JPH03198797A - Analyzing method - Google Patents

Abstract

PURPOSE:To accurately determine an aimed component by correcting a measured time variation rate as a physical quantity after mixing a reagent containing residual components necessary for an aimed reaction with a sample by the time variation rate after additionally mixing an auxiliary reagent for the reaction lacking said necessary residual components. CONSTITUTION:A reaction auxiliary reagent lacking at least one of components necessary for the aimed reaction is mixed with a sample and a time variation rate of a first physical quantity is measured. Next, a reagent containing residual components necessary for the aimed reaction not contained in said reaction auxiliary reagent is mixed with the first auxiliary reagent and a time variation of a second physical quantity is measured. Then, the time variation of the second physical quantity is corrected with said variation of the above-mentioned first physical quantity. By this method, an activating value of the aimed substance in a sample is able to be determined by a time variation rate of a physical quantity generated by an induced chemical reaction.

Description

[Detailed Description of the Invention] [Industrial Application Field] The present invention relates to an analysis method, and in particular to an automatic chemical analyzer for quantifying target components contained in a sample containing a large number of substances such as serum. The present invention relates to an analysis method suitable for adding a reaction reagent to a sample to be analyzed and quantifying a target component in the sample from changes in physical quantities caused by the induced chemical reaction.

[Conventional technology]

Two measurement methods have been used in conventional automatic chemical analyzers mainly used for clinical tests. In other words, (1) As in the enzymatic analysis of cholesterol and the analysis of total protein by the Biulet method, this is a method in which a sample and reagent are mixed, a reaction is caused, and the absorbance is measured after a certain period of time; generally, there is a colorimetric analysis method. (1) There are two methods called one-point method and end-point method, and (2) a method that tracks the change in absorbance after the addition of a trigger reagent, such as the reaction rate tracking method for glutamate oxaloacetate transaminase and lactate dehydratase. This method is generally called the reaction rate measurement method, initial velocity modification method, rate analysis method, or kinetic method.

[Problem to be solved by the invention]

However, when these conventional methods are applied to analytical chemistry such as the azobilirubin method for bilirubin, the hechirakinase method for glucose, the UV method for triglyceride 1-, and the UV method for urine enzyme nitrogen, other The effects of substances cannot be easily removed, and in order to remove them,
We had to measure its size, so we needed twice as much equipment,
Required twice as much sample and reagents.

To give a more detailed example, in analysis using the hexokinase method, which is the most specific analysis method for serum analysis, chemical reactions of the following formulas (1) and (2) proceed.

Hexokinase glucose + 鼎□-→ glucose-6-phosphate + A
DP... (1) Glu-o-6-lithic acid + NADP, episode 4 4. -7 Oyu, Lord O. , acid 0. (2) Here, ATP is adenosine 1-lyphosphate, ADP is adenosine diphosphate, NADP is nicotinamide adenine denucleotide 1-phosphate, 06P-D H is glucose 6-phosphate dehydrogenase, N
A I) PH is reduced nicotinamide adenine denucleotide phosphate ester.

In this analytical chemistry, NADP and NADP I
(The difference in absorption of
The change in absorbance at 340 nm in this system is proportional to the amount of glucose due to the fact that NADP has an absorption peak at 340 nm and has no absorption at that wavelength.

When this analytical chemistry is applied to the colorimetric analysis method described in (1) above, ordinary serum samples contain substances that absorb at 34-On m, such as pigments such as bilirubin and turbidity components, and these Accurate glucose quantification is not possible due to the large influence of Therefore, in the conventional technology, the magnitude of the influence of the mixed substances was measured and removed as follows. That is, a method has been adopted in which a sub-channel for a sample blank is provided in addition to the main analysis channel, the magnitude of the influence of interfering substances is measured in the sub-channel, and the data of the main channel is corrected accordingly. In this case, twice the mechanical equipment, double the amount of sample, and reagents for the ground specimen blank were required. In addition, in the rate analysis method of conventional technology (2) mentioned above, the reaction, the chemical reactions of equations (1) and (2) above, proceed at a very fast rate and are also strongly affected by temperature, so the accuracy is low. The drawback was that it was difficult to quantify accurately.

The present invention was made in order to eliminate the above-mentioned conventional drawbacks, and an object of the present invention is to provide an analysis method that can easily eliminate the interfering influence of components other than the target component mixed in a sample.

