JP2643428B2 - Biochemical analysis method - Google Patents

Description

The present invention relates to a biochemical analysis method. More particularly, the present invention relates to an analytical method useful for quantifying a predetermined component in a biochemical sample containing multiple components such as serum, plasma, urine, and lymph.

(B) Conventional technology Conventionally, as a method for analyzing a predetermined component in a biochemical sample as described above, one or two or more predetermined reaction reagents are mixed and reacted in a sample solution, and the sample solution generated by the reaction is mixed. A method of quantifying a predetermined component based on a change in the optical density value (absorbance value, fluorescence intensity value, etc.) or a rate of change during the reaction is known as a so-called endpoint method or rate method. Applied to the device.

In these specific analyses, the measurement of the change in the optical density value or the rate of change is based on the optical density value Ab of the reaction reagent blank liquid (solution containing no sample component and containing the same reaction reagent). Is performed as a baseline, and an appropriate quantification is performed.
(Optical density limit value) is uniformly performed within the optical density range.

For example, in the rate method, a plurality of optical densities in the course of the reaction from the start of the reaction to the saturation of the reaction are measured over time, and the optical density change rate is obtained from the relationship between the series of measured optical densities and time, and the quantitative Is performed. And
The optical density limit value A for obtaining the rate of change is, for example, around the optical density value before the reaction saturation, which is regulated by the amount of the substrate in the case of a reaction reagent using an enzyme.

Accordingly, since the measured optical density value for the portion exceeding the optical density limit value A is excluded from the data for the change rate measurement data, the occurrence of the quantitative error is prevented as much as possible.

On the other hand, in the end point method, the quantification is performed based on the difference between the optical density in the reaction completed state and the optical density Ab of the reagent blank. When the concentration exceeded the concentration limit value A, the quantification was determined to be inappropriate, and a retest was performed, thereby ensuring the reliability of the quantification.

(C) Problems to be Solved by the Invention However, in any analysis method using the optical density limit value A, the influence of optical interference components that can coexist in the sample, for example, bilirubin, hemoglobin, turbidity components, etc. Had been a problem.

In other words, even if the measured component concentration itself is within an appropriate range in terms of quantification accuracy, these interference components cause a change in the baseline of the optical density. The number of data of the actually measured optical density value within the limit value A that can be used for the calculation of the change rate is reduced, leading to a decrease in the quantification accuracy. In the case of the end point method, there are problems that the calculation is inconvenient, that the determination is inappropriate for quantification, that a retest is required after dilution, and that the quantification error due to the interference component is large.

Various proposals have hitherto been made in order to prevent adverse effects such as complicated quantification operations and reduced quantification accuracy due to such interference components. For example, Japanese Patent Application Laid-Open No. 57-82753 proposes a method of measuring the absorbance of a sample and a diluent (physiological saline or the first reagent) to correct the volume, and subtracting it from the absorbance obtained after the start of the reaction. I have. Also, JP-A-56-108941,
Nos. 56-104238, 56-104239, and 54-63785 add a sample and diluent (physiological saline or the first reagent of other reagents), measure at a specific wavelength, and calculate the interference component. Proposes a method of calculating and correcting the absorbance at the wavelength to be measured.

However, in the former method, a separate diluent is required, and the amount of the solution needs to be corrected. Further, it is necessary to consider the influence of swelling. . In addition, it could not be applied to the one-reagent system.

On the other hand, in the latter method, there is a problem that a separate channel is necessary for the apparatus, so that the processing capacity is reduced and the absorbance needs to be corrected.

The present invention has been made under such circumstances. Particularly, without performing independent measurement of the interference component, without performing the capacity correction, etc., the quantitative determination of the predetermined component is easily performed while preventing the adverse effect of the interference component. It is intended to provide a biochemical analysis method capable of performing the above.

(D) Means for Solving the Problems According to the present invention, a predetermined reaction reagent is mixed with a sample solution and reacted, and the optical density is measured over time. Calculating the change or rate of change of the optical density within the range of the predetermined optical density limit value A, and quantifying a predetermined component in the sample liquid based on the calculated value.
t _1, the optical density value at _{t 2 A s (1),} A s (2) and the reaction time was determined in the same manner for the standard sample solution t _1, an optical in T ₂ concentration value A _st (1), A _st (2) and the reagent blank mixture following equation from the optical density value a _b of (I): The optical density value A _I of the interference component in the sample solution is obtained based on the above, and the optical density change or change after correcting the series of actually measured optical density values or the optical density limit value A with the optical density value A _I. A biochemical analysis method is provided, wherein a rate is calculated.

In the method of the present invention, as shown in FIG.
Various biochemical measurement items in which the ratio b / a of the optical density at one reaction time is substantially constant (b ₁ / a ₁ ≒ b ₂ / a ₂ ) within the measurement concentration range of the component to be measured, for example, GOT, GPC (Rate method), TP
(Total protein), NEFA (free fatty acid), GLU (end point method) and the like. In addition,
Here, the reaction time refers to the elapsed time from the time of addition of the second reaction reagent when using a two-reagent-based reaction reagent or from the time of addition of the reaction reagent when using a one-reagent-based reaction reagent. .

