JPH02223859A - Biochemical analysis - Google Patents

Abstract

PURPOSE:To exactly determine a desired component by mixing a prescribed reaction reagent with a sample liquid to effect reaction, determining an estimated optical density value at the time of the start of the reaction by the regression equation obtd. by a method of least squares from the plural actually measured optical density values and determining an actually measured optical density value at the time of the end of the reaction. CONSTITUTION:The prescribed reaction reagent is mixed with the sample liquid. The absorbance data is determined from the right after the start of the reaction in an absorbance fluctuating region A and the quadratic regression equation y=at<2>+bt+c is determined by the method of least squares with the measurement groups from alpha to beta thereof. The value of t=0, i.e., y0 at the time of the start of the reaction is determined from this equation. The absorbance ys in the absorbance stationary region B after the end of the reaction is actually measured to determine ys-y0 and the concn. is calculated therefrom.

Description

DETAILED DESCRIPTION OF THE INVENTION (a) Field of Industrial Application This invention relates to a biochemical analysis method. For more details,
In particular, it relates to an analysis method useful for quantifying a predetermined component in a biochemical sample containing multiple components such as serum, plasma, urine, lymph fluid, etc.

(B) Conventional technology Conventionally, as a method for analyzing predetermined components in a biochemical sample as described above, one or more predetermined reaction reagents are mixed and reacted in a sample solution, and the components are analyzed after the reaction is completed. A method is known in which the concentration of a predetermined component is calculated based on the optical density value (absorbance value, fluorescence value, scattered light intensity value, etc.) of a sample liquid.

As a specific analysis method, the target component is quantified from the difference between the post-reaction optical density value of a blank solution with zero target component concentration and the post-reaction optical density value of the sample to be analyzed. This is known as the endpoint method.

(c) Problems to be Solved by the Invention However, in the above-mentioned endpoint method, the optical density value of the blank solution itself tends to fluctuate due to changes in the state of the reaction reagent, fluctuations in the output of the measuring device system, etc. It was necessary to measure the optical density value of the liquid and calibrate the baseline for quantitative determination, which was complicated. Furthermore, this method has the problem that large errors occur in quantitative values due to the influence of optical interference components such as bilirubin, hemoglobin, and turbidity components that may coexist in the sample.

In order to solve this problem, a so-called two-point method is also known in which the optical density during the reaction is measured a predetermined time after the start of the reaction, and the amount is quantified based on the difference between this value and the optical density after the completion of the reaction. In the two-point method, the optical density measurement point during the reaction is set immediately after the start of the reaction in order to ensure a sufficient difference from the optical density after the completion of the reaction and improve quantitative accuracy. Since the measured values are unstable, unexpected errors may occur. In addition, it was difficult to apply this method to items with extremely fast responses.

Furthermore, when using two types of reaction reagents, the first
After the reagent and sample solution are mixed (the reaction will not proceed in this state), the optical density is measured, a second reagent is added to start the reaction, and the optical density is measured after the reaction is complete. The method of quantitative determination is also known as the so-called sample blank method. However, with this method,
In order to secure the amount of liquid necessary for measurement, it is necessary to increase the amount of the first reagent, which reduces sensitivity and reduces the amount of liquid! There were problems such as the need to perform lkM correction.

This invention was made under such circumstances, and aims to provide a biochemical analysis method that can easily and accurately quantify a target component without preparing a blank solution or correcting the liquid volume. It is something.

(d) Means for Solving the Problems Thus, according to the present invention, a sample liquid is mixed with a predetermined reaction reagent and reacted, and the sample liquid is detected based on the change in optical density of the sample liquid accompanying this reaction. regression using reaction time and optical density value Yt as variables, based on the relationship between a plurality of actually measured optical density values in the optical density fluctuation region after the start of the reaction and their reaction times. The equation is determined by the least squares method, and based on this regression equation, the reaction start time (t
= 0), and then quantifying the predetermined component in the sample liquid based on the difference between this estimated optical density value Yo and the measured optical density value Y9 after the completion of the reaction. A biochemical analysis method is provided.

In this invention, first, a regression equation is determined by the least squares method from the relationship between a plurality of actually measured optical density values in the optical density fluctuation region and their reaction times, and from the obtained regression equation, the time at which the reaction starts, that is, reaction time 1= The optical density at 0 is calculated. Here, the time at which the reaction starts refers to the time when the second reaction reagent is added when a two-sample type reaction reagent is used, and the time when the second reaction reagent is added when a one-reagent type reaction reagent is used.

The regression equation may be a -order regression or a multidimensional regression such as quadratic or cubic regression, and the one that most closely approximates the profile of the optical density fluctuation region during the reaction of each target component is suitable. For ordinary biochemical analysis items, the quadratic regression equation (Y
=a t'+b t+c) or cubic regression equation (Y=at'
+bt'+ct+d) is suitable.

