 44.
to go into.

The operation of selecting and taking out one reaction tube 4 using this reaction tube supply device 6 will be explained with reference to FIG.

(A) shows the state at the time of selection, in which the reaction tube rack 24 is moved laterally in the direction of the arrow by the stepping motor 28, and the selected groove of one reaction tube rack 24 is at the position of the notch 42 of the partition plate. At this point, the reaction tube rack 24 stops.

Next, as shown in (B), the partition plate 40 is pulled up by the air cylinder 36, so that one reaction tube 4 at the tip of the reaction tube 4a disposed in the groove at the position of the notch 42 is removed. The partition plate 40 falls into the reaction tube storage section 44.

When the reaction tube rack 24 is moved laterally by the stepping motor 28, the connecting portion of the reaction tubes is broken and one reaction tube 4 is cut off. The reaction tube 4 that has been cut and stored in the reaction tube storage section 44 is pushed out by compressed air and stored in the reaction tube holder of the reaction line 2, as shown in FIG.

In this way, the reaction tube holder of 1 reaction line 2 has 6
Arbitrary reaction tubes 4 selected from the items are inserted one by one and moved along the reaction line 2.

The liquid sample analyzer shown in FIG. 4 is equipped with a microcomputer, and the part that controls the sampling means 50, the calibration curve creation means 58, and the data processing means 60 in FIG. 1 are realized by the microcomputer.

In order to create a calibration curve with this liquid sample analyzer, the first 41 standard solution stock solution 9 and the second standard solution stock solution a are placed in the same reaction tube.
= Dispense either f. In other words, when seven reaction tubes 4 of item A supplied to the reaction line 2 in succession reach the sample dispensing position, the first
Dispense 100 μΩ of the standard solution stock solution 9 into the reaction tube 4. The next reaction tube 4 contains the first standard solution stock solution 9 as a calibration point (2).
Dispense 95 μQ of the solution and 5 μΩ of the second standard solution stock solution a.

Stirring is performed by sucking and discharging the sample through a nozzle.

In the next reaction tube 4, as a calibration point (3), 90μ0 of the first standard solution stock solution 9 was added. Dispense 10 μQ of the second standard solution stock solution a. In this way, the amounts shown in Table 1 are dispensed in order, and when the seven stages of dispensing are completed, the next items B, C,
Repeat the same operation for D, E, and F in order.

After the calibration points have been measured, the unknown specimens Sl, S2°S3.
...... is dispensed and measured.

When using the effective value as the concentration of the calibration solution, use the effective value obtained by mixing the stock solution of the standard solution at the same dilution rate and measuring it with a more reliable calibration machine. i
R semi-liquid stock solution This value is attached.

The liquid sample analyzer to which the present invention is applied is not limited to the one shown in FIG.

(Effects of the Invention) In the present invention, a multi-stage calibration solution is prepared using two types of standard solution source solutions with compositions close to those of the specimen, so that measurements using a multi-item multi-point calibration curve can be performed with a small number of measurements. This can be achieved using the standard solution stock solution, making the operation easier.

For example, if the number of measurement items is N, then 2N or N+1 standard solution stock solutions are sufficient.

By using the effective value as the value of each correction point created by mixing, it is possible to correct errors caused by dilution.

[Brief explanation of drawings]

FIG. 1 is a block diagram showing the present invention, FIG. 2 is a flowchart showing an example of the operation, FIG. 3 is a diagram showing an example of a calibration curve to be created, and FIG. 4 is a configuration diagram showing one embodiment. FIG. 5 is a perspective view showing the reaction tube supply device in FIG. 4, FIG. 6 is a perspective view showing the connected reaction tubes, FIG. 7 is a perspective view showing the internal structure of the reaction tube take-out mechanism, and FIG. The perspective view which shows a tube storage part. FIG. 9 is a plan view showing the operation of the reaction tube take-out mechanism. 50... Sampling means, 52...
First standard solution stock solution, 54...Second standard solution stock solution, 56...Measurement section, 58...Calibration curve creation means, 6o... data processing means;

Claims (4)

[Claims] (1) In a liquid sample analyzer that samples and measures a liquid sample and quantifies its components based on a pre-prepared calibration curve, a first sample with a composition similar to that of the sample and a low concentration of the component to be detected is used. The sampling means is equipped with a standard solution stock solution and a second standard solution stock solution that has a composition similar to that of the specimen and has a high concentration of components to be detected and is prepared for each item. Liquid sample analysis characterized by having a function of creating calibration points at multiple concentration levels by sampling into a reaction tube at a ratio, and a calibration curve creation means for creating a calibration curve from the measurement results obtained by the sampling. Device. (2) The liquid sample analyzer according to claim 1, wherein the first standard solution stock solution is commonly used for each item. (3) a first standard solution stock solution containing a low concentration of the component to be detected;
It consists of a second standard solution stock solution prepared for each item with a composition similar to that of the sample and a high concentration of the component to be detected, and the predicted effective value when both standard solution stock solutions are mixed at the specified mixing ratio. Calibration fluid shown. (4) The calibration solution according to claim 3, wherein the first standard solution stock solution is commonly used for each item.