The present invention provides a method for calculating a change or a rate of change in optical density within a range of an optical density limit value A using a series of measured optical density values measured over time. The most characteristic feature is that A is corrected by the specific method described above.

Such correction is the reaction time t ₁ for the sample solution, optical in t ₂ concentration value A _s (1), A _s and (2), the reaction time obtained for a standard sample solution t _1, an optical in t ₂ concentration value A
_st (1), is performed using the optical density value A _b of A _st (2) and reagent blank solution. Here, the reaction times t ₁ and t ₂ are preferably set at intervals as long as possible in the region until the reaction is saturated.

The standard sample solution refers to a so-called standard solution containing the component to be measured and no interference component, and the concentration of the component to be measured in the solution is not particularly limited as long as the concentration is within the measurement concentration range. For example, it can be set to a standard value in a living body.

(E) Function The measurement principle is assumed on the assumption that a measurement curve satisfying the relationship shown in FIG. 2 is used to measure the sample solution and the standard sample solution, and the optical density change curve shown in FIG. 3 is obtained. Will be described.

First, the optical density value of the sample solution at the reaction times t ₁ and t _{2 is} represented by A
_s (1), and A _s (2), the optical density value of the standard sample solution A _st
(1), and A _st (2), the optical density value of the reagent blank mixture and A _b. Then the optical density due to interference components in the sample and A _I.

In this case, regardless of the magnitude of the concentration of the component to be measured in the sample liquid or the standard solution, the ratio of the optical density contribution by the component to be measured at t ₁ and t ₂ is constant. Therefore, the following equation is first satisfied from this relationship.

Therefore, from this relational expression, the following expression (I): Is led.

Therefore, the optical density value A _I due to the interference component can be calculated, and by using this value to correct a series of measured optical density values for the sample liquid, various inconveniences in the calculation and quantification due to the interference component can be eliminated. Alternatively, it is possible to perform appropriate analysis by the end point method or the like.

That is, in the case of the rate method, since the optical density value of the portion exceeding the optical density limit value A is not used for calculation, the number of data to be provided for the change rate measurement decreases and the accuracy of the quantification decreases. The number of data can be increased, and the quantification accuracy is improved. In addition, even when the optical density is reduced, contrary to FIG. 3, it is possible to eliminate the case where the data of the area not suitable for the change rate measurement is erroneously calculated.

On the other hand, in the case of the end point method, as shown by the measured values in FIG.
By weight, retest it is necessary, according to the method of the present invention, the amount corresponding the measured optical density values similarly A _I is subtracted. As a result, the optical density value at the time when the reaction is completed (the plateau region in the figure) is also within the range of the limit value A, and the quantification can be performed without re-examination. Limit value A
Since the error of the interference component can be easily removed even within the range, the quantification accuracy can be further improved.

Note that the correction may be relatively performed in relation to the limit value A, and thus the limit value A may be corrected.

(F) Embodiment FIG. 1 is an explanatory view of the configuration of an example of an automatic biochemical analyzer used for carrying out the method of the present invention. In FIG. 1, 1
Is a sample dispensing pump, 2 is a sample dispensing nozzle, 3 is a sample dispensing nozzle moving mechanism, 4 and 5 are standard sample containers and standard samples, 6 is a sample turntable, and 7 and 8 are sample containers and samples, respectively. , 9 is a reaction disk, 10, (10 ′, 1
0 ″) is the reaction cell, 11 is the first reagent dispensing pump, 12 is the first reagent
Reagent dispensing nozzle, 13 is a first reagent dispensing nozzle moving mechanism, 14
Is a reagent storage, 15 and 16 are first reagent containers and first reagents respectively, 17 is a spectroscope, 18 is a spectroscope moving mechanism, 19 is a control and data processing computer, 20 is a second reagent dispensing pump, 21
Is a 23rd 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.
Denotes a cleaning pump, 26 denotes a cleaning nozzle up / down mechanism, and 27 denotes a cleaning nozzle. In such an apparatus, a sample dispensing nozzle 2 connected to a sample dispensing pump 1 is moved by a sample dispensing nozzle moving mechanism 3 to aspirate a certain amount of a standard sample 5 from a standard sample container 4 and subsequently to suck a sample. A predetermined amount of the sample 8 is sucked from the sample container 7 set on the turntable 6, and the sample 8 and the standard sample 5 are dispensed into the reaction cell 10 arranged on the reaction disk 9. When the reaction disk 9 rotates and the reaction cell 10 advances one step, the first reagent dispensing nozzle 12 connected to the first reagent dispensing pump 11
The reagent is moved by the reagent dispensing nozzle moving mechanism 13 to aspirate a certain amount of the first reagent 16 from the first reagent container 15 set in the reagent storage 14, and then move to the reaction cell 10 'to perform the reaction. Dispense into cell 10 '. At this time, the absorbance (Yt) of the reaction cell of the item using the one-reagent-based reaction reagent is sequentially measured over time while the spectroscope 17 reciprocates around the same axis as the reaction disk 9 by the spectroscope moving mechanism 18. It is stored in the control and data processing computer 19 while measuring.

