JPH10313898A - Calibrator for measuring enzymatic activity and measurement of enzymatic activity using the same - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide the subject calibrator capable of reducing difference in measured values between different analytical instruments and increasing measuring accuracy by including plural enzymes having enzymatic active values not smaller than prescribed times based on upper limit value in standard area of each enzymatic analytical item of calibration curve-preparing object. SOLUTION: In a calibrator for measuring enzymatic activity containing an enzyme such as aspartic acid aminotransferase, alanineamino-transferase, creatinekinase or lactic acid dehydrogenase having well-known enzymatic active values, three or more kinds of enzymes having enzymatic active values of >=2 times respectively based on upper limit values of standard areas of enzymatic analytical items of calibration curve-preparing object are included. Furthermore, the upper limits of active values of three or more kinds of enzymes are preferably within the measuring effective range in using an analytical reagent corresponding to each enzymatic analytic item.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a calibrator and a measuring method suitable for use in measuring an enzyme activity of a biological sample by an automatic analyzer.

[0002]

2. Description of the Related Art Clinical tests on various analysis items are indispensable for diagnosing a disease state. In laboratory tests,
Although it is necessary to maintain high reliability of the measured values, there is a difference between facilities in which, when the same biological sample is measured, the measured value differs depending on the laboratory. The main cause of the difference between facilities is the difference in analyzers for measurement (difference between devices)
And differences in reagents used for measurement (difference between reagents).

[0003] In the measurement of enzyme activity of a biological sample such as blood, there is also a problem of differences between facilities, and various attempts have been made.
First, as a method of correcting the difference between devices, “3rd Summer Seminar of the Japanese Society of Clinical Chemistry, pp. 61-71, 19
In 1983, a method of determining a calibration coefficient (actually measured K factor) unique to an apparatus using a reaction indicator having a known concentration is known.

As a method of correcting the difference between samples,
There is known a method of further multiplying data output as a measurement result by a conversion coefficient between reagents (KR value). The KR value is
The relationship between the reagent for the reference measurement method and each of the other prescription reagents was investigated using a large number of patient samples. Can be converted. Regarding the results of studying the KR value of a commercially available reagent, see “Medical Inspection Vol. 42, No. 12, p. 1898-1905,
1993 ".

[0005]

Among the above-mentioned prior arts, the inter-apparatus difference correction method for obtaining the actually measured K factor can correct the dispensing system or optical system of each measuring apparatus, but the measurement temperature is low. Not included as a factor. Therefore, the user needs to further confirm the temperature in the reaction tank using a thermistor thermometer, and correct the temperature if the temperature deviates from the measured temperature.

As described above, in order to correct the difference between facilities in the enzyme activity measurement, it was necessary to measure the actually measured K factor, confirm the reaction temperature, and correct the data by the KR value at each facility. The reason why such a troublesome work was continued only for the enzyme item and the calibration by the calibrator was not so generalized was that an enzyme sample excellent as a calibrator was not prepared. This is because the work of preparing a calibrator did not proceed as expected due to the problem of long-term stability of enzyme activity, the difficulty of selecting an enzyme isozyme composition or the enzyme origin showing the same reactivity as human serum. However, the addition of stabilizers and preservatives has ensured long-term stability of activity, and many things have been elucidated about isozyme composition and enzyme origin. Became. In addition, the Japanese Society of Clinical Chemistry (JSCC) presented the JSCC standard method, which is the standard measurement method for enzyme activity measurement. The measured values were obtained via an enzyme calibrator that was priced by this method, and the measurement values were unified. The movement to pursue is becoming active.

By the way, many commercially available enzyme calibrators have their activity values set near the upper limit of the reference range.

[0008] Here, the reference range is a concept that has come to be used instead of the conventionally used concept of a normal value range, and is shared by each facility based on the recommendation of the International Clinical Chemistry Union (IFCC). This is a concept that indicates a compatible reference value. That is, with a healthy population as a population, a range between an upper limit and a lower limit that includes 95% of the entire range of the distribution of measured values obtained from each individual is referred to as a reference range. .

