JPH10104162A - Measurement of enzyme activity - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain an accurate enzyme activity value without performing re-measurement even with respect to a sample of high activity. SOLUTION: With respect to reaction to be measured, the correlation regression formula 1/VR=β/(A-a)+c of the reciprocal of the difference between absorbancy A and absorbancy (a) at the time of completion of enzymatic reaction, and the reciprocal of an absorbancy change relative value VR per unit time is preliminarily calculated and a correction value 1/VR is calculated from the absorbancy measured value of an unknown sample reaction soln. The correction value 1/VR is multiplied by an absorbancy change value dA/dt being the measured value to be corrected before an enzyme activity value is calculated.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for measuring the enzyme activity of a sample, and more particularly to a rate method for determining the enzyme activity value of a sample from a change in absorbance per unit time of a sample reaction solution under constant temperature conditions. The present invention relates to a method for measuring an enzyme activity by the method described above.

[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. A rate measurement is performed in which the amount of change is determined and the enzyme activity value is calculated from the amount of change in absorbance.

[0003] Since automatic biochemical analyzers for clinical use are used in hospitals, public health centers, test centers, and the like, there are samples in which enzyme components show abnormal values (generally high values for enzyme components) in measurement samples. I do. Automated biochemical analyzers define measurement conditions for measuring many samples with a certain level of accuracy. If a very high-concentration (high-activity) sample is encountered, it should be used before or early in the rate measurement time. As the concentration of the substrate as a reagent component decreases, a reaction rate (absorbance change) proportional to the amount of the enzyme cannot be obtained, and a situation occurs in which measurement cannot be performed. For such a high-concentration sample, treatment such as reducing the amount of the sample or diluting the original sample was performed, and a sample reaction solution was prepared again to measure the enzyme activity.

[0004]

However, when the sample reaction solution is prepared again and the enzyme activity is measured, the following inconvenience occurs. Reagents and samples are wasted. And when the sample runs out, it cannot respond. Also, some reagents are expensive and may be costly. This leads to a decrease in processing capacity. There are restrictions on reducing the sample volume. The procedure for diluting the original sample is complicated. When the dilution is performed manually, an error is likely to occur, and the dilution ratio needs to be corrected. If the dilution is to be performed automatically, complicated control is required, and the measurement results must be edited. Takes extra time to get results. Therefore, an object of the present invention is to enable accurate enzyme activity values to be obtained without preparing a test reaction solution again even for a highly active sample.

[0005]

According to the present invention, there is provided a method for measuring the enzyme concentration or enzyme activity of a sample, comprising the following steps (A) to (D). (A) For the reaction to be measured, the correlation regression equation of the reciprocal of the difference between the absorbance A and the absorbance a at the completion of the enzyme reaction at the measurement wavelength and the reciprocal of the relative change in absorbance per unit time VR 1 / VR = β / (A−a) + c (where β and c are slopes and constants of the correlation regression equation, respectively);

(B) With respect to the unknown sample reaction solution, the absorbance A ′ of the reaction solution is measured including the region where the rate measurement cannot be performed because the absorbance exceeds the measurable absorbance, and the change in absorbance per unit time is determined. Seeking step,

(C) Substituting the absorbance A 'of the unknown sample reaction solution in the reaction time range for determining the absorbance change value used for the measurement into A of the above correlation regression equation to obtain 1 / VR, Obtaining an average value of 1 / VR of

(D) The absorbance change value per unit time obtained above is multiplied by the average value of 1 / VR obtained above to correct the absorbance change value, and an unknown sample is determined based on the corrected absorbance change value. Determining the enzyme activity value of

The change in absorbance per unit time is (ΔA /
Δt), when the average value of 1 / VR is (1 / VR) B and the enzyme concentration or the enzyme activity value is C, C = (ΔA / Δt) × (1 / VR) B × K. Here, K is a device constant, and is expressed as follows. K = (1 / εL) × (W / w) ε: molar extinction coefficient of the reaction indicator. In the LDH activity measurement, NADH is a reaction indicator substance w: sample amount W: reaction solution amount (= sample amount + reagent amount)

[0010]

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.

[0011]

Embedded image

Reagents prepared based on this reaction principle (manufactured by International Reagents, reagent components: NADH 0.24 mM, pyruvate)
1.5 mM, Tris buffer 50 mM, pH 7.8)
The relationship between the NADH concentration of the reagent component and the LDH activity value in the serum sample was examined. Table 1 shows the results.

