JPS649571B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to an analysis method, and in particular to an analysis method suitable for use in 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 for quantifying target components in a sample from changes in physical property values caused by chemical reactions induced by adding a reaction reagent to the sample.

Two measurement methods have been used in conventional automatic chemical analyzers mainly used for clinical tests. (1) As in the enzymatic analysis of cholesterol and the analysis of total protein by the Buillet 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, colorimetric analysis or There are two methods called one-point method and end-point method, and (2) methods that track 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 called the measurement method, initial velocity method, rate analysis method, or kinetic method.

However, with these conventional methods, when analytical chemistry is applied, such as the azobilirubin method for bilirubin, the hexokinase method for glucose, the UV method for triglycerides, and the UV method for urine enzyme nitrogen, the effects of other substances mixed in the sample cannot be considered. They cannot be easily removed, and their size must be measured in order to remove their effects, so twice the equipment and twice the amount of samples and reagents are required.

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

Glucose + ATP hexokinase ――――――――→ Glucose-6-phosphate + ADP ……(1) Glucose-6-phosphate + NADPG6P-DH ――――――――→ NADPH + b-phosphoglucuronic acid ……( 2) Here, ATP is adenosine triphosphate, ADP
is adenosine diphosphate, NADP is nicotinamide adenine denucleotide phosphate,
G6P-DH is glucose 6-phosphate dehydrogenase, and NADPH is reduced nicotinamide adenine denucleotide phosphate ester.

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

When this analytical chemistry is applied to the colorimetric analysis method described in (1) above, ordinary serum samples contain substances that absorb at 340 nm, such as pigments such as bilirubin and turbidity components, and these have a large influence. Therefore, accurate glucose quantification cannot be performed. 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 subchannel 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 subchannel, and the data of the main channel is corrected accordingly. In this case, twice the mechanical equipment, twice the amount of sample, and other reagents for sample blanks 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 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 interference caused by components other than the target component mixed in a sample.

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, the first physical property value is measured, and then a reaction auxiliary reagent lacking at least one of the components required for the desired reaction that is not included in the reaction auxiliary reagent is added to the sample. A trigger reagent containing a residual component is added to the sample, a second physical property value is measured, and the second physical property value is measured.
The above object is achieved by correcting the physical property value of the sample with the first physical property value to eliminate the influence of the reaction between the reaction auxiliary reagent and components other than the target component in the sample.

The present invention has experimentally confirmed that in some types of chemical analysis methods, interference by coexisting substances is large, and as a means to remove this interference, the physical property value after the reaction is determined by the trigger before and after the addition of the trigger reagent. , for example, focusing on the fact that the difference in absorbance is the amount of change due only to the desired reaction and not due to other coexisting substances.

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 a plurality (for example, 40) reaction vessels 2 serving as measurement cells on its circumference, and can freely rotate around a rotation axis 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 pipette 7 and sampling probe 8, and dispensing of reagents is performed using dispensers 9 and 10. Spectrometer 1
1 is a multi-wavelength simultaneous photometry type using multiple detectors,
The row of reaction vessels 2 faces the light source lamp 12 when the reaction table 1 is in a rotating state.
It is installed so that the light beam 13 from the center passes therethrough.
The light beam 13 is arranged so as to pass through the center of, for example, the 30th reaction vessel 2, counting clockwise from the discharge position 25 when the reaction table 1 is in a stopped state. A drain pipe 26 and a cleaning liquid discharge pipe 27 are arranged between the position of the light beam 13 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 multiplexer 1
4, logarithmic conversion amplifier 15, A/D converter 16, central processing unit 17, read-only storage device 18, read/write storage device 19, printer 20, operation panel 2
1. It consists of a mechanism drive circuit 22 and is connected by a bus line 23.

The operating principle will be explained below with reference to the drawings. When a sample container 5 containing a sample to be measured, for example, serum, is supplied to the sampling position 31, the tip of the probe 8 of the pipette 7 is immersed into the sample container 5, and a certain amount of serum is aspirated into the probe 8. Hold. At the same time, the dispenser 9 sucks a certain amount of the reaction auxiliary reagent from the reagent 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 rotating intermittently in the clockwise direction, and the number of reaction vessels 2 that is one more than the total number of reaction vessels 2, for example, 41 reaction vessels 2, is placed on the reaction table 1. It rotates by the angle necessary to pass the discharge position 25, that is, 369 degrees, and then stops. Due to the rotation of the reaction table 1, the reaction container 2 containing the sample sampled in the sampling operation and the reaction auxiliary reagent comes to a position that has advanced clockwise by one pitch of the reaction container, or an angle of 9 degrees, from the discharge position 25. This means that it has stopped. During the rotation of the reaction table 1, all the reaction vessels 2 on the reaction table 1 pass through the light beam 13. Therefore, when each reaction container 2 passes through the light beam 13, light absorption is measured by the spectrometer 11,
From the output of the spectrometer 11, a signal of the currently required measurement wavelength is selected by the multiplexer 14, taken into the central processing unit 17 by the A/D converter 16, and stored in the read/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 specific sample to be measured is located in the reaction container 1 when the reaction table 1 is stopped.
Go clockwise one pitch at a time. Dispenser 10
For example, when the reaction table 1 is stopped, the piping is installed on the 15th reaction vessel 2 counting clockwise from the discharge position 25, and when looking at a specific sample to be measured, the auxiliary reaction starts at the discharge position 25. At the 15th cycle, a trigger reagent is added by the dispenser 10 to start the desired main reaction.
As the cycle progresses further, the position of the reaction container 2 with the reaction table 1 stopped is the luminous flux 13.
The sample in the reaction vessel 2 that lies between the light beam 13 and the discharge position 25 is the sample for which measurement has been completed;
6, the liquid is sucked and drained by a liquid draining device 28.
Further, a cleaning liquid (usually distilled water) is discharged from the cleaning device 29 through the cleaning liquid discharge pipe 27 . When the next reaction table 1 is stopped, the cleaning liquid in the reaction vessel 2 is drained for the last time in the same manner as described above, and the cycle further advances to the reaction vessel 2 again from the discharge position 25.
used as. The above operations are performed by the central processing unit 17 according to the program in the read-only storage device 18.
Each mechanism is controlled through the mechanism drive circuit 22. The operation panel 21 is used for operations such as inputting measurement conditions, starting measurement, and stopping measurement.