[0009] At the site of a clinical test, there is a case where an enzyme calibrator having an activity value about the upper limit of the reference range is used because it is desired to accurately measure the data of the patient near the reference range in order to check whether the data is normal or abnormal. Many. However, this does not allow the device to be calibrated with high accuracy.

For example, in the case of alanine aminotransferase (ALT), most commercially available ALT
The measurement reagent can measure up to at least about 1600 IU / l. However, the ALT activity value of the commercial enzyme calibrator considering only the reference range of 4-44 IU / l in the JSCC standard method is about 50 IU / l, which is -10 × 10 ⁻³ Abs./at the measurement wavelength of 340 nm. mi
It corresponds to the absorbance change of n.

The standard specification of a photometer built in an automatic analyzer for measuring a biological sample is about ± 1.0 × 10 ⁻³ Abs./min. In this case, it is assumed that the output of the photometer fluctuates within the specification range due to noise or the like during calibration of the calibration curve. In other words, the calibrator should normally be -1
The place to be measured as 0 × 10 ⁻³ Abs./min is −11 × 1
When measured as 0 ^-3 Abs./min, this results in a 10% error. When a test sample is measured with a device calibrated in this state, the measurement result may have a 10% measurement error. . Therefore, the error at each facility varies within a range of ± 10%, such as a -5% error at a certain facility and a 9% error at another facility, which is a large difference between facilities. Such a calibrator having a low activity value is easily affected by an optical system error in the apparatus, and a measurement error and a difference between facilities are likely to occur.

Next, consider the case where a calibration curve is prepared using a commercially available enzyme calibrator (ALT activity value 50 IU / l) to measure alanine aminotransferase (ALT). Because the device rounds off the decimal point and outputs it as an integer, if it is calculated as 49.5 IU / l inside the device, the measured value is output as expected at 50 IU / l, but calculated as 50.5 IU / l. Expected value 50 if
It is shifted by 1 IU / l from IU / l and is output as 51 IU / l. In either case, the value calculated inside the device deviates from the expected value by the same ratio as 0.5 IU / l, but only one of the final output results is 2.0% from the expected value.
Calibrators that are displaced and are affected by such errors in the calculation process are not preferred.

An object of the present invention is to improve the measurement accuracy of an automatic analyzer when measuring an enzyme activity value,
It is an object of the present invention to provide a calibrator and a method for measuring enzyme activity, which can reduce a difference in measured values between different analyzers.

[0014]

According to the present invention, there is provided a calibrator for measuring an enzyme activity which comprises at least three types of enzymes having an enzyme activity value which is at least twice the upper limit value of the reference range of each enzyme analysis item for which a calibration curve is to be prepared. Is included.

In this case, the upper limits of the activity values of the three or more enzymes are within the effective measurement range when the analysis reagent corresponding to each enzyme analysis item is used.

The enzyme contained in this calibrator is
Aspartate aminotransferase (AST),
Alanine aminotransferase (ALT), creatine kinase (CK), lactate dehydrogenase (L
D), alkaline phosphatase (ALP), gamma-glutamyltransferase (GGT), amylase (AMY), cholinesterase (CHE).

Further, the calibrator for measuring enzyme activity according to the present invention comprises aspartate aminotransferase (AST) and alanine aminotransferase (A
LT), creatine kinase (CK), lactate dehydrogenase (LD), alkaline phosphatase (ALP)
And three or more enzymes selected from gamma-glutamyltransferase (GGT), and the activity value of the contained enzyme is 250 to 800 IU / l for AST, A
LT 250-800 IU / l, CK 500-240
0 IU / l, LD: 400-800 IU / l, ALP: 700-3200 IU / l, and GGT: 200-10
00IU / l.

