JPH10213546A - Automatic analyzer for enzyme activation measuring - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain an enzyme activation value equal to a measured value under an optimal reaction condition by correcting an absorbed light intensity changing amount measured value per unit time by an average correction value when a partially deteriorated rate measuring reagent is used. SOLUTION: A reagent blank liquid is measured and, from its absorbed light intensities ARBm to ARBn , a ΔARB/Δt operation part 46 obtains an absorbed light intensity changing amount ΔARB/Δt per unit time. Also, a reagent reaction liquid is measured and, from its absorbed light intensities Asm to Asn , an absorbed light intensity changing amount operation part 44 obtains a Δ absorbed light intensity changing amount ΔAs /Δt per unit time. The absorbed light intensities Asm to Asn are corrected by a reagent blank correction operation part 42 to obtain absorbed light intensities A'sm to A'sn , correction values Fvm to Fvn corresponding to these are selected by a correction value selection part 32 and its average correction value Fv B is obtained by an average correction value calculation part 34. Then, an enzyme activation value calculation pat 36 substrats ΔARB/Δt from the absorbed light changing amount ΔAs /Δt, multiplies the average correction value Fv B by a K value from a K value storage part 48 to calculate an enzyme activation value C and outputs this.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a biochemical automatic analyzer, and more particularly to an automatic biochemical analyzer for measuring enzyme activity.

[0002]

2. Description of the Related Art In order to measure enzyme activity, a sample containing an enzyme and a reagent containing a substrate are mixed to prepare a sample reaction solution, and the absorbance of the sample reaction solution is measured to determine the absorbance per unit time. The amount of change is determined, and the enzyme activity value is calculated from the amount of change in absorbance.

[0003]

The measured value of the enzyme activity differs depending on the component concentration of the reagent used for the enzyme activity measurement. In other words, even when the same reagent is used, some of the components in the reagent are decomposed and degraded due to changes over time, so that the measured enzyme activity differs between the reagent immediately after preparation and the reagent after a lapse of time. become.

[0004] In addition, since clinical biochemical automatic analyzers are used in medical institutions such as hospitals and test centers, there are test samples whose enzyme components show abnormally high values. Automated biochemical analyzers define measurement conditions for measuring many samples with a certain degree of accuracy. If a sample containing a high concentration (highly active) enzyme is encountered, it must be used during or before the rate measurement time. At this stage, the substrate as a reagent component is reduced, and the conditions become non-optimal, so that a low activity value is obtained. In the case of an extremely high-activity sample, a non-steady-state reaction condition occurs, and a situation occurs in which measurement cannot be performed. In particular, an enzyme sample having an extremely high activity is measured by preparing a sample reaction solution again by reducing the amount of the sample or diluting the sample. The inventor has determined that the amount of change in absorbance per unit time when measuring the enzyme activity of the sample reaction solution depends on the absorbance reflecting the substrate concentration in the reaction solution, and that the amount of change in absorbance depends on the absorbance of the sample reaction solution. It has been found that measurement results can be corrected under appropriate reagent component concentration conditions (referred to as optimum conditions).

[0005] A first object of the present invention is to use a reagent for rate measurement in which a part of a substrate is degraded, or to encounter a sample with high activity under non-optimal or unsteady reaction conditions in enzyme activity measurement conditions. The purpose is to obtain an enzyme activity value equivalent to the measured value under optimal reaction conditions. The second object of the present invention is to prepare a sample reaction solution again by a method such as reducing the amount of a sample with an ultra-highly active enzyme sample or diluting the sample, and remeasurement to obtain a more accurate measurement value. It is an object of the present invention to provide a method for obtaining an appropriate sample amount or dilution rate for re-measurement in the case where the measurement is performed.

