JPS63289440A - Assay of component - Google Patents

Abstract

PURPOSE:To obtain a correct an highly reliable measured value, by a method wherein a first sample is added after the measurement of a blind detection value or a rate of change in blind detection of a measuring reagent and moreover, a second sample is added after a fixed time to measure a variation value or a rate of change separately. CONSTITUTION:First, a blind detection value A1 or a rate A2 of change in blind detection for a fixed time of a measuring reagent held in a container measurable by an optical or physical means are measured. Then, a first sample is added to the measuring reagent and a variation value B1 after a fixed time or a rate B 2 of change for a fixed time are measured. Moreover, a second sample is added and a variation value C1 after a fixed time and a rate C2 of change for a fixed time are measured to assay. In other words, measuring data for two samples are obtained in one container to avoid a shortcoming- difficult in the control of accuracy while maintaining advantage in a monotest method. This also allows the avoiding of effect on a measured value by an event which will accidentally take place in a discrete type automatic analyzer and of effect by a reagent or aging of a machine itself.

Description

[Detailed description of the invention] [Field of application of the invention] The present invention relates to a method for measuring components in a solution.

[Background of the Invention] In the field of clinical testing, a method for measuring components in a solution is often used, in which a substance to be measured is changed by a chemical reaction or an enzymatic reaction, and the change is measured using optical or physical means. However, recently, monotest type reagents using this method have been developed for emergency testing because it is possible to easily prepare only the required amount of measurement reagent compared to conventional measurement reagents. The frequency of use is increasing. The monotest type reagent referred to here refers to a measurement reagent in the form of a formulation for each sample.For example, the measurement reagent is dispensed into a suitable container for each measurement, and then freeze-dried. Prepare the measurement reagent solution by dissolving it in water or a special dissolving solution before use, add the sample to it, react it, and then measure the components in the solution by measuring its coloration or turbidity, etc. A reagent that can be used to In addition to the above-mentioned formulations, these monotest type reagents may be tablet-like, or so-called dry chemist formulations, such as filter paper, nonwoven fabric, film, etc., in which the measurement reagent is impregnated and dried, or coated and dried. Things like this can also be included in this category.

However, the measurement method (hereinafter referred to as the Monotest method) using a Monotest type reagent (hereinafter referred to as the Monoreagent) has serious drawbacks in terms of quality control. Ta. That is, in the current monotest method, when measuring one sample, at least three monoreagents (each used for reagent blind testing, sample measurement, and standard measurement) are required.
Therefore, variations in reagent blind testing of each monoreagent, color sensitivity during measurement, etc. must necessarily be within the error range, but in reality, variations in liquid volume,
Due to differences in storage conditions, etc., it is difficult to say that it is within the error range, and the only way to confirm this is to actually use the reagent, and whether or not the measurement is being performed accurately. It was virtually impossible to confirm.

In addition, currently methods for measuring components in solutions in which the substance to be measured is changed by a chemical reaction or an enzymatic reaction and the change is measured using optical or physical means are currently being carried out using automatic analyzers, especially a measuring method called the discrete method. In other words, it is applied to an automatic analyzer capable of processing multiple samples using a method in which each sample is reacted with a measurement reagent in a specific measurement cell and the change is measured. However, for example, it is possible to avoid the influence on measured values due to sudden dispensing errors of measurement reagents, sudden abnormalities in the optical system, dirt in individual cells, changes in measurement conditions due to scratches or differences in distortion, etc. Moreover, the color sensitivity during the reaction may change due to changes in the measurement reagent itself over time, and there may also be effects due to changes over time in the measuring system of the machine, so it is difficult to carry out measurements over a long period of time. If the method is continued, there are problems such as the need to repeat the measurement of the standard or a sample of known concentration at regular intervals in order to confirm whether accurate measurement values are being obtained.

[Purpose of the Invention] In view of the above-mentioned circumstances, the present invention avoids the disadvantage of difficulty in accuracy control while retaining the advantages of the mono test method, and also provides a method for handling unexpected problems in discrete automatic analyzers. It is an object of the present invention to provide a method that can obtain accurate and reliable measured values by avoiding the influence of events that occur on measured values and the influence of changes in reagents or the machine itself over time.