Further, the enzyme activity measuring method according to the present invention measures a change in the absorbance of a reaction solution relating to an enzyme analysis item of a test sample, and uses a calibrator for measuring the enzyme activity based on a calibration curve obtained in advance. In the method for measuring the enzyme activity value of a test sample, three or more types of calibrators containing three or more types of enzymes having enzyme activity values twice or more the upper limit of the reference range of each enzyme analysis item are used. It is characterized in that a calibration curve of an analysis item corresponding to each of the enzymes is created.

[0019]

DESCRIPTION OF THE PREFERRED EMBODIMENTS The enzyme used as a calibrator for measuring enzyme activity is preferably derived from human cells, human organs, animal cells, animal organs or microorganisms. These enzyme calibrators are prepared by adding human pool serum prepared by collecting human serum, delipidated human serum, or 6% bovine serum albumin solution, and adjusting the specific gravity and viscosity of the final solution to the same level as those of pooled serum. It is provided in the form of a prepared solution.

The calibrators according to the present invention include aspartate aminotransferase (AST), alanine aminotransferase (ALT), creatine kinase (CK), lactate dehydrogenase (LD), alkaline phosphatase (ALP), and γ-glutamyltransferase (GGT ), Amylase (AMY),
It contains three or more enzymes selected from cholinesterase (CHE), particularly preferably six enzymes. The enzyme activity value in the calibrator should be within the range that is not less than twice the upper limit of the reference range of each analysis item (test target item) and not more than the maximum value of the measurable limit based on the JSCC standard method. And a calibration curve for a plurality of enzyme analysis items is created using a single calibrator. According to the JSCC standard method, the enzyme activity value of the calibrator according to the present invention is 250 to 8 for AST.
00 IU / l, ALT 250-800 IU / l, CK
Is 500 to 2400 IU / l, LD is 400 to 800 I
U / l, ALP is 700-3200 IU / l, and GGT is 200-1000 IU / l. Such an enzyme reference sample is used not only as a calibrator but also as a control sample. Hereinafter, examples to which the present invention is applied will be described in detail.

(1) Preparation of Enzyme Calibrator for Calibration Curve A 0.2 mol / l phosphate buffer (pH = 7.0) is prepared. Sodium chloride, sodium azide, bovine serum albumin, pyridoxal phosphate, N-
Acetyl-L-cysteine (NAC), ethylenediaminetetraacetic acid (EDTA) and magnesium chloride are added and dissolved.
The concentration of each additive was 0.9% by weight of sodium chloride, 2mmol / l of sodium azide, 6% by weight of bovine serum albumin,
Pyridoxal phosphate 0.5 mmol / l, N-acetyl-L
Cysteine (NAC) 15 mmol / l, ethylenediaminetetraacetic acid (EDTA) 1 mmol / l, magnesium chloride 1.5 mmol / l.

AST, ALT, CK, LD, ALP, GGT manufactured by Sigma are added to a phosphate buffer containing each additive and dissolved. When the prepared sample was measured by the JSCC standard method, the enzyme activity value was AST 250 to 800.
IU / l, ALT 250-800 IU / l, CK500
~ 2400 IU / l, LD400 ~ 800 IU / l, A
LP700-3200 IU / l, GGT200-100
Add each enzyme to 0 IU / l.

The above-mentioned calibrator is an example including six types of enzymes. However, by preparing a calibrator including three or more types of enzymes, the calibration of three or more items can be performed simply by setting a small number of calibrators in the automatic analyzer. Can be used for line creation. As the three kinds of enzymes, for example, AST, ALT, and LD are selected and prepared in the same manner as in the above-mentioned example, but are not limited to such a combination of enzymes.

The value published as the reference range is 8 to 38 IU / l for AST. ALT is 4-4
4 IU / l. CK is 29-203 for children (C)
IU / l, female (F) 29-145 IU / l, male (M) 58-348 IU / l. LD is 106-
211 IU / l. ALP is 104-338 IU /
l. GGT is 5-50 IU / l for women (F) and 5-83 IU / l for men (M). In these numerical values, the value described above is the lower limit, and the value described later is the upper limit. The calibrator according to the present invention is prepared so as to exhibit an enzyme activity value substantially twice or more the upper limit of the reference range in each enzyme analysis item.