A conventional method for re-measuring an enzyme sample having an extremely high activity
One method is to derive from the relationship between the rate measurement time of the sample reaction solution and the absorbance for determining the unsteady reaction conditions. This is based on the fact that the rate reaction shows a substantially constant reaction rate in a steady-state reaction, so that the relationship between the sample amount and the rate measurement time can be estimated, and the sample amount condition at the time of re-measurement can be estimated. However, a change in the substrate concentration of the measurement reagent, that is, a difference between the production lots of the original reagent at the time of preparation or a change over time after the preparation lowers the substrate concentration in the reagent, that is, the sample reaction solution, and changes the measurable concentration range. For this reason, it is necessary to determine the re-measurement sample amount or the dilution ratio with a margin in consideration of these changes, but there is a disadvantage that the degree of the margin cannot be accurately determined by the conventional method.

As another conventional method, there is a method in which a sample amount or a dilution rate at the time of re-measurement is determined in advance. This is based on the fact that the enzyme activity measurement conditions (mainly the substrate concentration in the reagent and the rate measurement time), the type of the sample and the enzyme to be measured are determined, and the concentration range to be measured can be roughly estimated. However, determining the sample amount or dilution ratio at the time of re-measurement in advance is not always the optimal condition from the viewpoint of measurement accuracy, and performing unnecessary dilution work lowers the processing capacity of the automatic analyzer. And other problems.

[0008]

In order to achieve the first object, according to the present invention, as shown in FIG. 1, an absorbance measurement value Asm of a sample reaction solution for enzyme activity measurement within a rate measurement time is used.
An absorbance change calculator 44 for calculating the absorbance change ΔAs / Δt per unit time from Asn, and an absorbance change per unit time under optimal reaction conditions from the absorbance of the sample reaction solution for enzyme activity measurement. A correction value storage unit 30 that stores the correction values Fv of the sample reaction solution, a correction value selection unit 32 that selects the respective correction values Fv corresponding to the measured absorbances Asm to Asn of the sample reaction solution from the correction value storage unit 30, Average correction value calculation unit 3 for calculating the average value FvB of the selected correction values
4 and the measured absorbance change per unit time ΔAs / Δt calculated by the absorbance change calculator 44 by the average correction value FvB calculated by the average correction calculator 34 to calculate the enzyme activity C. And an enzyme activity value calculating section 36 for outputting the result.

In order to achieve the second object, as shown in FIG. 2, a reagent blank measurement value storage section 38 for storing the reagent blank solution absorbance A RB, a sample amount v, a reagent amount R 1,
R2, limit absorbance AL, end absorbance AF and measurement time Δt
A rate measurement condition storage unit 50 for storing rate measurement conditions including (tm to tn); an enzyme activity value C calculated by the enzyme activity value calculation unit 36; and a reagent blank measurement value storage unit 3.
Re-measurement for calculating the re-measurement sample volume vs. or the dilution rate of the re-measurement sample using the reagent blank measurement value A RB stored in No. 8 and the rate measurement condition stored in the rate measurement condition storage unit 50 And a sample amount / dilution ratio calculation unit 52.

[0010]

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS In FIG.
A reagent blank measurement value storage unit 38 for storing RBf, ARBm to ARBn is provided. A RBf is the absorbance of the reagent blank before the addition of the rate reaction starting solution. Further, a sample reaction liquid measurement value storage section 40 for storing the absorbance measurement values Asf, Asm to Asn of the sample reaction liquid is provided, and the absorbance Asm 'to Asn' corrected for the sample blank is calculated by the following equation (1). A sample blank correction calculation unit 42 is provided. Asf is the absorbance of the sample reaction solution before the addition of the rate reaction starting solution, R1 and R2.
Is the liquid volume of the first reagent and the liquid volume of the second reagent, respectively. Asm '= Asm- (Asf-ARBf) (v + R1) / (v + R1 + R2): Asn' = Asn- (Asf-ARBf) (v + R1) / (v + R1 + R2) (1)

The correction value selection unit 32 calculates a plurality of measured absorbance values Asm to Asn based on the sample reaction solution absorbances Asm 'to Asn' obtained by correcting the reagent blank according to the above equation (1).
The respective absorbance measured values Asm 'to
Select the correction value corresponding to Asn '. Also, the reagent blank value A stored in the reagent blank measurement value storage unit 38
A ΔARB / Δt calculating unit 46 for calculating a variation ΔARB / Δt per unit time of RBm to ARBn is also provided.