[0013]

[Table 1]

The data in Table 1 show the results of the measurement of the time course of the LDH reaction and the change in absorbance for the reaction of the high LDH sample. The reaction time course is shown schematically in FIG. [1 / (A340-0.048)] in Table 1 indicates a measurement wavelength of 340.
The absorbance 0.048 at the time when the LDH reaction was completed (the NADH in the reaction solution was completely eliminated) from the absorbance A340 at nm.
This is the reciprocal of the value obtained by subtracting (not shown in the table, but actually measured value). v (reaction rate: change in absorbance per minute) is the absorbance An-1 and An-1 before and after the reaction time of 12 seconds.
The difference of +1 converted per minute, [1 / v] is the reciprocal thereof. VR is a relative value based on v at 0.2 mM (absorbance 1.26) or higher, which is regarded as the optimum NADH concentration for LDH reaction, and [1 / VR] is its reciprocal. Here, v when A340 = 1.286 was used as a reference. Therefore, [1 / VR] is used to correct the measurement result (absorbance change value ΔA / Δt per minute) of the sample reaction solution having the non-optimal NADH concentration to the measurement value of the sample reaction solution having the optimum NADH concentration. This is a relative correction value.

FIG. 2 illustrates the data of Table 1. The horizontal axis is [1 / (A340-0.048)], and the vertical axis is [1 / v] and [1
/ VR]. [1 / VR] and [1 / (A340-0.04
8)] is obtained as 1 / VR = 0.696 / (A-0.048) +0.454. For the LDH reaction, 1 / VR is calculated from the measured absorbance using this correlation regression equation, and the average value in the reaction time range used for the measurement is calculated to correct the measurement result of the unknown sample reaction solution.

Next, Table 2 and Table 3 show the results obtained by determining the enzyme activity value by applying to a high LDH sample. Table 2 shows three types of high L
For the DH sample, the absorbance A340 at a measurement wavelength of 340 nm was measured, and the relative correction value 1 / VR corresponding to each absorbance was determined as shown in FIG.
Is a result obtained from the correlation regression equation.

[0017]

[Table 2]

Table 3 shows absorbance change values ΔA / Δt in each of three reaction time ranges based on the measurement results.
The figure shows the result of calculating the average value (1 / VR) B of 1 / VR, correcting ΔA / Δt by multiplying ΔA / Δt by (1 / VR) B, and calculating the activity value C. The activation value C is represented by the following equation. C = (ΔA / Δt) × (1 / VR) B × K

[0019]

[Table 3]

In order to verify the results obtained, each LDH sample was diluted 1/10 and measured as a sample having an optimum NADH concentration. The activity values of Samples I, II and III were 1245 U, respectively. / L, 1902U / L, 628
It became 0U / L. The results in Table 3 are close to the results of dilution measurements, indicating that the correction according to the present invention is an effective alternative to dilution re-measurement.

[0021]

According to the present invention, by using the correction value 1 / VR obtained from the correlation regression equation, the correct enzyme activity value can be obtained without performing dilution re-measurement, even if the measurement result is obtained using a sample under non-optimal conditions. Can be derived. As a result, the following effects can be achieved. (1) The measurement results can be immediately used for patient diagnosis. In the case of an ultra-high concentration sample, the accuracy may be as low as 10 to 30%, but from a clinical point of view, it is often not necessary to precisely measure the amount of enzyme in the high concentration region. Rather,
Since a rough value may be used, obtaining the measurement result is prioritized. (2) It is possible to cope with a shortage of the sample. Also, reagents can be saved. (3) When the need for re-measurement arises, the optimum measurement conditions, that is, the sample amount can be known.

[Brief description of the drawings]

FIG. 1 is a diagram showing a time course of a reaction according to one embodiment.

FIG. 2 shows a correlation regression equation of a reaction according to one embodiment.

Claims (1)

[Claims] 1. A rate method for obtaining an enzyme concentration or an enzyme activity value of a sample from a change in absorbance per unit time of a sample reaction solution under a constant temperature condition, wherein the rate method includes the following steps (A) to (D). Characteristic enzyme activity measurement method. (A) For the reaction to be measured, the correlation regression equation of the reciprocal of the difference between the absorbance A and the absorbance a at the completion of the enzyme reaction at the measurement wavelength and the reciprocal of the relative change in absorbance per unit time VR 1 / VR = β / (A−a) + c (where β and c are the gradients and constants of the correlation regression equation, respectively). (B) For the unknown sample reaction solution, the rate measurement exceeds the rate-measurable absorbance and the rate measurement is not possible. Measuring the absorbance A 'of the reaction solution including the region where it became possible to determine the change in absorbance per unit time; and (C) determining the change in absorbance used for the measurement. The absorbance A ′ of the solution was substituted into A of the above correlation regression equation to obtain 1 / VR, and 1 / VR in the reaction time range was obtained.
(D) correcting the absorbance change value by multiplying the absorbance change value per unit time determined above by the average value of 1 / VR determined above, and correcting the corrected absorbance change value Determining the enzyme concentration or enzyme activity value of the unknown sample based on