Multiple measurement points (t, yt) to determine the above regression equation
can be set at any point in the fluctuation range between the start of the reaction and the end of the reaction, and it is preferable to increase the number of samples as much as possible. However, since an inflection point may actually exist just before the end of the reaction, it is not appropriate to use it as a measurement point. Therefore, it is suitable to set a plurality of measurement points between the time immediately after the start of the reaction and the time when the optical density reaches a certain percentage of the reaction, for example, 90%.

Estimated optical density value Yo obtained in this way at 1=0
The concentration of the target component is calculated by multiplying the difference value between Y8 and the optical density value Y8 after the reaction is completed by a predetermined conversion value, or by comparing and contrasting it with a calibration curve prepared in advance.

(e) Effect In the present invention, the estimated optical density Yo at the start of the reaction is determined by a regression equation for the optical density variation region based on the change in optical density during the reaction, and this optical density Y is determined by the estimated optical density Yo at the start of the reaction. Using the values of Ys and the optical density after the completion of the reaction, the target component is quantified as if by a two-point method using the optical density at the time of 1=0.

The optical density at the time of t=0 is not actually measured, but is obtained by regression from the measured values at multiple points, so it is compared to the conventional two-point method that uses the actually measured optical density at the beginning of the reaction. In this way, adverse effects on the measured values due to fluctuations in the initial stage of the reaction can be avoided or reduced, and at the same time, a decrease in sensitivity can be avoided even when the reaction is fast.

(f) Example FIG. 1 is an explanatory diagram of the configuration of an example of an automatic biochemical analyzer used to carry out the method of the present invention. In Fig. 1, l is a sample dispensing pump, 2 is a sample dispensing nozzle, 3 is a sample dispensing nozzle moving mechanism, 4.5 is a standard sample container and a standard sample, respectively, 6 is a sample turntable, and 7.8 is a sample dispensing nozzle moving mechanism. 9 is a reaction disk, io, (to
", to") is a reaction cell, 11 is a first reagent dispensing pump,
12 is a first reagent dispensing nozzle, 13 is a first reagent dispensing nozzle moving mechanism, 14 is a reagent storage, 15.16 is a first reagent 16 and a first reagent, respectively. 7 is a spectrometer, 18 is a spectrometer moving mechanism, 19 is a control and data processing computer, 2
0 is the second reagent dispensing pump, 2■ is the second reagent dispensing nozzle,
22 is a second reagent dispensing nozzle moving mechanism, 23 and 24 are a second reagent container and a second reagent, respectively, 25 is a cleaning pump,
26 is a cleaning nozzle up/down mechanism, and 27 is a cleaning nozzle.

In this device, a sample dispensing nozzle 2 connected to a sample dispensing pump 1 is moved by a sample dispensing nozzle moving mechanism 3, sucks a certain amount of a standard sample 5 from a standard sample container 4, and then A certain amount of sample 8 is aspirated from the sample container 7 set on the turntable 6, and the reaction disk 9
A sample 8 and a standard sample 5 are dispensed into a reaction cell 10 placed in a chamber. When the reaction disk 9 rotates and the reaction cell IO advances by one step, the first reagent dispensing nozzle 12 connected to the first reagent dispensing pump 11 is moved by the first reagent dispensing nozzle moving mechanism 13, Reagent storage 14
A predetermined amount of the first reagent 16 is sucked from the first reagent container 15 set therein, and then moved to the reaction cell lO' and dispensed into the reaction cell lO'. At this time, for the reaction cell of the item using the -reagent type reaction reagent, the spectrometer 17 is reciprocated around the same sleeve as the reaction disk 9 by the spectrometer moving device 111B, and the absorbance (Yt) is sequentially measured over time. It is stored in the control and data processing computer 19 as it is being measured.

Next, when the reaction cell 10 reaches the position of the reaction cell 10'', the second reagent dispensing nozzle 21 connected to the second reagent dispensing pump 20 moves up to the second reagent dispensing nozzle moving mechanism 22, and moves to the reagent storage 14. A certain amount of the second reagent 24 is aspirated from the second reagent container 23 set in the chamber, and then moved to the reaction cell lO'', where a two-reagent type reaction reagent is used. After the addition of the second reagent, the reaction cell 10'' is connected to the cleaning pump 25 and the absorbance Yt at each position is maintained as described above until the reaction cell 10'' is connected to the cleaning pump 25 and advances to the position of the cleaning nozzle 27, which is moved up and down by the cleaning nozzle up and down mechanism 26. is measured and stored in the computer 19. and,
Control and data processing computer! 9 simultaneously controls the operation of each part synchronously! For reagent system items, the absorbance data is based on the time change of absorbance data Yt after dispensing the first reagent (after the start of the reaction), and for the two reagent system items, the absorbance data is based on the absorbance data after dispensing the second reagent (after the start of the reaction). Based on the time change of Yt, find a regression equation for the absorbance variation region, and based on this, calculate the estimated absorbance value Yo at 1=0,
Next, the concentration of each predetermined component is calculated based on the difference between this Yo and the absorbance value Ys in a steady state (end point) after the reaction is completed.

