JPH10282000A - Enzyme activity measurement - Google Patents

Abstract

PROBLEM TO BE SOLVED: To detect and reject an abnormal value for attaining accurate measured results by sequentially-calculating an gradient between respective photometry points in reacting process to obtain respective relative values, and comparing them with preset permissible values for confirmation of the linearity of a change in an absorption value. SOLUTION: In this enzyme activity measurement, such as GOT, which permits enzyme to react in a container disposed on a rotating disc and obtains the enzyme activity by the change of absorbancy, absorbancy measuring is conducted at a prescribed number of photometry points of a reaction time course. Gradient (absorbancy variance per unit-hour) is computed sequentially from the photometry point lm where calculation preset as analysis parameter is started, through points lm+1 , lm+2 ,..., the point ln where the calculation is completed. A relative value is calculated by a prescribed method for respective calculated gradients, and comparative discrimination with permissible value preset arbitrarily is conducted. By rejecting the absorbancy at the point which is discriminated to be abnormal compared with the permissible value, and obtaining absorbancy changing speed by the method of least squares from remaining data, it is possible to calculate the enzyme activity.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] The present invention relates to a method for measuring enzyme activity.

[0002]

2. Description of the Related Art Enzyme activity measurement methods in biochemical tests are based on:
As shown in JP-A-57-33956 and JP-A-58-4918, the activity of an enzyme is determined by reducing the amount of one of the reactants in a series of reactions involving the target enzyme as a decrease in absorbance. And a method of increasing the absorbance of an enzyme by determining the activity of the enzyme by regarding the increase in a kind of reactant in a series of reactions involving the target enzyme as a decrease in the absorbance. The principle of the measurement method will be described below by taking GOT as an example of the measurement system in the reduction method.

[0003] GOT measurement principle

[0004] That is, the measurement principle of the absorbance reduction method for determining the activity of GOT is based on a series of reactants, NAD.
The amount of decrease in H is measured as a method of decreasing absorbance at 340 nm, and the activity value of the target enzyme is determined from the decrease rate. Next, an example of actual measurement will be described with reference to a GOT reaction time course in FIG. Each time the reaction container serving as the optical cell into which the test sample and the reagent are dispensed rotates and passes through the optical axis of the photometer, the absorbance at the main wavelength of 340 nm and the sub wavelength of 405 nm is measured. Subsequently, the photometry point (l
~ M) by the method of least squares (NADH → NAD)
Rate of change to ⁺ : change in absorbance per unit time)
, And the value is multiplied by a calibration coefficient to determine the GOT activity value. That is, as in the case of the GOT, the enzyme activity measurement method is a method for determining the rate of change in absorbance by the least squares method, and thus has the following check function. One is a function that compares the slope of the first few points and the slope of the last few points between photometric points, and checks whether the rate of decrease or increase in absorbance is progressing linearly. Is a function to check whether the reaction has reached a saturated state when the enzyme activity is high by setting the limit value of

[0005] Fig. 3 shows an example in which a defective measurement result is generated despite checking by this method. 3 (a) and 3 (b) are examples of a time course when dust enters the reaction solution and the absorbance increases only at one point, and FIG. 3 (c) shows the deterioration of the halogen lamp as the light source. It is an example of a time course when the absorbance varies.
In (a), a data alarm is generated by checking the linearity of the device, but in (b) and (c), although the time course is clearly abnormal, it is determined to be normal and an incorrect measurement result is output. This is an example. This can be said to have occurred because the check of the linearity of the reaction in the prior art only checked the slope of the first few points and the slope of the last few points of the photometry point. In addition, since the slope at the time of checking is obtained by the least square method, it is determined that the light is normal despite abnormalities as in the example of FIG. Therefore, it resulted in inaccurate measurement results.

[0006]

That is, in the conventional enzyme activity measurement method, the slope of the first several points between the photometric points and the slope of the last several points are compared, and the rate of decrease or increase in the absorbance is linear. It was not possible to accurately detect abnormalities in absorbance between photometric points because it was confirmed whether or not the process had proceeded. This resulted in inaccurate measurement results.

It is an object of the present invention to accurately confirm whether the rate of decrease or increase in absorbance is linear, to detect and reject abnormal absorbance to obtain correct measurement results, An object of the present invention is to improve inspection work efficiency.