[Components of the invention In order to achieve the above object, the present invention consists of the following configuration.

"In the measurement of components, (a) first measure the blinded value A1 of the measurement reagent or the blinded rate of change A2 over a certain period of time; (b) then add the first sample to the measurement reagent. (c) Add a second sample to this and measure the fluctuation value C2 after a certain period of time or the rate of change B2 in a certain period of time. A method for quantifying a component, characterized by quantifying it by measuring the rate of change C2 of the component. The blinded value A or '-blind rate of change A2 over a certain period of time is first measured, and then the first sample is added to the measurement reagent, and the change after a certain period of time is measured. By measuring the value B1 or the rate of change B2 over a certain period of time, further adding a second sample to this, and measuring the fluctuation value CI after a certain period of time or the rate of change C2 over a certain period of time, that is, By obtaining measurement data for two samples in one container, it retains the advantages of the monotest method, avoids the disadvantage of difficult accuracy control, and allows automatic analysis of the discrete F method. The present invention was accomplished by discovering that it is possible to obtain accurate and reliable measured values by avoiding the effects of unexpected events on the measured values in the machine and the effects of changes in reagents or the machine itself over time. reached.

In the present invention, the measurement is performed by reaction in a solution, and the concentration of the component to be measured is known (the concentration is defined as Sl).

) is used as a standard to measure the concentration s2 of the component to be measured in the second sample, S2 is obtained by equation-1 or 2.

Equation-1, the amount of sample collected (1), and ■3 is the amount of sample collected (1) of the second sample.
), AH, A2+ BI+ B2+ C1t C
2+ Si.

S2 is the same as above. ] In addition, when performing dry chemistry or under the conditions of 1 > Vl: (V++ V2 + V3) > 0, 95, errors due to liquid volume can be almost ignored, so in these cases, formula-1 or formula-1 can be used as an approximate formula. S2 can be obtained by Equation-3 or 4 instead of 2.

If A1 or A2 is negligibly small compared to Equation-3 or Equation-3, A1 or A2 can be further omitted from Equation-3 or Equation-4.

On the other hand, when the sample whose concentration of the component to be measured is known is the second sample (its concentration is S2), formulas -1, 2, 3, 4
In place of , the following formulas-1' and 2' are used.

By using 3' and 4', it is possible to similarly determine the concentration S1 of the component to be measured in the first sample.

The advantage of Equation 3' and Equation 4' is that as long as the amounts of the first and second samples are kept constant (for example, if they are collected with the same pipette, the amount of each pipette collected is the same). There is no particular problem even if the value differs from the displayed value.It is clear from the above that there is no particular problem in the measurement even if the liquid volume of the input measurement reagent varies slightly. Samples to be used are not particularly limited as long as they are liquid, but in the field of clinical testing, examples include biological fluids such as blood, serum, plasma, urine, and spinal fluid. Examples include substrates such as glucose, cholesterol, free fatty acids, triglycerides, bile acids, and uric acid, as well as lactate dehydrogenase, α-hydroxybutyrate dehydrogenase, cholinesterase, monoamine oxidase, amylase, alkaline phosphatase, transaminase, and leucine aminopeptidase. Examples include enzymes such as γ-glutamyl transpeptidase, but needless to say, they are not limited to these.In addition, as samples with known concentrations of the components to be measured, commercially available standard solutions or controlled Although it is convenient to use serum or the like, there is no particular problem in using the above-mentioned samples whose concentration has been assayed based on the serum.

Examples of optical or electrical measuring means used in the present invention include, but are not particularly limited to, measuring electric potential, conductivity, absorbance, turbidity, amount of reflected light, fluorescence, chemical or biological luminescence, etc. It's not something you can do.

The measurement reagent used in the present invention is not particularly limited as long as it reacts with the component to be measured contained in the sample as described above and causes the optical or electrical change as described above. For example, as long as it is a measurement reagent commonly used in the field of clinical testing, it may be of a solution type or one for dry chemistry.