The K value for obtaining the enzyme activity value from the measured change in absorbance of the sample reaction solution is stored in the K value storage section 48. The enzyme activity value calculation section 36 calculates the K value as shown in the following equation (2). From the measured absorbance change per unit time ΔAs / Δt calculated by the absorbance change calculator 44, ΔAR
After performing a correction for subtracting ΔARB / Δt calculated by the B / Δt calculation unit 46, the average correction value FvB calculated by the average correction value calculation unit 34 and the K value stored in the K value storage unit 48 are calculated. Then, the enzyme activity value C is calculated and output. Enzyme activity value C = {(ΔAs / Δt) − (ΔARB / Δt)} FvB · K (2)

The operation of the invention of FIG. 1 proceeds as shown in the flowchart of FIG. That is, the reagent blank solution is measured, and its absorbances ARBf, ARBm to ARBn and the amount of change in absorbance per unit time ΔARB / Δt are obtained. The absorbances Asm to Asn of the sample reaction solution are measured, and the change in absorbance per unit time ΔAs / Δt is determined by the least squares method.
Is required. Then, based on the measured absorbances Asm to Asn of the sample reaction solution, the sample reaction solution absorbances Asm 'to Asn' obtained by correcting the sample blank values based on the above equation (1) are obtained. Further, the correction values Fvm to Fvn corresponding to the sample reaction solution absorbances Asm 'to Asn' obtained by correcting the sample blank values are selected, and the average value FvB is calculated. Then, the enzyme activity value C is calculated by the above equation (2) and output.

In order to perform re-measurement using the calculated sample amount for re-measurement vs or the dilution ratio of the sample for re-measurement, FIG.
As shown in, the control unit 54 that prepares the re-measurement sample according to the calculated re-measurement sample amount vs or the dilution ratio of the re-measurement sample, and performs re-measurement using the prepared re-measurement sample. And an absorbance change calculator 56 for calculating the absorbance change ΔAss / Δt from the result of the calculation, and calculates and outputs the enzyme activity value CR from the calculated absorbance change ΔAss / Δt according to the following equation (3). An enzyme activity value calculator 58 is provided. Enzyme activity value CR = {(ΔAss / Δt) − (ΔARB / Δt)} K · Kf (3) Here, Kf is a K value correction coefficient at the time of re-measurement, and is expressed as follows. Kf = (v / vs) (R1 + R2 + vs) / (R1 + R2 + v)

The sample amount vs at the time of re-measurement is obtained as follows. The enzyme activity value C is generally calculated by the following equation (4). C = {(ΔAs / Δt) − (ΔARB / Δt)} (V / v · ε · L) (4) V: total reaction solution amount (= sample amount + reagent solution amount) v: sample amount ε: enzyme reaction Molar extinction coefficient at the measurement wavelength of the reaction indicator substance in L

By substituting V = (R1 + R2 + v) into the equation (4), it can be modified as the following equation (5), and the amount (vs) of the sample for remeasurement at the enzyme activity value C can be known. Here, the sample amount v in the equation (4) is represented by vs.

(Equation 1)

(ΔAs / Δt) in the equation (5) is a rate of change in absorbance per unit time (usually 1 minute) in a steady state of the rate reaction (a state in which the reaction proceeds at a substantially constant reaction rate). However, the steady state of the rate reaction in which the absorbance decreases is when the absorbance of the reaction solution is higher than the rate measurement limit absorbance AL, and the upper limit is the absorbance ARB of the reagent blank solution.
(The absorbance of the reagent blank at the reaction time tn
). If the absorbance range (ARBn-AL) at which the rate can be measured when this rate measuring reagent is used is to be used to the utmost, this can be expressed as ΔAs in equation (5).
Can be considered as

Similarly, for the rate measurement time at the time of re-measurement, since the highest accuracy is obtained when the time between (tn-tm) is utilized to the maximum, Δt in equation (5) is equal to (tn-tm). Can be substituted to calculate the sample amount vs for the remeasurement. Therefore, the expression (5) can be modified as the following expression (6).

(Equation 2)

The change in absorbance of the reagent blank is very small compared to the change in absorbance of the reaction solution, and ΔARB / Δt = 0.
Can be expressed as in the following equation (7).