The prepared enzyme sample is measured and valued according to the JSCC standard method. Then, after the container is placed in a container having a predetermined capacity, the container is covered with a lid and hermetically sealed, and a label indicating the value of each enzyme activity is attached to the outer wall of the container. This indicated value becomes the known activity value of each enzyme.

(2) Example of Creating a Calibration Curve for Enzyme Analysis Items by Automatic Analyzer The enzyme calibrator prepared as described above is used for calibration of an automatic analyzer as shown in FIG.

First, a configuration example of the automatic analyzer will be described.
In FIG. 1, on a sample disk 8, a large number of sample containers 12B accommodating a test sample composed of a biological sample such as serum are arranged in an annular shape on an outer peripheral side, and standard samples of various analysis items are arranged on an inner peripheral side. The sample containers 12A containing the enzyme calibrators are arranged in a ring. A row of reaction vessels 4 for reacting a sample and a reagent is arranged in a ring on a reaction disk 5 forming a reaction line.
Reagent bolts 9A containing first reagents corresponding to a plurality of analysis items are arranged on the reagent disk 10A for the first reagent, and the second reagent disk 10B for the second reagent corresponding to the plurality of analysis items is arranged on the reagent disk 10B for the second reagent. A reagent bottle 9B containing a reagent is arranged.

The sample dispensing nozzle 15 attached to the movable arm of the sample dispensing mechanism 1 is connected to a sample syringe pump 16. First reagent dispensing mechanism 2A
The reagent dispensing nozzle 21A attached to the movable arm of the second reagent dispensing mechanism 2B and the reagent dispensing nozzle 21B attached to the movable arm of the second reagent dispensing mechanism 2B are connected to the syringe pump 22 for reagent. The reaction disk 5 is provided with a light source 61 for white light and a multi-wavelength photometer 6, and the light beam from the light source 61 is traversed by the rows of the reaction vessels 4. Data measured by the photometer 6 is converted by the analog / digital converter 62 and input to the microcomputer 11 as a control unit via the interface 30. The operation of each mechanism of the automatic analyzer is controlled by the microcomputer 11. The control program is stored on a floppy disk 31 as an external memory.

The analysis parameters necessary for executing the analysis operation are input from the operation unit 13 and displayed on the CRT 33 as a screen display device. The sample container 12A of the enzyme calibrator prepared according to the above (1) is set on the sample disk 8, and AST, ALT, CK, LD, AL
P and GGT are input from the operation unit 13, and the known activity value of each enzyme in this calibrator is input. The microcomputer 11 stores these data in the storage unit in association with the set position of the sample container 12A on the sample disk 8. The number of calibrators required for preparing a calibration curve may be only one, but a plurality of calibrators having different enzyme activity values may be used as needed. AST, A on reagent disks 10A, 10B
Labels indicating reagent information are attached to the outer walls of the reagent bottles 9A and 9B necessary for the reaction of each analysis item of LT, CK, LD, ALP, and GGT. The reagent information is read from the label by the identification reading device (for example, a bar code reader) 37, and the reagent information is set together with the set position of the corresponding reagent on each reagent disk.
1 storage unit.

When the sample disk 8 is rotated and the sample container 12A containing the enzyme calibrator is positioned at the sample suction position, the sample dispensing mechanism 1
Sucks a predetermined amount of enzyme calibrator for AST analysis from the sample container 12A into the nozzle 15 and discharges it into one reaction container 4 on the reaction disk 5. Next, the reaction disk 5 is rotated, and the sample dispensing mechanism 1 similarly dispenses the same enzyme calibrator for the ALT analysis into the next reaction container 4 and subsequently, the same enzyme calibrator for the LD analysis is used for the reaction disk 5. Dispense into another reaction container 4 above. The same calibrator is similarly dispensed for the other three enzyme items. Each time the calibrator is dispensed, the reaction disk 5 is rotated by one rotation and the distance of one container.