[0008]

In order to achieve the above object, the present invention uses the following technical means.

[0009] (1) the inclination of between the metering point (l _m to l _n), the slope of l _m and l _{m + 1,} the slope of l _m and l _{m + 2,}
..., the slope between l _m and l _n, the slope between l _{m + 1} and l _{m + 2} , ..., l
the inclination of the _{m + 1} and l _n, and sequentially providing the function of calculating.

(2) A function of calculating a relative value for each calculated slope is provided, and a comparison is made with an arbitrarily set allowable value.

(3) When an abnormality is detected by (2), a function is provided for rejecting the absorbance at the photometry point.

(4) A function is provided for outputting a data alarm when the absorbance at a normal photometry point is equal to or less than an arbitrarily set number of photometry points.

(5) When the absorbance at a normal photometry point is equal to or more than an arbitrarily set number of photometry points, a function is provided for calculating a slope by the least squares method and calculating an enzyme activity value of a target enzyme.

[0014]

FIG. 1 shows an embodiment of an automatic chemical analyzer using the present invention. The apparatus comprises a sample disk 2 on which a plurality of sample cups 1 can be mounted, a sampling mechanism 4 having a sample probe 3 for collecting a predetermined amount of sample, and a reagent pipetting mechanism 5 for dispensing a plurality of reagents.
a, 5b, reagent disks 6a, 6b, a reaction disk 8 holding a plurality of direct photometric reaction vessels 7, a stirring mechanism 9
a, 9b, a reaction vessel cleaning mechanism 10, a photometer 11, a central processing unit (microcomputer) 12 for controlling the entire mechanical system, and the like. The reaction disk 8 holding a plurality of reaction vessels is controlled to perform a half-rotation + 1 rotation of the reaction vessel every cycle to temporarily stop the reaction vessel. That is, at the time of stopping for each cycle, the reaction vessels 7 of the reaction disk 8 are stopped in such a manner that they have advanced counterclockwise one reaction vessel at a time. As the photometer 11, a multi-wavelength photometer having a plurality of detectors is used.
When the reaction disk 8 is in a rotating state, the rows of the reaction vessels 7 pass through the light beam 14 from the light source lamp 13. Position of light beam 14 and sample discharge position 15
Between them, a reaction vessel cleaning mechanism 10 is provided. Further, a multiplexer 16 for selecting a wavelength, a logarithmic conversion amplifier 17, an A / D converter 18, a printer 19, a CRT 20,
It comprises a reagent dispensing mechanism driving circuit 21 and the like, all of which are connected via an interface 22 to the central processing unit 12.
It is connected to the. This central processing unit also performs control of the entire apparatus including control of the entire mechanical system and data processing such as concentration or enzyme activity value calculation. The operation principle of this configuration will be described below.

When the start switch on the operation panel 23 is pressed, the washing of the reaction vessel 7 is started by the reaction vessel washing mechanism 10, and the water blank is measured. This value serves as a reference for the absorbance measured in the reaction vessel 7 thereafter. One cycle of operation of the reaction disk 8, ie, anti-rotation +
When the process proceeds to the sample discharge position 15 by repeating the operation of causing one reaction container to temporarily stop, the sample cup 1 moves to the sampling position. Similarly, two reagent disks 6
a and 6b also move to the reagent pipetting position. During this time, the sampling mechanism 4 operates, and for example, the sample amount of the analysis item A is sucked from the sample cup 1 by the sample probe 3 and then discharged to the reaction container 7. On the other hand, when the sampling mechanism is discharging the sample to the reaction container 7, the reagent pipetting mechanism 5a
Starts operation and analysis item A installed on reagent disk 6a
Is aspirated by the reagent probe 24a. Next, the reagent probe 24a moves onto the reaction container 7 and discharges the sucked reagent. Then, the inner wall and the outer wall of the probe are washed in the probe washing tank to prepare for the next reagent B of the next analysis item B. Photometry is started after the addition of the first reagent. The photometry is performed when the reaction disk 8 rotates and the reaction container 7 crosses the light flux 14. When the reaction disk rotates 2 rotations + 2 reaction vessels after the addition of the first reagent, the stirring mechanism 8a operates to stir the sample and the reagent. When the reaction container 7 has been rotated from the sample dispensing position by 25 rotations + 25 reaction containers, that is, to the second reagent dispensing position, the second reagent is added from the reagent probe 24b, and thereafter, stirring is performed by the stirring mechanism 8b. Will be The reaction disk 8 traverses the light beam 14 one after another by means of the reaction disk 8 and the absorbance is measured each time. These absorbances are measured 50 times in total in a reaction time of 10 minutes. After the photometry, the reaction vessel 7 is washed by the reaction vessel washing mechanism 10 and is ready for the next sample measurement. The measured absorbance is converted into a concentration or an enzyme activity value by the central processing unit 12, and the analysis result is output from the printer 19.

