JP2002031641A - Standard solution containing protein measuring method for substance to be measured by using it, reaction- indication-substance acqueous solution containing protein and calculation method for calibration factor using it - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a standard solution which can obtain the measured value of a substance to be measured equal to that in a case where a calibration is performed by serum, blood plasma, standard serum or standard blood plasma by a method wherein the dispensing amount of the standard solution in an analyzer is equal to that of a sample such as the serum, the blood plasma or the like, to provide a measuring method for the substance to be measured, to provide a reaction-indicating-substance acqueous-solution in which the dispensing amount of the reaction-indicating-substance acqueous solution in the analyzer is the same as an intrinsic dispensing amount, by which a correct calibration factor not containing an error can be calibrated and which can obtain the precise measured value of the object to be mea sured and to provide a calculation method for the calibration factor. SOLUTION: In the standard solution, a protein is contained in water or an aqueous solution together with the substance to be measured. In the measuring method for the substance to be measured, the calibration is performed by the standard solution. In the reaction-indicating- substance aqueous solution, the protein is contained in the water or the acqueous solution together with a reaction-indicating substance. In the calculation method for the calibration factor, the calibration factor is calculated by the reaction-indicating-substance aqueous solution.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a standard solution containing a protein together with a substance to be measured in water or an aqueous solution,
And a method for measuring a substance to be measured, which is calibrated using the standard solution.

[0002] The present invention also relates to a reaction indicator aqueous solution in which a protein is contained in water or an aqueous solution together with a reaction indicator, and to a method for calculating a calibration coefficient for performing calibration using the reaction indicator aqueous solution.

[0003] The present invention is particularly useful in the fields of medicine, analytical chemistry, and life sciences, and is particularly useful in the field of clinical testing.

[0004]

2. Description of the Related Art At present, measurement of a substance to be measured contained in a sample using a chemical reaction is widely used. For example, a reaction between an enzyme and a substance serving as a substrate of the enzyme, a complex formation reaction, an antigen-antibody reaction (immunological reaction), a binding reaction of a nucleic acid with a nucleic acid complementary thereto, a hydrophobic binding reaction, or other chemical reactions Measurement of a substance to be measured using a reaction or the like is performed.

[0005] In the quantitative measurement of these substances to be measured, usually, calibration is performed using a standard substance to determine the concentration of the substance to be measured (or the activity value of the substance to be measured) contained in the sample. Or the calibration coefficient (K
Using the factor), the concentration of the substance to be measured (or the activity value of the substance to be measured) contained in the sample is determined.

(2) This standard substance, when used for measurement, is usually used as it is for calibration using a standard solution supplied in a liquid form as it is, or a standard solution supplied in a solid form. The substance is dissolved in a solvent such as water to make a standard solution, which is used for calibration.

(3) Calibration with a standard solution to measure a substance to be measured will be specifically described below, taking the case of measuring the creatinine concentration in a serum sample as an example.

[0008] To 4 µL of a serum sample containing creatinine, which is a substance to be measured, 210 µL of the first creatinine measuring reagent (including creatinase, sarcosine oxidase, and an aniline derivative) is added.
Let stand for a minute and let it react.

Next, the second creatinine measuring reagent
70 μL of reagents (including creatininase, 4-aminoantipyrine and peroxidase) were further added,
Allow to react at 37 ° C.

About 5 minutes after the addition of the second reagent, the main wavelength 54
The absorbance (As) at 6 nm and the auxiliary wavelength 700 nm is measured.

[0011] The measurement is performed as described above using water as a sample instead of the serum sample, and the absorbance (Arb) is measured to obtain a reagent blank value. Then, absorbance (ΔAs = As−Arb) obtained by subtracting the reagent blind value is obtained.

Next, 5.00 g was used instead of the serum sample.
Using a standard solution of creatinine, which is a creatinine aqueous solution having a concentration of / dL, as a sample, the measurement is performed as described above, and the absorbance (Astd) at the time of measuring the standard solution is measured. And also in this case, the absorbance (ΔAstd
= Astd-Arb).