As the intermittent rotation of the reaction disk 5 is repeated thereafter, each reaction vessel 4 corresponding to each enzyme analysis item is
Is temporarily stopped at the first reagent addition position and the second reagent addition position, the reagent corresponding to each analysis item is dispensed to each reaction container 4 by the reagent dispensing mechanisms 2A and 2B, and each analysis item is Reaction proceeds in the reaction vessel. After the reagent is added to each reaction container, the mixture is stirred by the stirring mechanisms 3A and 3B. The reaction solution in the reaction vessel is measured when the plurality of reaction vessels cross the light flux of the photometer 6,
The photometric signal is converted to absorbance. For each enzyme analysis item of the enzyme calibrator, the amount of change in absorbance is calculated by the microcomputer 11 from a plurality of photometric data obtained by traversing the luminous flux a plurality of times for the reaction solution in the same reaction vessel in which the enzyme reaction is in progress. AST, ALT, CK, LD,
A calibration curve for each analysis item of ALP and GGT is calculated. After that, the sample container 1 is
The reagent corresponding to the analysis item is added to the test sample dispensed from 2B to the reaction container 4, the reaction solution is measured, the change in absorbance of the test sample is determined, and the calibration previously prepared The microcomputer 11 calculates the enzyme activity value in the test sample for each enzyme analysis item based on the line. The calculated enzyme activity values as the measured values (analysis results) of the test samples are displayed on the CRT 33 and the printer 35 in association with the respective test sample numbers and analysis items.
The reaction container 4 for which the photometry has been completed is washed by the washing mechanism 7 and used for the subsequent analysis of the sample.

FIG. 2 is a diagram showing an example of a calibration curve obtained based on the above-mentioned operation, wherein the horizontal axis indicates the enzyme activity value and the vertical axis indicates the amount of change in absorbance. The example of FIG. 2 is an example of an LD calibration curve obtained from a measured value of a calibrator containing an LD having an enzyme activity value of 600 IU / l and a blank measured value, but the same applies to the other five types of enzymes. Using a calibrator, a calibration curve having similar linearity is obtained.

Such an enzyme calibrator can also be used as a sample for quality control of an analyzer, and every time a predetermined number of test samples are measured, for example, every 300 samples, or every predetermined time. Then, for example, every two hours, the enzyme calibrator is measured together with the blank, and the quality is controlled.

(3) Inferring the difference between the apparatuses The influence on the measurement error was examined by using a commercially available calibrator having an LD activity value of 200 IU / l and the enzyme calibrator (LD activity value of about 600 IU / l) prepared in the above (1). Was. It is assumed that the specification of the absorbance change noise level of the photometer of the automatic analyzer is within ± 1.0 × 10 ⁻³ Abs./min. First, in a commercially available calibrator, when the noise or the like of the photometer is almost zero, the change in absorbance at an LD activity value of 200 IU / l corresponds to 26 × 10 ⁻³ Abs./min. However, if the noise of the photometer influences within the range of the specification, the absorbance change amount of this calibrator is 25 × 10 ^{−3 to} 27 ×.
It can be 10 ^-3 Abs./min. This results in -3.
A measurement error of 8 to + 3.8% results.

Assuming that the calibrator is carried out using this commercial enzyme calibrator with two automatic analyzers, the maximum difference between the apparatuses is 7.6%. On the other hand, the enzyme calibrator according to the present invention has a change in absorbance of 78 × 10 ⁻³ Abs./mi when the noise or the like of the photometer is almost zero.
n. If the photometer noise is affected within the specified range, the absorbance change amount is 77 × 10 ^{−3 to} 79 × 10 ⁻³ Abs./min. This results in a measurement error of -1.3 to + 1.3%, and the difference between the devices can be reduced to a maximum of 2.6%.

Similarly, the difference between AST and ALT is considered.