The present invention is particularly advantageous when applied to measuring instruments controlled by computers, etc., and may be manufactured as a specialized instrument for carrying out the present invention, or may be used for conventional automatic analysis for clinical tests. The present invention may be implemented by adding program software to a machine or the like to implement the present invention.

In addition, in automatic analyzers using the discrete method,
It is also possible to apply the present invention to quality control for each batch. In other words, when measuring a component by measuring absorbance, for example, the absorbance of the standard can be measured separately in advance, divided by the concentration of the component to be measured in the standard, and stored as a specific standard coefficient. Among the values obtained by measuring each batch according to the method of the invention, the concentration value of the component to be measured can be obtained by simply multiplying the absorbance obtained from the sample whose concentration of the component to be measured is unknown by the standard coefficient. In this case, the absorbance obtained from a sample with a known concentration of the component to be measured can be used to check whether a value within a certain range is obtained. In other words, sudden dispensing errors in the measurement reagent, sudden abnormalities in the optical system, changes in measurement conditions due to differences in dirt, scratches, or distortion in individual cells, and the influence on measurement values, as well as the measurement reagent itself. This makes it possible to avoid the effects of changes in color sensitivity during reaction due to changes over time, or changes over time in the measuring system of the machine.

For example, when measuring a component by measuring absorbance, the absorbance of the standard is measured in advance and then divided by the concentration value of the component to be measured in the standard and stored as a specific standard coefficient. If the two absorbance data obtained by the method of the present invention are multiplied by the standard coefficient, assuming that the and the second sample are the same, it is possible to perform two measurements with the amount of reagent that could be used for one measurement on one sample using the conventional method. Another advantage is that measurements can be taken and the reliability of the measured values is increased.

The present invention will be explained in more detail with reference to Examples below.
The present invention is not limited to these examples.

[Example] Example 1. Measurement of glucose in serum (measuring reagent) Glucose oxidase 70 units Peroxidase 3000 units Mutarotase 6 units 4-aminoantipyrine'10I118 phenol
50mg phosphorus W phosphorus potassium 0.
54g dipotassium phosphate 2.76g
The above substance was dissolved in purified water, the pH was adjusted to 7.4, and the total amount was adjusted to 1001 to prepare a measurement reagent.

(Sample) An aqueous solution with a glucose concentration of 100 mg/dl was used as the first sample, and 10 human serum samples were used as the second sample.

(Operation method) Measurement cell of spectrophotometer (layer length: l (1 mm, width: lO
+nw+.

At a height of 42++u++), exactly 31 samples of the measurement reagent were sampled, and after heating at 37°C for 2 to 3 minutes, the absorbance A1 at a wavelength of 505nw+ was measured. Next, exactly 0.021 of the first sample was added and mixed well, followed by a 5-park reaction at 37°C, and the absorbance B1 at a wavelength of 505t++++ was measured.

Furthermore, after adding exactly 0.02 ml of the second sample and mixing well, the mixture was allowed to react at 37° C. for 5 minutes, and the absorbance C1 at a wavelength of 505 nm was measured.

(Calculation of glucose concentration) - Calculation method-1 In the above equation-1, v, = 3. o, V2=V3”0.02
．． Substituting s, = loo, A obtained by the above operation method
, + B 1 and C1 to obtain the glucose concentration S2 in the second sample.

-Calculation method-2 The glucose concentration S2 in the second sample was obtained by substituting S = 100 into Equation-3 and substituting AI, B1, and C1 obtained by the above operation method.

Example 2 The same measurement reagent and sample as in Example 1 were used, and the same operations were carried out, except that the sample was collected using a single pipette (the amount of measurement reagent collected was approximately 31, and the sample was not verified). A by law
I, Bit c, was determined, and the glucose concentration S2 in the second sample was obtained using the same calculation method-2.

Comparative example 1. Measurement of glucose in serum using conventional methods (Measurement reagents and samples) The same material as in Example 1 was used.

(Procedure) Accurately collect a sample into a 0.02ml test tube, add exactly 31ml of measurement reagent, react at 37°C for 5 minutes,
The absorbance at 0.05 rv (the absorbance obtained by the first sample was designated as E' S ld, and the absorbance obtained by the second sample was designated as Es) was measured. FBI was obtained by performing the same operation using purified water instead of the sample.