With the above operation, if the stop time of reaction table 1 in one cycle is 9.5 seconds and the rotation time is 20.5 seconds, then
When focusing on a specific sample, the reaction process of that specific sample is measured 30 times every 29.5 seconds, 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 concentration calculations, etc. The data is processed and output 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 hexokinase method. According to the experimental results, the reaction process for analyzing glucose in serum by the hexokinase method proceeds as shown in FIG. Here, the trigger reagent is a hexokinase solution, and the reaction auxiliary reagent is a solution containing ATP, NADP, and G6P-DH in the above formulas (1) and (2).

As shown in FIG. 3, the change in the absorbance of the reaction solution over time shows the absorbance of only the reagent at the zero point of the reaction time, that is, the absorbance a of the reagent blank, as well as the absorption of the coexisting substances of the serum itself. Furthermore, coexisting substances may cause
A side reaction that converts NADP to NADPH takes place for a short time until a certain absorbance b is reached. Therefore, when the trigger reagent is added using the dispenser 10 at the reaction time of 7.5 minutes, the reactions of equations (1) and (2) occur and proceed rapidly, and almost complete after 1 to 2 minutes, and the absorbance increases. c.
reach. 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. Also, when this reaction is applied to the device described above, it is possible to
30 measurements are taken every 29.5 seconds and stored in memory. Therefore, most simply, (c-b) can be found as the difference between the 14th data b and the 30th data c.
(b-c) can be multiplied by a density conversion coefficient using 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, a case will be described in which the apparatus according to the above example is applied to analysis of glutamate oxalacetate transaminase (GOT) by the rate method. The reactions of GOT analyzed by the rate method are expressed by the following chemical formulas (3) and (4), and according to the experimental results, the reactions of serum GOT proceed as shown in FIG.

L-aspartic acid + α-ketoglutarate GOT ――――→ Oxalacetic acid + L-glutamic acid ……(3) Oxalacetic acid + NADHMDH ――――→ Malic acid + NAD ……(4) Here, MDH is maleate dehydrogenase .

Here, the trigger reagent is α-ketoglutaric 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, equation (4) progresses due to oxalacetic acid in the serum, and other substances other than GOT that oxidize NADH to NAD may be present in the serum, and reactions other than GOT occur. The process proceeds as shown below. These reactions have sample-specific magnitudes, ie, they vary in magnitude from patient to patient. Therefore, when the trigger reagent is added using the dispenser 10 at the reaction time of 7.5 minutes, the GOT reaction formula (3) and the MDH are conjugated to it.
Reaction (4) occurs, and the absorbance decreases as shown in Figure 4. Here, in the conventional method, the change in absorbance per unit time after the addition of the trigger reagent is
I was trying to determine the activity value of GOT, but I found that NADH in the serum was not found.
When substances other than GOT, which change from GOT to NAD, are present, these side reactions cause errors in the measured values. In this example, {(f-g)-(d-e)} is calculated by setting the 10th absorbance data to d, the 14th absorbance data to e, the 16th absorbance data to f, and the 20th absorbance data to g. , can be multiplied by a unit conversion coefficient according to 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.

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, and FIG. 4 is a diagram showing the reaction process of glutamate oxaloacetate transaminase analysis by the rate method to which the present invention is also applied. 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 target component in the sample is quantified from changes in physical property values caused by the induced chemical reaction,
First, a reaction auxiliary reagent lacking at least one of the components necessary for the desired reaction is added to the sample, and the first physical property value is measured. In addition, a trigger reagent containing residual components necessary for the desired reaction is added to the sample, a second physical property value is measured, the second physical property value is corrected by the first physical property value, and the reaction is performed. An analysis method characterized by eliminating the effects of reactions between auxiliary reagents and components other than the target components in the sample.