Next, the obtained absorbances (ΔAs and ΔAs
Calibration is performed by substituting td) into the following equation to determine the creatinine concentration contained in the serum sample.

The concentration of creatinine (mg / dL) contained in the serum sample = [ΔAs ÷ ΔAstd] × 5.00
(Mg / dL)

(3) Regarding the measurement of the concentration of the substance to be measured (or the activity of the substance to be measured) contained in the sample using the calibration coefficient, the activity of the alkaline phosphatase in the serum sample is measured. The case will be specifically described below.

Measurement of Change in Absorbance 4 μL of a serum sample containing alkaline phosphatase, which is a substance to be measured, contains 2 parts of the first reagent (including 2-ethylaminoethanol) of the alkaline phosphatase measurement reagent.
Add 40 μL and place at 37 ° C. for 5 minutes.

Next, 60 μL of a second reagent (including disodium 4-nitrophenylphosphate) as a reagent for measuring alkaline phosphatase is further added thereto, and the mixture is placed at 37 ° C.

Then, the amount of 4-nitrophenol released by the action of alkaline phosphatase on the substrate, disodium 4-nitrophenylphosphate, is measured.

That is, the main wavelength 405 nm and the sub wavelength 50
At 5 nm, the amount of change in absorbance from about 1 minute 30 seconds to about 5 minutes after the addition of the second reagent is measured, and the amount of change in absorbance per minute (As) is determined.

The measurement is performed as described above using water as a sample instead of the serum sample, and the change in absorbance per minute (A
rb) is measured and used as a reagent blind value. Then, the absorbance change amount per minute (ΔAs =
As-Arb) is determined.

Calculation of Calibration Coefficient The calibration coefficient is calculated before measuring the alkaline phosphatase activity value in the serum sample.

That is, the absorbance derived from 4-nitrophenol is measured, and the concentration of the target substance (or the activity value of the target substance) is measured by using a calibration coefficient. The calibration coefficient is calculated using an aqueous solution containing phenol.

First, the reaction indicator substance BmM (mmo)
1 / L) of 4 μL of an aqueous solution of a reaction indicator comprising an aqueous solution containing 4-nitrophenol, 240 μL of the first reagent of the alkaline phosphatase measurement reagent was added,
Place at 37 ° C. for 5 minutes.

Next, 60 μL of the second reagent of the alkaline phosphatase measurement reagent was further added thereto, and
Place at 5 ° C. for 5 minutes.

Thereafter, the main wavelength 405 nm and the sub wavelength 505
absorbance (A) at about 5 minutes after the addition of the second reagent in nm.
k).

The measurement is performed as described above using water as a sample instead of the reaction indicator aqueous solution, and the absorbance (Akrb) is measured to obtain a reagent blank value. Then, the absorbance (ΔAk = Ak−Akrb) obtained by subtracting the reagent blank value is obtained.

From the absorbance (ΔAk) obtained by subtracting the reagent blank value of this BmM aqueous solution of the reaction indicator, the actually measured calibration coefficient in the above measurement is calculated.

Calibration coefficient (K) = (B ÷ ΔAk) × 10 ⁶

Calculation of the Activity Value of the Substance to be Measured The activity value of alkaline phosphatase contained in the sample is calculated from the amount of change in absorbance per minute (ΔAs) and the calibration coefficient (K) determined earlier.

Activity value of alkaline phosphatase contained in the sample [international unit (IU)] = ΔAs × K

(4) The use of an analyzer to measure a substance to be measured contained in a sample has been widely used in recent years. However, in calibration with a standard solution or calculation of a calibration coefficient with an aqueous solution of a reaction indicator, the following is required. This is a problem.