The AST activity value of the commercially available calibrator is 10
At 0 IU / l, the change in absorbance was −20 × 10 ⁻³ Abs.
/ Min. Here, the noise of the photometer (± 1.0 × 10 ⁻³⁾
Abs./min), the change in absorbance is −21 ×
10 ⁻³ to −19 × 10 ⁻³ Abs./min, which is −
A measurement error of 4.8 × 10 ⁻³ to + 5.3 × 10 ⁻³ % is caused.
On the other hand, the ALT activity value of the enzyme calibrator according to the present invention is 300 IU / l, and the change in absorbance thereof is −60 × 1.
0 ^-3 Abs./min. Noise of photometer (± 1.0 × 10
^-3 Abs./min), the change in absorbance is -61.
× 10 ^-3 to -59 × 10 ^-3 Abs./min, which is-
A measurement error of 1.6 × 10 ⁻³ to + 1.7 × 10 ⁻³ % is obtained.
The difference between the apparatuses can be reduced to about one third of the commercially available calibrator.

The ALT activity value of the commercially available calibrator is 80 IU / l, and the change in absorbance is -16 × 10 ^-3.
Abs./min. Here, the noise of the photometer (± 1.0 × 1
0 ^-3 Abs./min), the change in absorbance is -1.
7 × 10 ⁻³ to −15 × 10 ⁻³ Abs./min, which results in a measurement error of −5.9 × 10 ⁻³ to + 6.7 × 10 ⁻³ %. On the other hand, the ALT of the enzyme calibrator based on the present invention
The activity value is 250 IU / l, and the change in absorbance is -50.
× 10 ⁻³ Abs./min. Photometer noise (± 1.0
(× 10 ⁻³ Abs./min), the change in absorbance is −51 × 10 ⁻³ to −49 × 10 ⁻³ Abs./min, which is −2.0 × 10 ⁻³ to +2. The measurement error is 0.0 × 10 ⁻³ %, and the difference between the apparatuses can be reduced to about one third of the commercially available calibrator.

As described above, it can be inferred that when the calibration is performed using the enzyme calibrator according to the present invention, the analyzer can be calibrated with high accuracy and the difference between the devices can be further reduced.

(4) Experimental Results A survey was conducted using the enzyme calibrator according to the present invention, and the effect of correcting differences between apparatuses was examined. The number of analyzers for which the survey was conducted was 70, and the model was Hitachi 7150.
Type, Hitachi 7070 type, Hitachi 7170 type, Hitachi 7020
, Hitachi 7250, Hitachi 7350, and Hitachi 7450.

The survey sample is an enzyme calibrator according to the present invention, a calibrator sample manufactured by Company A, and a human pool serum as a test sample. The enzyme activity values of the enzyme calibrator according to the present invention according to the JSCC standard method are AST (420 IU / l) and ALT (288 I
U / l), LD (673 IU / l), CK (654 IU)
/ L), ALP (801 IU / l), GGT (440I
U / l). On the other hand, the enzyme activity value of a sample manufactured by Company A according to the JSCC standard method was AST (103 IU / l), A
LT (83 IU / l), LD (258 IU / l), CK
(272 IU / l), ALP (459 IU / l), GG
T (154 IU / l). For 70 automatic analyzers installed in different clinical laboratories, the enzyme sample according to the present invention and the sample manufactured by Company A were used as calibrators, and human pooled sera as test samples were measured. The degree of convergence of the difference between the human pool serum measurements between the two calibrators was measured. The measurement reagent used was a reagent for daily routine at each facility.

The results of the survey are shown in Table 1. Data exceeding the average value of ± 2.7 SD was rejected in advance.
The difference between devices can be determined from CV (%). CV (%)
Indicates the degree of data variation, and the smaller this value is, the smaller the data variation in all facilities is, and it can be said that the difference between apparatuses has converged. As can be seen from Table 1, the difference between the devices obtained by the measurement values obtained from the enzyme calibrator according to the present invention is smaller than that of the commercially available calibrator. N is the number of measuring devices.

[0043]

[Table 1]

(5) Accuracy control An enzyme calibrator and a company A calibrator sample of the same lot as the sample used for the survey experiment (4) were used as control samples, and these were measured twice a day for 20 days. , Quality control was implemented. A commercially available JSCC-compliant reagent was used as a measurement reagent.