(Calculation of glucose concentration) Glucose concentration (g/d+) was determined using the following formula.

Glucose concentration = (Es-Eat) ÷ (Estd-EB
I) Glucose concentrations in the human serum samples obtained in X100 Example 1.2 and Comparative Example 1 are shown in Table 1.

Table 1 As is clear from the results in Table 1, no significant difference was observed between the values obtained by the method of the present invention and those obtained by the conventional method.

Example 3. Measurement of lactate dehydrogenase activity in serum (measurement reagent) Dipotassium phosphate 770II 1g Potassium phosphate 122g Lithium pyruvate 6.7mg Nicotinamide adenine dinucleotide (reduced form)
Dissolve 14mg of the above substance in purified water and make po 7
．． 6, and the total amount was set to 1001, which was used as a measurement reagent.

(Sample) As the first sample, a human serum with known activity of lactate dehydrogenase (hereinafter abbreviated as LDH) (activity value: 300 Ll) was prepared.
A human serum 10 sample was used as the second sample.

(Operation method) Measurement cell of spectrophotometer (layer length: 10 mw++ width; lO
am.

Accurately collect 21 samples of the measurement reagent at a height of 42 mm, and
1 at a wavelength of 340 nm after heating at ℃ for 2 to 3 minutes.
The rate of change A2 in absorbance per minute was measured. Then the first
Add exactly 0.05w+1 of the sample and mix well, and while heating at 35° C., the rate of change in absorbance B2 per minute at a wavelength of 340 nm was measured. Furthermore, set the second sample to exactly 0.
．． Add 05ml and mix well, and while heating at 35℃, change rate of absorbance per minute at wavelength 340nwl C
2 was measured.

(Calculation of LDH activity) The above formula-2 V'=2.0. V2=V3=0.05
．． Substituting 5I=300, A2. obtained by the above operation method.
By substituting B2 and C2, LDH activity S2 in the second sample was obtained.

Example 4. Measurement of LDH activity Example 3 except that the pH of the reagent used in Example 3 was 7.5 (measurement sensitivity usually changes due to pH fluctuations).
A2+ 82. was measured using the same measurement reagents and samples and the same procedure. Find C2 and use the same calculation method to calculate the second
LDH activity S2 in the sample was obtained.

Comparative example 2. LDH-UV Te5twak is commercially available as a reagent for measuring LDH activity using a conventional method.

(manufactured by Wako Pure Chemical Industries, Ltd.) in the same sample as in Example 3 according to the standard operating method described in the product manual.
Activity was determined.

L in human serum samples obtained in Examples 3 and 4 and Comparative Example 2
The DH activity values are also shown in Table-2.

5 Table 2 As is clear from the results in Table 2, if the method of the present invention is used, even if there is some kind of trouble with the measurement reagent,
It can be seen that there is almost no effect on the measured value of the target component.

Example 5. Measurement of total cholesterol in serum (measurement reagent) Cholesterol esterase 200 units Cholesterol oxidase 40 units Peroxidase 600 units Ascorbate oxidase 500 units Doaminoantipyrine
4mg3.5-dimethoxy-N-ethyl-
N-(2-hydroxy-3-sulfopropyl)aniline' sodium salt 30I118 Triton X-1
00100mg2-'(N-morpholino)ethanesulfonic acid't,tgSodium hydroxide
Dissolve 0.15g of the above substance in purified water and reduce p■ to 6
．． 1, and the total amount was set to 1001, which was used as a measurement reagent.

(Sample) As the first sample, 100 g of cholesterol was dissolved in 10 ml of isopropanol, and 20 χ Triton
100 aqueous solution was added to make the total 4110 (1 ml), and a human serum 10 sample was used as the second sample.・
(Operation method) Measurement cell of spectrophotometer (layer length: 10 mm, width: 10 m
m.