When calibration is performed using a standard solution containing the substance to be measured in water or an aqueous solution, and the measurement of the substance to be measured is performed by an analyzer, the measured value obtained differs from the original value, An error occurs in the measured value of the target substance.

This is because the smaller the amount of the sample such as serum or plasma in the analyzer (that is, the amount of the standard solution), the greater the degree of this positive error.

The amount of the standard solution dispensed by the analyzer and
The volume of the sample such as serum or plasma should be exactly the same [4 μL in the above (2)], but actually the volume of the standard solution is smaller than the volume of the sample such as serum or plasma. The injection volume is different, and the calibration target (Calibration) is different from the amount of the substance measured in this standard solution. It is considered that the substance concentration (measurement target substance activity value) is different from the original value.

In addition, when the calibration coefficient is calculated with the reaction indicator aqueous solution in the analyzer, the same as when the calibration is performed with water or the standard solution contained in the aqueous solution,
The obtained calibration coefficient differs from the original value, and an error occurs in the measured value. If the calibration coefficient is different from the original value, the concentration of the substance to be measured (or the activity value of the substance to be measured) obtained by multiplying the calibration coefficient by the absorbance value obtained by the measurement is also different from the original value. Will be different and an error will occur.

In this case, the smaller the amount of the sample such as serum or plasma in the analyzer (that is, the amount of the aqueous solution of the reaction indicator), the greater the degree of the positive error.

Even in the case of this reaction indicator aqueous solution, the dispensed amount of the reaction indicator aqueous solution in the analyzer differs from the originally dispensed amount. When the absorbance of the reaction indicator aqueous solution is measured with an amount of the reaction indicator different from the amount of the reaction indicator, the calculated calibration coefficient is considered to be different from the original value. .

In the analyzer, the reason why the dispensed amount of the standard solution is different from the dispensed amount of the sample such as serum or plasma, and the reason that the dispensed amount of the reaction indicator aqueous solution is different. This is presumed to be due to the difference between the composition of the sample such as serum or plasma (matrix) and the composition of the standard solution composed of the aqueous solution (matrix), or the composition of the reaction indicator aqueous solution (matrix). You.

Japanese Patent Publication No. Hei 7-104344 discloses a standard solution for clinical test or a reaction instruction for setting a calibration coefficient in which the viscosity and the specific gravity are adjusted with polyethylene glycol having an average molecular weight of 400 or less so as to be equivalent to serum. A substance solution is described, which is said to reduce the coefficient of variation and improve the accuracy of the sample collection amount. Japanese Patent Application Laid-Open No. 9-89899 describes a standard solution for clinical test or a reaction indicator solution for setting a calibration coefficient having the same viscosity and specific gravity as a sample serum containing ethylene glycol. It is alleged that the variation coefficient becomes smaller and the sample collection amount accuracy becomes better.

However, these publications disclose a standard solution for a clinical test or a reaction for setting a calibration coefficient in which the viscosity and the specific gravity are adjusted by adding polyethylene glycol having an average molecular weight of 400 or less or ethylene glycol so as to be equivalent to serum. Although there is a statement that the indicator substance solution has a small coefficient of variation and that the accuracy of the sample collection volume is good, that is, that the precision of measurement has been improved, the dispensed volume of the standard solution or the reaction indicator aqueous solution in the analyzer. There is no description that an accurate measurement value without errors is obtained without a difference from the dispensed volume of a sample such as serum or plasma, that is, the measurement accuracy is improved.

The specific gravity of the human pooled serum and the standard solution of Company K were similar, and the amount of the sample collected was almost the same. Therefore, physical properties such as surface tension pointed out by Haga et al. Are also involved. It was suggested that there was a possibility that the possibility was high. "," In spite of differences in viscosity and specific gravity of various sera, the amount collected was almost constant regardless of the type "[Tsuchiya et al., Japan Journal of Japan Society of Clinical Laboratory Automation, Vol. 17, No. 5, 627
632, 1992], there is a difference between the dispensed amount of the sample such as serum or plasma and the dispensed amount of the standard solution or the aqueous solution of the reaction indicator in the analyzer. The error in the measured value or the calibration coefficient is caused not only by the difference in viscosity and specific gravity from serum or plasma but also by the complex composition (matrix) of the sample such as serum or plasma. Other factors can be considered to be involved.