Table 2 shows the results. Using the calibrator according to the present invention could reduce the measurement error, and all of the six enzymes had smaller variations than those using the calibrator manufactured by Company A.

[0046]

[Table 2]

[0047]

According to the present invention, a calibration curve for an automatic analyzer can be prepared using a calibrator containing three or more enzymes so as to be at least twice the upper limit of the reference range. The measurement accuracy of the automatic analyzer when measuring the enzyme activity value can be improved, and the difference in the measured values between different analyzers can be reduced.

[Brief description of the drawings]

FIG. 1 is a schematic diagram showing the entire configuration of an automatic analyzer to which the present invention is applied.

FIG. 2 is a diagram showing an example of a calibration curve created based on the present invention.

[Explanation of symbols]

1: sample dispensing mechanism, 2A, 2B: reagent dispensing mechanism, 4
... Reaction vessel, 5 ... Reaction disk, 6 ... Multi-wavelength photometer, 8
... Sample disk, 11 ... Microcomputer, 1
2A, 12B ... sample containers.

────────────────────────────────────────────────── ───

[Procedure amendment]

[Submission date] February 16, 1998

[Procedure amendment 1]

[Document name to be amended] Statement

[Correction target item name] 0003

[Correction method] Change

[Correction contents]

[0003] In the measurement of enzyme activity of a biological sample such as blood, there is also a problem of differences between facilities, and various attempts have been made.
First, as a method for correcting apparatus difference is "3rd Japan Society of Clinical Chemistry Summer Seminar Book, Chapter 68-71, pp. 19
In 1983, a method of determining a calibration coefficient (actually measured K factor) unique to an apparatus using a reaction indicator having a known concentration is known.

[Procedure amendment 2]

[Document name to be amended] Statement

[Correction target item name] 0012

[Correction method] Change

[Correction contents]

[0012] Next consider the case of measuring the commercial enzyme calibrator (ALT activity value 50 IU / 1) and as a quality control sample alanine aminotransferase (ALT). Since the device rounds off the decimal point and outputs it as an integer, if it is calculated as 49.5 IU / 1 inside the device, the measured value is output as expected at 50 IU / 1,
Expected value 50 IU if calculated as 50.5 IU / 1
The output is 51 IU / 1 shifted by 1 IU / 1 from / 1.
In either case, the value calculated inside the device deviates from the expected value by the same ratio as 0.5 IU / 1, but in the final output result, only one deviates from the expected value by 2.0%. Thus, a sample affected by such an error in the calculation processing is not preferable for the precision control of the apparatus .

[Procedure amendment 3]

[Document name to be amended] Statement

[Correction target item name] 0030

[Correction method] Change

[Correction contents]

The sample disk 8 is rotated, the sample container 12A containing the enzyme calibrator is positioned at the sample suction position, the sample dispensing mechanism 1,
The sample container 12A inhalation shi a predetermined amount of the enzyme calibrator for AST analysis nozzle 15 from ejecting the reaction disk one reaction vessel 4 on 5. Next, the reaction disk 5 is rotated, and the sample dispensing mechanism 1 similarly dispenses the same enzyme calibrator for the ALT analysis into the next reaction container 4 and subsequently, the same enzyme calibrator for the LD analysis is used for the reaction disk 5. Dispense into another reaction container 4 above. The same calibrator is similarly dispensed for the other three enzyme items. Each time the calibrator is dispensed, the reaction disk 5 is rotated by one rotation and the distance of one container.