(Equation 3)

The operation of the invention of FIG. 2 proceeds as shown in the flow chart of FIG. That is, after the enzyme activity value C is obtained in the same manner as the flowchart of FIG. 3 showing the operation of the invention of FIG. 1, the sample amount vs for the re-measurement is calculated according to the equation (7).

If the calculated sample amount vs is equal to or more than the minimum sample amount (vmin) at which the sample of the analyzer can be sampled, re-measurement is performed with the sample amount. The absorbance change ΔAss / Δt is calculated by the least squares method for the rate measurement absorbances Assm to Assn by the re-measurement, and the enzyme activity value CR is calculated by the equation (3) based on the change.

If the calculated sample volume vs is smaller than the minimum sample volume vmin, the original sample cannot be sampled, so the original sample is diluted at a dilution ratio (vs / v), and the diluted sample is sampled. The sample is sampled by the amount v and subjected to re-measurement. The sampling amount at this time is v, but since the original sample is diluted by [vs / v] times, the sample amount in the reaction solution for re-measurement is vs (= v × (vs / v
v)).

[0023]

EXAMPLE As an example of the enzymatic reaction, a case where the activity of LDH (lactate dehydrogenase) shown below is measured will be described as an example.

Embedded image

FIG. 5 explains the outline of the LDH activity measurement by the rate method. Referring to FIG. 5 and FIG.
An embodiment of the present invention will be described. The enzyme reaction is L
Take the activity measurement of DH. The reagent blank solution is measured, and its absorbances ARBf, ARBm to ARBn and the amount of change in absorbance per unit time ΔARB / Δt are obtained. The absorbances Asm to Asn of the sample reaction solution are measured, and the absorbance change amount ΔAs / Δt per unit time is obtained by the least square method.
Then, based on the measured absorbances Asm to Asn of the sample reaction solution, the sample reaction solution absorbances Asm 'to Asn' obtained by correcting the sample blank values based on the above equation (1) are obtained.

Further, correction values Fvm to Fvn corresponding to the sample reaction solution absorbances Asm 'to Asn' corrected for the sample blank value are selected, and the average value FvB is calculated. Then, the enzyme activity value C is calculated by the above equation (2) and output.
Table 1 shows the relationship between the absorbance of the sample reaction solution (36 seconds after the start of the reaction) and the change in absorbance per unit time (ΔA / Δt) for 24 to 48 seconds after the start of the reaction.

[Table 1]

Since a constant reaction rate was obtained with a sample reaction solution having an absorbance of 1.2 or more, this was considered to be the optimum condition. Less than 0.6 was defined as unsteady reaction conditions.
In addition, a correction coefficient Fv for the relative reaction rate, that is, the enzyme activity value under the optimum condition was determined. The correction coefficient Fv is 1.00 for a measured value having an absorbance of 1.2 or more, and Fv> 1.00 for a sample reaction solution having an absorbance of less than 1.2.

FIG. 6 shows the absorbance of the LDH sample reaction solution and the LDH.
It shows the relationship between the reaction rates and plots the results in Table 1.

Table 2 shows the absorbance of the sample reaction solution which could not be measured in Table 1 from the relation curve prepared in FIG.
The reaction rate is read in detail to determine the correction coefficient Fv.

[Table 2]

Table 3 shows that the LDH high activity (high concentration) samples
LDH activity measuring reagents with different DH concentrations (a)-
FIG. 6 shows the activity value measured using (c) and the corrected activity value based on the average corrected value FvB for Fv at each absorbance of the sample reaction solution within the rate measurement time.

[Table 3]

Table 4 shows the measured activity values and the corrected activity values of the samples 1 to 3 in the same manner as in Table 3, using the reagents for measuring the LDH activity at various NADH concentrations.

