JP3027061B2 - Reaction measurement method - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a reaction measuring method suitable for use in a biochemical analyzer.

[0002]

2. Description of the Related Art In a biochemical analyzer, as shown in FIG. 3, a sample such as serum and a predetermined reagent are mixed in a transparent reaction cell 1 to perform a color-forming reaction. White light is emitted, and the transmitted light is separated by the spectroscope 3 to determine the absorbance of the dye, and the concentration of the substance to be measured is determined based on the absorbance.

Conventionally, as a method for obtaining the concentration of a substance to be measured, two methods, an end point method and a rate method, have been adopted. As shown in FIG. 4, the endpoint method is a method used when the amount of the dye generated by the color reaction is saturated with the elapse of the reaction time, and after the sample and the reagent are sufficiently reacted. This is a method for determining the concentration of the substance to be measured based on the final absorbance A. On the other hand, the rate method is a method used when the amount of a dye produced by a color reaction increases with the lapse of reaction time, such as when measuring enzyme activity in a sample,
In this case, as shown in FIG. 5, a linear function y = at + b is approximated by the least square method to the absorbance y measured during the reaction, and the substance to be measured is determined based on the value of the coefficient a obtained as a result. Is required.

[0004]

However, the conventional concentration measuring method has a problem in accuracy. That is, in the concentration measurement by the end point method, good results can be obtained if the reaction time is sufficiently long. However, in the biochemical analyzer, the concentration measurement of a predetermined substance must be performed for many samples. Therefore, it is not possible to spend much time in measuring the concentration of one substance. As a result, before the color reaction is completed, the absorbance is measured at the time shown by a point P in FIG. In many cases, the absorbance at that time must be determined as the final absorbance A to determine the concentration of the substance, and the accuracy has been a problem.

In the concentration measurement by the rate method, the absorbance at the measurement point is theoretically plotted on one straight line as shown in FIG. 5, but in actuality it is shown in FIG. 6 due to various factors. In some cases, a curve is drawn as shown, and in such a case, it is difficult to approximate the linear function as described above, and the accuracy of the density obtained as a result is not good.

An object of the present invention is to solve the above-mentioned problem, and an object of the present invention is to provide a reaction measuring method capable of accurately determining the concentration of a substance to be measured.

[0007]

In order to achieve the above object, a reaction measuring method of the present invention comprises reacting a sample with a reagent in a cell, irradiating the cell with light, and measuring transmitted light. A reaction measurement method for measuring the amount of a predetermined substance contained in a sample, wherein the relationship between the absorbance of the reaction and time is determined by the least squares method using the measured data, and the final absorbance is A,
The initial absorbance of the reaction was B, the reaction rate constant was K, the time was t, and the absorbance at the time of measurement by the reaction between the sample and the reagent was y, which was approximated by y = A + ( ^BA ) / e ^Kt , and the result was obtained. The amount of the substance to be measured is determined based on the final absorbance A, the initial reaction absorbance B, and / or the reaction rate constant K.

[0008]

In the present invention, the relationship between the absorbance and the time of the reaction is determined for the substance which should be measured by the end-point method and also for the substance which should be measured by the main body rate method. ^Is approximated by y = A + ( ^BA ) / e ^Kt by the least square method using the data of Here, y is the measured absorbance, A is the final absorbance, B is the initial absorbance of the reaction, K is the reaction rate constant, and t is the measurement time. Then, the concentration of the substance to be measured is determined based on the determined final absorbance A, initial reaction absorbance B, and rate constant K.

[0009]

Embodiments will be described below with reference to the drawings.
FIG. 1 is a flowchart of the process of the reaction measurement method according to the present invention. First, a reagent is mixed with a sample in a reaction cell, and photometry is performed a predetermined number of times within a predetermined measurement time. As a result, a predetermined number of combinations of the absorbance y _n (n = 1, 2,..., M) and the time t _n are obtained. Then, using these measurement data, the relationship between the absorbance y and the time t is approximated by y = A + ( ^BA ) / e ^Kt by the least square method. This formula is derived from the reaction kinetics, where A is the final absorbance, B is the initial absorbance of the reaction, K
Is the reaction rate constant.

When the above equation is obtained as described above, the concentration of the substance to be measured is calculated next. If the substance is a substance to be measured by the end point method, Then, the concentration is calculated by multiplying (AB) obtained by subtracting the initial absorbance B of the reaction from the final absorbance A (AB) by a predetermined coefficient.

When the substance is a substance to be measured by the rate method, there are the following two methods for calculating the concentration. First, it is possible to calculate the concentration by differentiating the above equation with time to obtain a rate of change in absorbance with time, substituting a predetermined time t _O therefor, and further multiplying by a predetermined coefficient. The time to be used as t _O can be determined experimentally or empirically.