[Procedure amendment 4]

[Document name to be amended] Statement

[Correction target item name] 0037

[Correction method] Change

[Correction contents]

The AST activity value of the commercially available calibrator is 10
At 0 IU / 1, the amount of change in absorbance was −20 × 10 ⁻³ Abs.
/ Min. Here, the noise of the photometer (± 1.0 × 10 ⁻³⁾
Abs./min), the change in absorbance is −21 ×
10 ⁻³ to −19 × 10 ⁻³ Abs./min, which is −
4. lead to 8 to + 5. 3% of the measurement error. On the other hand, the ALT activity value of the enzyme calibrator according to the present invention is 300 IU /
At 1, the change in absorbance is −60 × 10 ⁻³ Abs./min. When the noise of the photometer (± 1.0 × 10 ⁻³ Abs./min) is affected, the amount of change in absorbance is −61 × 10 ⁻³ to −59.
× becomes 10 ^-3 Abs./min, this -1. 6 ~ + 1.7%
And the difference between the apparatuses can be reduced to about one third of the commercially available calibrator.

[Procedure amendment 5]

[Document name to be amended] Statement

[Correction target item name] 0038

[Correction method] Change

[Correction contents]

The ALT activity value of the commercially available calibrator is 80 IU / 1, and the change in absorbance is -16 × 10 ^-3.
Abs./min. Here, the noise of the photometer (± 1.0 × 1
0 ^-3 Abs./min), the change in absorbance is -1.
7 × becomes ^{10 -3 ~-15 × 10 -3} Abs./min, which leads to -5. 9 to +6. 7% measurement error. On the other hand, the ALT activity value of the enzyme calibrator according to the present invention is 250 IU
/ 1, the change in absorbance is −50 × 10 ⁻³ Abs./min.
It is. Noise of photometer (± 1.0 × 10 ⁻³ Abs./min
), The change in absorbance is −51 × 10 ⁻³ to
-49 × becomes 10 ^-3 Abs./min, which -2. 0 ~ +
The measurement error was 2.0 % , which was about 3 % of that of the commercial calibrator.
The difference between devices can be reduced to one-half.

──────────────────────────────────────────────────の Continued on the front page (51) Int.Cl. ⁶ Identification code FI C12Q 1/44 C12Q 1/44 G01N 21/75 G01N 21/75 A

Claims (5)

[Claims] 1. An enzyme activity measuring calibrator containing an enzyme having a known enzyme activity value, wherein at least three types of enzyme activity values which are at least twice the upper limit value of the reference range of each enzyme analysis item for which a calibration curve is to be prepared. A calibrator for measuring enzyme activity, comprising an enzyme. 2. The calibrator according to claim 1, wherein
An enzyme activity measurement calibrator characterized in that the upper limit of the activity values of the three or more types of enzymes is within the effective measurement range when using an analysis reagent corresponding to each enzyme analysis item. 3. The calibrator according to claim 1, wherein
The enzymes contained are aspartate aminotransferase (AST), alanine aminotransferase (A
LT), creatine kinase (CK), lactate dehydrogenase (LD), alkaline phosphatase (ALP),
A calibrator for measuring enzyme activity, which is selected from gamma-glutamyltransferase (GGT), amylase (AMY), and cholinesterase (CHE). 4. A calibrator for measuring an enzyme activity containing an enzyme having a known enzyme activity value, comprising aspartate aminotransferase (AST), alanine aminotransferase (ALT), creatine kinase (CK),
Contains and contains three or more enzymes selected from lactate dehydrogenase (LD), alkaline phosphatase (ALP), gamma-glutamyltransferase (GGT), amylase (AMY) and gamma-glutamyltransferase (GGT) The activity value of the enzyme was 250-800 IU / l for AST and 250 for ALT.
~ 800 IU / l, CK is 500 ~ 2400 IU / l,
LD 400-800 IU / l, ALP 700-32
00IU / l and GGT 200-1000 IU / l
A calibrator for measuring an enzyme activity, characterized in that: 5. A method for measuring the change in absorbance of a reaction solution relating to an enzyme analysis item of a test sample, and measuring an enzyme activity value of the test sample based on a calibration curve previously obtained by using a calibrator for measuring enzyme activity. In the method, the analysis items corresponding to each of the three or more enzymes are obtained by using a calibrator including three or more enzymes having an enzyme activity value twice or more the upper limit of the reference range of each enzyme analysis item. A method for measuring enzyme activity, which comprises preparing a calibration curve.