JPS6181774A - Apparatus for determination of enzymatic activity - Google Patents

Abstract

PURPOSE:To determine the enzymatic activity of even a specimen having abnormal enzymatic activity only by single determination procedure, by measuring the time wherein the absorbance variation reaches a preset level, calculating the absorbance variation of the specimen per unit time from the measured time, and determining the enzymatic activity from the calculated value. CONSTITUTION:The light 4 having a specific wavelength emitted by the spectrophotometer 3 and transmitted through the specimen 2 is detected by the detector, and when the detected signal reaches a preset level, a measurement-terminating signal is transmitted to operate the calculation means 7. The time (t) wherein the absorbance variation of the specimen 2 from the start of the reaction reaches the preset variation level (DELTAAt) stored in the calculation means 7 is measured by the measuring means 6, and the ratio DELTAAt/t (absorbance variation of the specimen 2 per unit time) is calculated by the calculation means 7 to determine the enzymatic activity of the specimen 2. The enzymatic activity is calculated by the formula DELTAAt/t1 in the case of abnormal reaction, and DELTAAt/t2 is the case of normal reaction B.

Description

DETAILED DESCRIPTION OF THE INVENTION (a) Field of Industrial Application This invention relates to an enzyme activity measuring device, and more particularly to an enzyme activity measuring device suitable for measuring the amount of enzyme in a sample.

(b) Prior Art Generally, reaction systems are known in which an enzyme in a sample and a substrate for the enzyme come into contact to cause a reaction, thereby producing a reaction product. In this reaction system, for example, if the substrate does not have light absorption properties and the reaction product has light absorption properties, the amount of enzyme in the sample, that is, the enzyme activity value, can be measured from the rate at which the absorbance increases during the reaction. .

Conventionally, when measuring enzyme activity values using the above principle, a reaction time that allows most enzyme activity values in different samples to be measured is set empirically in advance, and the amount of change in absorbance during that reaction time is measured. was being measured.

However, if the reaction time is predetermined in this way, it is possible to measure samples such as biological samples whose enzyme activity values may have a normal value and an abnormal value (high value) of more than two orders of magnitude. In this case, the absorbance of a specimen having an abnormal value may reach the measurement limit of the enzyme activity device before the reaction time elapses, making it impossible to measure the enzyme activity value.

In this case, the sample was diluted and measured again.
For example, in cases where it is difficult to leave a sample in advance for re-measurement, such as when the amount of the sample is small, there is an inconvenience that the sample cannot be re-measured.

(c) Purpose This invention was made in view of the above circumstances, and its main purpose is to change the amount of absorbance change per unit time from the time it takes for the amount of change in absorbance during the reaction to reach a preset amount of change in absorbance. The purpose of the present invention is to determine the enzyme activity value of a specimen and to enable the enzyme activity value of the specimen to be measured in - times without re-measurement, even if the specimen has an abnormal enzyme activity value.

(D) Structure This invention is an enzyme activity measurement method that measures the amount of enzyme in a specimen by bringing an enzyme in a specimen into contact with a substrate for the enzyme and measuring the amount of change in absorbance of the resulting reaction product. In the apparatus, when the amount of change in absorbance during the reaction reaches a preset absorbance change N (ΔAt), the arrival time (
t), and if the ΔAt is not reached even after a preset time (T) has elapsed, a measuring means for measuring the amount of change in absorbance (ΔA) at that T time, and a measuring means for measuring the ΔAT and t measurement signal. Based on ΔA T / T or ΔAt/t
This is an enzyme activity measuring device comprising a calculating means for calculating.

That is, the present invention differs from the conventional method in that the amount of enzyme in the sample is measured from the time taken until the amount of change in absorbance reaches a preset amount of change in absorbance.

(e) Examples The present invention will be described in detail below based on examples shown in the drawings. Note that this invention is not limited thereby.

In Fig. 1, the enzyme activity measuring device (t) is used to measure sample (2).
Transmitted light of a specific wavelength from the spectroscope (3) (4)
is detected by a detector (5), and the detection signal is measured to measure the enzyme activity value in the sample.

Calculating means (7) comprising measuring means (6) for measuring time are electrically connected to the detector (5). Measuring means (
6) is a clock that oscillates clock pulses, and the detector (
The calculation means (7) is operated based on the detection signal of step 5), and the calculation means (7) is operated based on the measurement time end signal that occurs when the detection signal reaches a preset value.

Specific examples of the calculation means (7) include a microcomputer, an operational amplifier, and the like.

Next, the operation of the above device (t1) will be explained.

First, a preset absorbance change amount (ΔAt) is input to the calculation means (7). Next, the time from the start of the reaction of sample (2) until the amount of change in absorbance reaches ΔAt (
t) is measured by the measuring means (6). Then, the calculation means (
In 7), ΔAt/t is calculated to measure the amount of change in absorbance of the sample (2) per unit time. Thereafter, the enzyme activity value in the sample (2) is determined in the same manner as before. .

In Figure 2, in the reaction (A) where the enzyme activity value is an abnormal value,
ΔA t / t 1, and ΔA / t in the normal value reaction (B).
12 is calculated. Note that the middle part of the figure shows the absorbance measurement limit at the same level as the conventional one.

By configuring the device (t1) as described above, even if a sample has an abnormal enzyme activity value, the enzyme activity value can be measured in one go without diluting the sample, thereby eliminating the need to waste the sample. disappears.

In addition, in the case of a sample with a very slow reaction rate, such as an abnormal sample with an extremely low amount of enzyme, or a blank sample used to determine the amount of change in only the reagent, it may take a long time to reach Δ^L. Since it is not practical, in case of such a case,
The reaction time limit (T) is set in advance, and the sample (2
) When the reaction time reaches time T during measurement, the amount of change in absorbance (ΔAT) at time T is measured. Then, ΔAT/T is calculated to measure the enzyme activity value in the sample.

Also, if T is set for a long time, the enzyme activity value will be small.
In other words, it is possible to improve the measurement sensitivity (measurement accuracy) even for samples that react slowly.

(F) Effect This invention is configured to measure the amount of enzyme in a sample by calculating the amount of change in absorbance of the sample per unit time from the time until the amount of change in absorbance reaches a preset amount of change in absorbance. Therefore, even if the sample has an abnormally high enzyme activity value, there is no need to dilute the sample and re-measure it.
The amount of enzyme in the sample can be measured in - times of measurement, and even if the amount of enzyme in the sample is small, that is, the reaction is slow, the enzyme amount can be accurately measured.

[Brief explanation of drawings]

FIG. 1 is an explanatory diagram of the main part configuration showing one embodiment of the enzyme activation device according to the present invention, and FIG. 2 is a graph of the amount of change in absorbance. (t)...-enzyme activation device, (2)--one sample, (6)
...-Measuring means, (7)--Calculating means. tJ2 Figure tl t2TIME

Claims (1)

[Claims] 1. Enzyme activity measurement in which an enzyme in a specimen is contacted and reacted with a substrate for the enzyme, and the amount of change in absorbance of the resulting reaction product is measured to measure the amount of enzyme in the specimen. In the device, the amount of change in absorbance during the reaction is determined by the amount of change in absorbance set in advance (
When ΔAt) is reached, the arrival time (t) is measured, and if the ΔAt is not reached even after a preset time (T), the amount of absorbance change (ΔA
An enzyme activity measuring device comprising a measuring means for measuring ΔA_T) and a calculating means for calculating ΔA_T/T or ΔAt/t based on the ΔA_T and t measurement signal.