Accurately collect 31 samples of measurement reagent at height; 42+nw+),
After heating at 37° C. for 2 to 3 minutes, absorbance A1 at a wavelength of 600 RUW was measured. Then the first sample is set to exactly 0
021 was added, mixed well, and reacted at 37° C. for 5 minutes, and then the absorbance B1 at a wavelength of 600 nn+ was measured. Furthermore, add exactly 0.02+wl of the second sample and mix well.
After reacting at 37°C for 5 minutes, the absorbance C1 at a wavelength of 600r++s was measured.

(Calculation of total cholesterol concentration) Substituting S = 100 into the above formula-3, A1. obtained by the above operation method. 'B, and C1 were substituted to obtain the total cholesterol concentration S2 in the second sample.

Comparative example 3. Measurement of total cholesterol in serum by conventional method (measurement reagent and sample) The same ones as in Example 5 were used.

(Procedure) Collect a sample into a 0.02w+1 test tube, add exactly 31ml of measurement reagent, react at 37°C for 5 minutes, and measure the absorbance at a wavelength of 600n (absorbance obtained with the first sample). was defined as E Sld, and the absorbance obtained by the second sample was defined as ES.) was measured. FBI was obtained by performing the same operation using purified water instead of the sample.

(Calculation of total cholesterol concentration) String cholesterol concentration (w+g/d I ) using the following formula:
The total cholesterol concentration in the sample is also shown in Table 3.

Table 3 As is clear from the results in Table 3, no significant difference was observed between the values obtained by the method of the present invention and those obtained by the conventional method.

[Effects of the Invention] The present invention has remarkable effects as described below, and greatly contributes to this industry.

(1) In the measurement method of the present invention, even if the amount of sampled reagent varies, it has no or little effect on the measured value of the target component.

(2) In the measurement method of the present invention, even if the sensitivity of the measurement reagent during measurement changes, it has no or little effect on the measured value.

(3) In the measurement method of the present invention, even if the reagent blindness of the measurement reagent changes, the measurement value is not affected at all or hardly.

(4) In the measurement method of the present invention, during continuous measurement of a large number of samples, the amount of the measurement reagent suddenly fluctuates, or the measurement reagent itself or the measurement system of the machine changes over time. Even in cases where there is no effect on the measured concentration of the target component,
Furthermore, it is possible to detect the degree of variation.

(5) In the measurement method of the present invention, by monitoring the measurement sensitivity obtained with a sample whose concentration of the component to be measured is known,
It is possible to monitor the operating status of the device or abnormalities in the measurement reagent for each sample.

(6) In the conventional measurement method, at least three containers are used (each used for reagent blind testing, sample measurement, and standard measurement).
However, according to the method of the present invention, this can be carried out using one container and reagents for one measurement, which is economical.

Patent Applicant Wako Pure Chemical Industries, Ltd. Procedural Amendment 1. Indication of the case 1988 Patent No. 032775 2, Method for quantifying name components of the invention 3, Person making the amendment Relationship with the case Patent applicant 541 Address 3-10 Doshomachi, Higashi-ku, Osaka-shi, Osaka Contact information Patent Division (Tokyo) 03-270-8571 Name Wako Pure Chemical Industries, Ltd. 5 Column for detailed description of the invention in the specification to be amended.

6. Contents of the amendment (1) "It is obvious" written on page 9, line 14 of the specification.
After that, insert a new line and insert the following text.

``It goes without saying that if the blind value A1 of the designated reagent or the rate of change Aλ over a certain period of time is negligibly small, or if it is known, the measurement of A1 or Ai can be omitted. That's it.''

Claims (3)

[Claims] (1) In the measurement of components, (a) First, the blinded value A_1 or the blinded rate of change A_2 over a certain period of time of the measuring reagent is measured, (b) Next, the first After adding the sample, change value B_1 after a certain period of time or rate of change B_ in a certain period of time
(c) further adding a second sample to this, and quantifying by measuring the fluctuation value C_1 after a certain period of time or the rate of change C_2 in a certain period of time. . (2) The quantitative method according to claim 1, wherein the measurement reagent is provided from a monotest type reagent. (3) The quantitative method according to claim 1, wherein the measuring means is optical or electrical means.