As a second method, two specific times t _P1 and t _P2 (> t _P1 ) are substituted into the above equation, and the absorbances y _P1 and y _P2 at that time are obtained, and (y _P2 −y _P1 ) /
(T _P2 -t _P1) performs operation determined time rate of change of the absorbance at time t _P1 ~t _P2 of, there is a method to determine the concentration it is multiplied by a predetermined coefficient. The time to be used as t _P1 and t _P2 may be determined experimentally or empirically. Employment of the first method or the second method is optional.

In the above example, the reaction rate constant K is determined from the measured data. However, it is known that the value of K is constant if the reaction system is constant. It is also possible to determine K by performing measurement using a sample and treat it as a constant when measuring a sample to be measured.

Next, the configuration of a biochemical analyzer employing the reaction measurement method of the present invention will be described with reference to FIG.
Note that the same components as those in FIG. 3 are denoted by the same reference numerals.

In FIG. 2, a specimen and a predetermined reagent are mixed in a reaction cell 1, and white light is emitted from a light source 2 to the mixture. The light transmitted through the reaction cell 1 is separated by the spectroscope 3, and the absorbance is measured for each wavelength. The measured absorbance value is converted to an analog / digital converter (AD
C) digitized by 4 and paired with the measurement time and written to memory 5;

The arithmetic unit 6 is composed of an appropriate processor and its peripheral circuits. The relation between the absorbance y and the time t is calculated by the least square method using the absorbance and the time value written in the memory. Approximate by A + ( ^BA ) / ^eKt . Here, A is the final absorbance, B is the initial absorbance of the reaction, and K is the reaction rate constant.

Whether the substance to be measured now is to be measured by the end point method or the substance to be measured by the rate method is registered in advance in the calculation unit 6, and the substance to be measured by the end point method is registered in advance. , The concentration is calculated by multiplying the obtained value of the final absorbance A by a predetermined coefficient, and if the substance is to be measured by the rate method, the above equation obtained by the least squares approximation is calculated as Differentiation is performed, a predetermined time set in advance is substituted for the obtained value, and the obtained value is multiplied by a predetermined coefficient to calculate the density.

When the substance to be measured is a substance to be measured by the rate method, the two times t _P1 and t _P2 (> t substituting _P1), the absorbance y _P1, y _P2 at that time _{determined, (y P2 -y P1) /} (t P2 -t P1) time rate of change of absorbance at performs operation time t _P1 ~t _P2 of , And a predetermined coefficient may be multiplied to obtain the density.

Although the embodiments of the present invention have been described above, the present invention is not limited to the above embodiments, and various modifications are possible. For example, in the above-described embodiment, the light transmitted through the reaction cell is spectrally detected by the spectroscope 3 and detected. However, a spectroscope is disposed between the light source 2 and the reaction cell 1 so that the separated light is reflected on the reaction cell. And may be configured to be detected by transmitting through.

[0020]

As is clear from the above description, according to the present invention, the data obtained at a plurality of measurement points were used to approximate the above equation derived from the reaction kinetics, and the result was obtained. Since the concentration is obtained based on the value of the coefficient, the concentration of the substance to be measured can be obtained with high accuracy.

[Brief description of the drawings]

FIG. 1 is a flowchart for explaining a process of a reaction measurement method of the present invention.

FIG. 2 is a diagram showing a configuration example of a biochemical analyzer employing the reaction measurement method of the present invention.

FIG. 3 is a diagram for explaining biochemical analysis.

FIG. 4 is a diagram for explaining concentration measurement by an end point method.

FIG. 5 is a diagram for explaining concentration measurement by a rate method.

FIG. 6 is a diagram for explaining a problem of a conventional concentration measuring method.

[Explanation of symbols]

DESCRIPTION OF SYMBOLS 1 ... Reaction cell, 2 ... Light source, 3 ... Spectroscope, 4 ... ADC, 5
... Memory, 6...

────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Miyoshi Tanabe 3-1-2 Musashino, Akishima-shi, Tokyo Japan Electronics Co., Ltd. (58) Field surveyed (Int. Cl. ⁷ , DB name) G01N 21 / 75 G01N 31/00

Claims (1)

(57) [Claims] 1. A reaction measurement method comprising reacting a sample and a reagent in a cell, irradiating the cell with light, measuring transmitted light, and measuring the amount of a predetermined substance contained in the sample. The relationship between the absorbance of the reaction and the time is measured by the least squares method using the measured data, and the final absorbance is A, the initial reaction absorbance is B, the reaction rate constant is K, the time is t, and the measurement is performed by the reaction between the sample and the reagent. The amount of the substance to be measured based on the final absorbance A, the initial reaction absorbance B and / or the reaction rate constant K obtained by approximating y = A + ( ^BA ) / e ^Kt with the absorbance at the time as y. A reaction measurement method characterized by determining