[Means to solve the problem]

The present invention is an analysis method in which a reaction reagent is added to a sample to be analyzed, and a target component in the sample is quantified from changes in physical property values caused by the induced chemical reaction. A reaction auxiliary reagent lacking at least one of the components is added to the sample to measure the first physical property value, and then, the reaction auxiliary reagent lacking at least one component not included in the reaction auxiliary reagent is added to the sample.
A 1-Riger reagent containing residual components necessary for the desired reaction is added to the sample, a second physical property value is measured, and the second physical property value is corrected by the physical property value of the above-mentioned physical property value, and the reaction is performed. The above objective is achieved by eliminating the effects of reactions between the auxiliary reagent and components other than the target components in the sample.

- The present invention has experimentally confirmed that in certain chemical analysis methods, there is a large interference from coexisting substances, and as a means to remove this interference, 1- After the reaction is completed by triggering before and after the addition of Riger reagent. This was done by focusing on the fact that the difference in physical property values, such as absorbance, is due only to the desired reaction and is not caused by other coexisting substances.

〔Example〕

Hereinafter, the present invention will be explained in detail based on examples.

FIG. 1 is a plan view showing the configuration of an embodiment of the present invention.

The reaction table 1 has multiple pieces (for example, 40 pieces) on its circumference.
It has a reaction vessel 2 that also serves as a measurement cell, and can freely rotate around a rotation shaft 3. The sample table 4 has a plurality of sample containers 5 on its circumference and can freely rotate around a rotation axis 6. Pipetting of the sample is performed using a pipetter 7 and sampling probe 8, and dispensing of reagents is performed using dispensers 9 and 10. The spectrometer 11 is a multi-wavelength simultaneous photometry type using multiple detectors, and the light source lamp 1
2, the row of reaction vessels 2 is disposed so that a light beam 13 from a light source lamp 12 passes through the row of reaction vessels 2 when the reaction table 1 is in a rotating state. The luminous flux 13 is counted clockwise from the discharge position 25 when the reaction table 1 is in a stopped state,
For example, it is arranged so that it passes through the center of the 30th reaction vessel 2. A drain pipe 26 and a cleaning liquid discharge pipe 27 are arranged between the position of the light beam 1.3 and the discharge position 25, and are connected to a drain device 28 and a cleaning device 29, respectively.

FIG. 2 is a block diagram showing the electrical system of this embodiment. The entire electrical system consists of a multiplexer 14, a logarithmic conversion amplifier 15, an A/I) converter 16, and a central processing unit 17.
, a read-only storage device 18, a continuous write storage device 9, a printer 20, an operation panel 21, and a mechanical quick-acting circuit 22, which are connected by a pass line 23.

The operating principle will be explained below according to the figures. A sample container 5 containing a sample to be measured, for example, serum, is located at a sampling position 31.
When the serum is supplied, the tip of the probe 8 of the pipetter 7 is immersed into the reagent container 5, and a certain amount of serum is aspirated and retained within the probe 8.

At the same time, the dispenser 9 sucks a certain amount of the reaction auxiliary reagent from the sample storage container 24. Thereafter, the probe 8 moves to the discharge position 25 of the reaction table 1, and discharges the serum held by the probe 8 into the reaction container 2 that has been transferred to the discharge position 25, and at the same time, the dispenser 9 is used to assist the reaction. Dispense the reagent. By the above operation, the sample to be measured is mixed with the reaction auxiliary reagent in the reaction container 2, and the first reaction is started.