Next, when the reaction cell 10 comes to the position of the reaction cell 10 ″, the second reagent dispensing nozzle connected to the second reagent dispensing pump 20.
21 moves up to the second reagent dispensing nozzle moving mechanism 22 and moves,
A certain amount of the second reagent 24 is aspirated from the second reagent container 23 set in the reagent storage 14, and then moved to the reaction cell 10 ″ to use the reaction cell of the item using the two-reagent reaction reagent.
After the addition of the second reagent, the reaction cell 10 "is connected to the washing pump 25, and moves to the washing nozzle 27 which moves up and down by the washing nozzle up-and-down mechanism 26. absorbance Y _t is stored in the Konbyuta 19 is measured in. At the same time, the control and data processing computer 19 controls the operations of the respective parts synchronously, and at the same time, absorbs the absorbance data A at the reaction times t ₁ and T ₂ from the first reagent dispensing (after the start of the reaction) for the items of one reagent. _s (1), A _s (2)
And the two-reagent system items, absorbance data A _s (1), at reaction times t ₁ and T ₂ after dispensing the second reagent (after the start of the reaction).
A _s (2) and reaction time previously determined for the standard sample solution
t _1, t ₂ absorbance data A _st (1) in, A _st (2) and to calculate the optical density value A _I of the interference component from the formula (I) the optical density value A _b of the reagent blank solution as a parameter and then the absorbance (Y _t) is corrected by this a _I, performed each rate measurement or endpoint measurement within the predetermined limits absorbance a on the basis of this derived absorbance data, quantitative values of each measurement component Is converted and measured.

 Hereinafter, data when actually performed will be described.

Measurement of Total Protein (TP) A solution containing 5.5 g / dl (optimal measurement range) of total protein was used as a sample solution, and a dilution series of hemoglobin (interference component: Hb) was added to the sample solution. 50
The measurement was performed on a sample prepared to be 0 mg / dl. As a reaction reagent, a one-reagent TP measurement reaction reagent (manufactured by Wako Pure Chemical Industries, Ltd.) was used.

The final absorbance (average value of n = 5) at this time is shown below.

As shown in the results, since hemoglobin becomes a positive interference component of the absorbance when measuring the total protein, it can be seen that the respective measured absorbance values increase in accordance with the amount thereof.
In the biochemical analyzer, the total protein is measured by the end point method.

Therefore, first, the absorbance of the sample solution at an Hb concentration of 500 mg / dl was corrected by the method of the present invention.

The Hb-free solution containing 4 g / dl of total protein was used as the standard sample solution.

For the sample solution and the standard sample solution, t ₁ = 0.3 min, T ₂
= The absorbance at 6.0 minutes was measured. The results were as follows.

Using the above absorbance values and the reaction reagent blank value (16 mAbs), the absorbance A _I of the interference component was calculated by the formula (I), and a value of A _I = 58.4 mAbs was obtained. Therefore, by subtracting this value from the final absorbance of the sample solution, the corrected absorbance of the sample solution from which the liquid crystal of the interference component was excluded is 143.1 mAbs.
The results of the same absorbance correction for each of the other sample solutions having different Hb concentrations by the formula (I) in the same manner are shown in the following table (n = 5).

Incidentally, the absorbance of the sample having a Hb concentration of zero is 139.1 mAb.
s.

As described above, according to the method of the present invention, it is possible to remove the optical influence of the interference component regardless of the amount of the interference component.
As a result, it can be seen that the total protein as the target component can be accurately quantified using the corrected absorbance, and the number of retests can be reduced.

When the same correction was performed using a solution containing 10 g / dl of total protein as a standard sample solution, the corrected absorbance was 136.5 to 14
The value was 3,5 mAbs, and almost the same result was obtained.

(G) Effects of the Invention According to the present invention, it is possible to easily eliminate the adverse effect of the interference component and perform the quantification of the predetermined component without performing independent measurement of the interference component, volume correction, and the like. In the method, the complexity of the retest can be reduced, and in the rate method, the measurement accuracy can be further improved.

[Brief description of the drawings]

FIG. 1 is a configuration explanatory view illustrating an apparatus for carrying out the biochemical analysis method of the present invention, and FIGS. 2 and 3 are explanatory views of the principle of the method of the present invention.

Claims (1)

(57) [Claims] 1. A sample solution is mixed with a predetermined reaction reagent to cause a reaction, and the optical density thereof is measured with time, and then within a predetermined optical density limit value A in a series of actually measured optical density values obtained. Calculating the change or change rate of the optical density in the above, and quantifying a predetermined component in the sample liquid based on the calculated value, the optical density value A _{s at} the reaction time t ₁ , t ₂ for the sample liquid (1) a _s (2), the optical density value a at reaction time t _1, t _2, which is determined in the same manner for the standard sample solution
_{st (1), A st (} 2) and the reagent blank mixture following equation from the optical density value A _b of (I): The optical density value A _I of the interference component in the sample solution is obtained based on the above, and the optical density change or change after correcting the series of actually measured optical density values or the optical density limit value A with the optical density value A _I. A biochemical analysis method comprising calculating a ratio.