[Table 4]

In FIG. 5, the absorbance A at the measurement point m is shown.
Although sHm is a value under the steady-state reaction condition, the absorbance (marked by x) after the next measurement point (m + 1) is a value of the non-steady-state reaction solution, and these values accurately determine the activity value of LDH. Since it is not reflected, it has been considered meaningless to calculate the change in absorbance (ΔAs / Δt). Therefore, conventionally, such a highly active sample was determined to be “measurement impossible”, and a re-measurement was newly performed using a diluted sample. However, in the present invention, it is possible to obtain a value equivalent to the rate measurement result under the optimal reaction condition even for the reaction solution of the high activity value sample in the non-steady state.

FIG. 7 shows a state in which re-measurement is performed with a small amount of a highly active sample according to the second invention. The absorbance after the measurement point (m + 1) in the original high-activity sample is the absorbance of the unsteady reaction solution. However, in the remeasurement, the reaction time showing the absorbance under the steady-state reaction condition is reduced by reducing the sample volume in the reaction solution. It becomes longer, and a more accurate enzyme activity value can be obtained. here,
It is characterized in that the sample amount at the time of re-measurement is automatically calculated based on the measured value of the enzyme activity value of the highly active sample.

[0033]

According to the present invention, a somewhat deteriorated reagent can be used, so that the reagent can be used effectively.
In the first aspect of the present invention, the amount of change in absorbance per unit time of the sample reaction solution for enzyme activity measurement is corrected based on the absorbance of the sample reaction solution for enzyme activity measurement. Since a rate measurement value can be obtained even for a highly active enzyme sample that deviates from the reaction conditions, the frequency of retests can be reduced, and reagents and samples are not wasted. In the second aspect of the present invention, when the measurement result is found to be a high-concentration sample, and the re-measurement for obtaining a more accurate enzyme activity value is performed, the sample sampling amount or dilution rate at the time of the re-measurement is automatically adjusted. The re-measurement can be performed easily and with an appropriate sample amount.

[Brief description of the drawings]

FIG. 1 is a block diagram showing a first invention.

FIG. 2 is a block diagram showing a second invention.

FIG. 3 is a flowchart showing the operation of the first invention.

FIG. 4 is a flowchart showing the operation of the second invention.

FIG. 5 is a diagram showing a reaction time course in LDH activity measurement by a rate method.

FIG. 6 is a diagram showing the relationship between the absorbance of an LDH sample reaction solution and the LDH reaction rate.

FIG. 7 is a diagram showing a reaction time course of remeasurement according to the present invention.

FIG. 8 is a graph showing a change over time in absorbance at a measurement wavelength of 340 nm after preparation of an LDH reagent.

[Explanation of symbols]

 2,30 Correction value storage unit 4,32 Correction value selection unit 6,36 Enzyme activity value calculation unit 18,44 Absorbance change amount calculation unit 34 Average correction value calculation unit 38 Reagent blank measurement value storage unit 50 Rate measurement condition storage unit 52 Re-measurement sample volume / dilution ratio calculator

Claims (1)

[Claims] 1. An automatic analyzer for rate measurement for determining an amount of an enzyme in a sample from a change in absorbance per unit time under a constant temperature condition, comprising: a reagent blank measured value storage section for storing a reagent blank solution absorbance; A rate measurement condition storage unit that stores the rate measurement conditions including the reagent amount, the limit absorbance, the end point absorbance, and the measurement time, and the absorbance change per unit time from the absorbance measurement value within the rate measurement time of the sample reaction solution for enzyme activity measurement And a correction value storage unit that stores a correction value for obtaining an amount of change in absorbance per unit time under optimal reaction conditions from the absorbance of the sample reaction solution for enzyme activity measurement. A correction value selection unit that selects a correction value corresponding to the measured absorbance of the sample reaction solution; an average correction value calculation unit that calculates an average value of the selected correction values; An enzyme activity value calculation unit that calculates an enzyme activity value by correcting the measured absorbance change amount per unit time calculated by the amount calculation unit with the average correction value calculated by the average correction value calculation unit; Using the enzyme activity value, the reagent blank measurement value stored in the reagent blank measurement value storage unit, and the rate measurement condition stored in the rate measurement condition storage unit, the sample amount for remeasurement or the sample for remeasurement An automatic analyzer comprising: a remeasurement sample amount / dilution ratio calculation unit for calculating a dilution ratio of the sample.