When the above-mentioned sampling operation is completed, the reaction table 1 starts to rotate intermittently in the clockwise direction, and a number of reaction vessels 2, for example 4 reaction vessels 2, which is one more than the total number of reaction vessels 2,
One reaction vessel 2 rotates by an angle necessary for passing through the discharge position 25, that is, 369 degrees, and then stops. A reaction container 2 containing a sample sampled in the sampling operation by the rotation of the reaction table 1 and a reaction auxiliary reagent
It comes to a position that has advanced clockwise from the discharge position 25 by one pitch of the reaction container, that is, an angle of 9 degrees, and has stopped. While the reaction table 1 is rotating, the reaction table 1 is rotated. Therefore, when each reaction container 2 passes through the light beam 13, light absorption is measured by the spectrometer 11, and from the output of the spectrometer 11, a signal of the currently required measurement wavelength is selected by the multiplexer 14, and the A/D converter 16 The data is taken into the central processing unit 17 and stored in the continuous write storage device 19. If the time period during which the reaction table 1 is rotated and stopped is, for example, 30 seconds, the above operation is repeated with 30 seconds as one cycle. As the cycle progresses, the position of the sampled sample to be measured advances clockwise by one pitch of the reaction container when the reaction table 1 is stopped. For example, when the reaction table 1 is in a stopped state, the piping of the dispenser 10 is connected to the discharge position 25.
The trigger reagent is placed on top of the 15th reaction vessel 2 in the clockwise direction, and when looking at a specific sample to be measured, the trigger reagent is added by the dispenser 10 at the 15th cycle from the start of the auxiliary reaction at the discharge position 25. The desired main reaction is initiated. The cycle continues and the reaction table',
The position of the reaction vessel 2 when the light is stopped is the luminous flux 1.
The sample in the reaction container 2 which is between the light beam 13 and the discharge position 25 beyond 3 is the sample whose measurement has been completed, and is suctioned and drained by the liquid drain device 28 through the liquid drain pipe 26. Further, a cleaning liquid (usually distilled water) is discharged from the cleaning device 29 through the cleaning liquid discharge pipe 27 . When the reaction table 1 is next stopped, the cleaning liquid in the reaction container 2 is drained for the last time in the same manner as described above, and the cycle is further advanced to be used again as the reaction container 2 from the discharge position 25. The above-mentioned operations are controlled by the central processing unit 17 through the mechanism stirring circuit 22 in accordance with the program in the read-only storage device 18. The operation panel 21 is used for operations such as inputting measurement conditions, starting measurement, and stopping measurement.

Assuming that the stop time of reaction table 1 in one cycle is 9.5 seconds and the rotation time is 20.5 seconds in the above operation, when focusing on a specific sample, the reaction process of that specific sample is 29.5 seconds.
Measurements are taken 30 times every second, and measurement data for a total of 14 minutes and 45 seconds is stored in the read/write storage device 19. The central processing unit 17 operates according to the program in the read-only storage device 18, extracts necessary data from the 30 measurement data in the read/write storage device 19 according to the scheduled program, and performs processing such as concentration calculation. and output it to the printer 20.

Example 1 Here, a case will be described in which the apparatus according to the above example is applied to analysis of glucose by the hekyrakinase method. According to the experimental results, the reaction process in the analysis of glucose in serum by the Hekyrakinase method proceeds as shown in FIG. Here, the l.Riger reagent is a hexokinase solution, and the reaction auxiliary reagents are ATP and NAD in the above formulas (1) and (2).
This is a solution containing P, 06P-DH.

As shown in FIG. 3, the change in the absorbance of the reaction solution over time shows the absorbance of only the reagent, i.e., the absorbance a of the reagent blank, at the zero point of the reaction time, plus the absorption of the coexisting substances of the serum itself.

Furthermore, NADP in the reagent can be converted to NAD by coexisting substances.
A side reaction that changes the pH occurs briefly and a certain absorbance is reached. Therefore, at the reaction time of 7.5 minutes, the pipettor 10
When the trigger reagent is added according to the formula (1), (2
) The reaction of the formula occurs and rapidly progresses, almost completing after 1 to 2 minutes and reaching absorbance C. Hexokinase, which is an analytical chemical trigger, has high specificity and acts only on glucose, and the absorbance difference (c-b) is proportional to the true amount of glucose in serum. Moreover, when this reaction is applied to the apparatus mentioned above, it takes 29.5 minutes from sample transfer.
Thirty measurements are taken every second and stored in memory. Therefore, the simplest way is (c-b) is the 14th data b and 30
It is obtained as the difference between the data C for the second time. (b-c)
can be multiplied by a density conversion coefficient according to a predetermined program and output to the printer 20.

According to this example, glucose in serum can be easily quantified without interference from mixed components.

Example 2 Here, the apparatus according to the above example was used for the reaction of glutamic acid 1-2-oxalacetic acid transaminase (G○'F).
We will discuss the case where this method is applied to analysis using the method.

The reaction of GOT analysis using the rate method is the following chemical formula (3)
It is expressed by the formula and (4), and according to the experimental results, serum GO
The reaction of T proceeds as shown in FIG.