The principle of this measurement is shown in Figure 2. That is, for both the first reagent system and the second reagent system, a quadratic regression equation ( Y=a
t"+bt+c) (solid line L in the figure), and from this equation, find the value Y at t=0 (at the start of the reaction). Then,
From the absorbance Ys in the constant absorbance region B after the reaction, this Y
The concentration is calculated by subtracting o (estimated absorbance) and multiplying this subtracted value by a predetermined conversion coefficient f.

Below, data from actual implementation will be explained.

Measurement of uric acid A dilution series of hemoglobin was added to the uric acid-containing sample (8,019/12), and the final hemoglobin concentration was o, too.
.

200.300, 400.500x9/d12 and measured using the above-mentioned biochemical analyzer.

As a reaction reagent, a two-reagent uric acid reaction reagent (manufactured by Yatron Co., Ltd.) was used, and 10 μQ of the sample and 200 μe of the first reagent were mixed, and then 200 μQ of the second reagent was added to start the reaction. The measurement cycle was performed 20 seconds and 38 seconds after the addition of the second reagent, and then 14 times at 24 second intervals (16 cycles in total). A linear regression equation ('/=a t+b) is calculated using the least squares method based on the relationship between the obtained absorbance and time, and from this, each 1=
The absorbance value (b) at 0 was determined. A sample blank was also measured using water instead of the reagent. These results are shown in the table below.

It can be seen that the physical (optical) effects of interference components can be compensated for in this way.

(Left space below) The uric acid concentration was calculated by determining the difference between each of the above absorbance values (b) and the absorbance values after the completion of each reaction, and multiplying this by each concentration conversion constant.

The results of measurements using the conventional endpoint method and this method are shown below.

It is considered that the chemical influence (on the reaction) of the interfering component hemoglobin can be accurately measured in this way.

Measurement of apoprotein A hemoglobin dilution series was added to the sample containing apoB 130i9/df2 in the same manner as in the uric acid measurement method described above, and the hemoglobin concentration was 0.100.200, 300, 40.
The sample was prepared to give 0,500 decay/dQ and was measured. As a reaction reagent, a two-reagent system ApoB reaction reagent (manufactured by Daiichi Chemicals (Aji)) was used, and after mixing 20μQ of the sample and 350μQ of the first reagent, 50μQ of the second reagent was added to start the reaction. .

A quadratic regression equation (Y = a, t" + b j + c) is determined by the least squares method using the relationship between the obtained absorbance and time, and from this, the absorbance value (c) at t = 0 is determined, and this absorbance value (c ) and the absorbance value after the completion of each reaction and multiplied by a concentration conversion constant to calculate the apoBQ concentration.The measurement results using the conventional sample blank method and this method were as follows. .

In this case, by adopting the present method, reagent blank measurement can be omitted, so that expensive reagents can be saved.

(G) Effects of the Invention According to the present invention, a target component can be easily and accurately quantified without preparing a blank liquid or correcting the liquid volume. It should also be possible to prevent or reduce the adverse effects on measured values due to interfering components and reaction instability, as in the conventional end-point method and two-point method.

[Brief explanation of the drawing]

Fig. 1 is a configuration explanatory diagram illustrating an apparatus for carrying out the biochemical analysis method of this invention, Fig. 2 is an explanatory diagram of the principle of the method of this invention, and Fig. 3 is the same (absorbance obtained in Example). It is a graph diagram showing the relationship between time and time. Reaction start Figure 2 Elapsed time (t) □

Claims (1)

[Claims] 1. Mixing a predetermined reaction reagent with a sample liquid and reacting it, and measuring the concentration of a predetermined component in the sample liquid based on the change in optical density of the sample liquid accompanying this reaction. Therefore, from the relationship between the plurality of actually measured optical density values in the optical density fluctuation region after the start of the reaction and their reaction times, a regression equation using the reaction time and the optical density value Y_t as variables is determined by the least squares method, and this The estimated optical density value Y_o at the start of the reaction (t=0) is determined based on the regression equation, and then this estimated optical density value Y
A biochemical analysis method, characterized in that a predetermined component in the sample liquid is quantified based on the difference between _o and the actually measured optical density value Y_s after completion of the reaction.