[0016] In this operation, the absorbance is measured, l _{m + 1} from the metering point l _m to initiate operation set as analysis parameter as in the example shown in FIG. _{4, l m + 2, l} m + 3, ...
.., The slope (the amount of change in absorbance per unit time) is sequentially calculated between the photometric points l _{n at} which the calculation ends. First calculating the gradient of _{l m} points and l _{m + 1} points of the starting point seeking .DELTA.A1, followed by l _m and l _{m + 2} (ΔA2),
l _m and _{l m + 3 (ΔA3),} l m and l _{m + 4} (ΔA4),
.., And the slope of l _m and l _n (ΔAn) is calculated. Further l
_{m + 1} and l _{m + 2} (ΔB1), l _{m + 1} and l _{m + 3} (ΔB2), l
_The slopes of _{m + 1} and l _{m + 4} (ΔB3),..., l _{m + 1} and l _n (ΔXn) are calculated. That is, calculates an inclination in sequence from l _m point to l _n points. Subsequently, relative values (ΔA2 / ΔA1, ΔA3 / ΔA) are calculated for the calculated slopes.
1, ΔA4 / ΔA1,... ΔAn / ΔA1,.
ΔX1) is calculated, and a comparison is made with an arbitrarily set allowable value. Table 1 shows examples performed at photometering points 30 to 40 of the GOT.

[0017]

[Table 1]

If the allowable value of the relative ratio is assumed to be 0.8 to 1.2, it can be determined that 37 points and 39 points are abnormal. Then, the absorbance at 37 points and 39 points is rejected, and the slope (NADH → NAD) is calculated by the least squares method.
⁽ Rate of change to ⁺ ) is obtained, and the value is multiplied by a calibration coefficient to obtain the GOT activity value. According to the present invention, by detecting an abnormal absorbance which cannot be detected conventionally and rejecting the absorbance, a correct measurement result can be obtained and the working efficiency of a biochemical test can be improved.

[0019]

As described above, the slope between each photometric point in the reaction process is sequentially obtained, and the comparison with a predetermined allowable value is performed to detect the abnormal absorbance and to detect the abnormal absorbance. When the detection is detected, the absorbance at the photometric point is rejected and the amount of change in the absorbance is calculated, so that a correct measurement result can be obtained and the work efficiency of the biochemical test can be improved.

[Brief description of the drawings]

FIG. 1 is an explanatory view showing one embodiment of an automatic chemical analyzer according to the present invention.

FIG. 2 is a characteristic diagram showing an example in GOT.

FIG. 3 is a characteristic diagram showing an example of a failure in GOT.

FIG. 4 is an explanatory diagram showing an example of a method of obtaining a tilt in the present invention.

[Explanation of symbols]

DESCRIPTION OF SYMBOLS 1 ... Sample cup, 2 ... Sample disk, 3 ... Sample probe, 4 ... Sampling mechanism, 5 ... Reagent pipetting mechanism, 6 ... Reagent disk.

Claims (2)

[Claims] A reaction solution is formed by dispensing a sample and a reagent into a plurality of reaction vessels arranged on a circular disk, and the disk rotates to pass the reaction vessel through an optical path of a photometer. In an automatic chemical analyzer that measures the absorbance of the reaction solution in accordance with the above, the amount of change in absorbance between each photometric point in the reaction process of the sample is sequentially obtained, and a comparison with a preset allowable value is performed, and abnormal determination is performed. A method for measuring enzyme activity, comprising detecting absorbance. 2. An enzyme activity measuring method according to claim 1, wherein a function is provided for rejecting an abnormal absorbance when an abnormal absorbance is detected, and a slope is determined from the remaining absorbance to determine an enzyme activity.