Oxalacetic acid + NAD
...(4) Here, M D H is maleate dehydrogenase.

Here, the trigger reagent is α-glutaric acid, and the reaction auxiliary reagent is a solution containing L-aspartic acid, NADH, and MDH in formulas (3) and (4).

As shown in FIG. 4, the absorbance of the reaction solution shows the overlapped absorbance of the first reagent and serum immediately after the 0 point of reaction time. After that, formula (4) may proceed due to oxalacetic acid in the serum, and other substances other than GOT that oxidize NAD H to NAD may be mixed in the serum.
Reactions other than GOT occur and proceed as shown in Figure 4. These reactions have sample-specific magnitudes, ie, they vary in magnitude from patient to patient. Therefore, when the 1-Riger reagent is added using the dispenser 10 at the reaction time of 7.5 minutes, the GOT reaction formula (3) and the M
The DH reaction (4) occurs, and the absorbance decreases as shown in FIG. Here, in the conventional method, the activity value of GOT was determined from the change in absorbance per unit time after the addition of the trigger reagent, but the serum contains substances other than GOT that convert NAD H to NAD. If there is
These side reactions caused errors in the measured values. In this example, the 10th absorbance data is d, the 14th absorbance data is e, the 16th mouth absorbance data is f, and the 20th absorbance data is g, ((f g) - (d - e)). It can be calculated, multiplied by a unit conversion coefficient using a scheduled program, and output to the printer 20.

According to this example, GOT in serum can be easily quantified without interference from mixed components. When measuring oxygen activity with an automatic analyzer, if noise data is included when determining the rate of change in absorbance, or slope, shown in Figure 4 before and after adding the trigger reagent, it is necessary to include the noise data. There is a possibility that the slope obtained by this method will deviate greatly from the original slope. Therefore, it is necessary to discard such data when calculating the slope. As shown in FIG. 2, the data corresponding to the first physical property value and the data corresponding to the second physical property value stored in the read/write storage device 19 are extracted and stored in the read-only storage device 18. When calculating according to the program, since the extracted data is the data stored in memory, it is possible to remove noise data from the data and improve the reliability of the measurement results. can.

In each of the above embodiments, the present invention was applied to an automatic chemical analyzer having a turntable, so in a conventional automatic chemical analyzer, the analysis process was simply slightly changed without adding any measuring instruments. Although the present invention was able to be carried out by only making changes, it is clear that the scope of application of the present invention is not limited to this, and can be similarly applied to a flow cell type automatic chemical analyzer or a belt conveyor type automatic chemical analyzer. .

In addition, in all of the above examples, the present invention was applied to analysis using absorbance, but the scope of application of the present invention is not limited to this, and is applicable to analysis methods that measure and analyze general physical property values. It is clear that the same applies.

As explained above, according to the present invention, analysis is performed from the reaction state before the addition of a trigger reagent specific to the target component and after the addition of the trigger reagent. It has the excellent effect of being able to be easily and accurately carried out without interference.

[Brief explanation of drawings]

FIG. 1 is a plan view showing the configuration of an automatic chemical analyzer to which an embodiment of the analysis method according to the present invention is applied, FIG. 2 is a block diagram showing the electrical system in the automatic chemical analyzer, and FIG. FIG. 4 is a diagram showing the reaction process of glucose analysis by the hexokinase method to which the present invention is applied; FIG. be. DESCRIPTION OF SYMBOLS 1... Reaction table, 2... Reaction container, 4... Sample table, 5... Sample container, 7... Pipettor, 8
... Probe, 9, 10... Dispenser, 11... Spectrometer, 12... Light source lamp, 13... Luminous flux.

Claims (1)

[Claims] 1. In an analysis method in which a reaction reagent is added to a sample to be analyzed and the activity value of a target substance in the sample is measured from the time rate of change of a physical quantity generated by the induced chemical reaction, first, the reaction reagent is added to the sample to be analyzed. At least one of the necessary ingredients
The rate of change over time of the first physical quantity is measured by mixing the sample with a reaction auxiliary reagent that lacks the reaction auxiliary reagent. 1. An analysis method comprising: mixing reagents containing reagents; measuring the rate of change over time of a second physical quantity; and correcting the rate of change over time of the second physical quantity by the rate of change of the first physical quantity.