[0042]

SUMMARY OF THE INVENTION In view of the above problems in the prior art, the present invention provides a method of calibrating a substance to be measured using a standard solution containing water or an aqueous solution, and measuring the substance to be measured by an analyzer. When performing this, a difference occurs in the dispensed volume of the standard solution compared to the dispensed volume of the sample such as serum or plasma, and the measured value obtained thereby differs from the original value, and the It is an object to eliminate the occurrence of errors in measured values.

That is, according to the present invention, the amount of the standard solution dispensed in the analyzer is equivalent to that of a sample such as serum or plasma.
It is an object of the present invention to provide a standard solution capable of obtaining a measured value of a measurement target substance equivalent to that obtained by calibration using serum, plasma, standard serum, or standard plasma, and a method for measuring the measurement target substance.

Further, according to the present invention, when the calibration coefficient is calculated by using the reaction indicator aqueous solution, the dispensed amount of the reaction indicator aqueous solution is different from the original dispensed amount. It is an object of the present invention to solve the problem that an error occurs due to a difference from an original value, and an error occurs in a measured value of a substance to be measured.

That is, according to the present invention, the dispensed amount of the reaction indicator aqueous solution in the analyzer is the same as the original dispensed amount, and a correct calibration coefficient without errors can be calculated. It is another object of the present invention to provide a reaction indicator aqueous solution from which a measured value of a substance can be obtained and a method for calculating a calibration coefficient.

[0046]

Means for Solving the Problems The present invention includes the following inventions. (1) A standard solution comprising a protein and water or an aqueous solution together with a substance to be measured. (2) The standard solution according to the above (1), wherein the concentration of the protein is 4 to 8%. (3) The standard solution according to (1) or (2), wherein the protein is albumin. (4) The standard according to any one of (1) to (3) above, wherein the amount of the standard solution to be dispensed becomes equivalent to serum, plasma, standard serum, or standard plasma by containing the protein. liquid. (5) The method according to any one of the above (1) to (4), wherein the inclusion of a protein provides a measurement value equivalent to that obtained by calibration using serum, plasma, standard serum, or standard plasma. Standard solution. (6) The standard solution according to any one of the above (1) to (5), which is a standard solution when measuring the substance to be measured by an analyzer. (7) A method for measuring a substance to be measured, wherein in the measurement of the substance to be measured, the protein is calibrated together with the substance to be measured using a standard solution containing water or an aqueous solution. (8) The concentration of the protein contained in the standard solution is 4 to 8
%, The method for measuring a substance to be measured according to the above (7). (9) The method according to (7) or (8), wherein the protein contained in the standard solution is albumin. (10) The above-mentioned (7) to (7), wherein the calibration is performed using a standard solution containing a protein together with the substance to be measured, so that the dispensed amount of the standard solution becomes equivalent to serum, plasma, standard serum, or standard plasma. The method for measuring a substance to be measured according to any one of 9). (11) By performing calibration using a standard solution containing a protein together with a substance to be measured, a measurement value equivalent to that obtained by performing calibration using serum, plasma, standard serum, or standard plasma is obtained. Any one of ~ (10)
The method for measuring a substance to be measured described in (1). (12) The measurement of the substance to be measured is performed by an analyzer.
The method for measuring a substance to be measured according to any one of (7) to (11). (13) A reaction indicator aqueous solution, wherein the protein is contained in water or an aqueous solution together with the reaction indicator. (14) The reaction indicator aqueous solution according to (13), wherein the protein concentration is 4 to 8%. (15) The above-mentioned (1), wherein the protein is albumin.
The reaction indicator aqueous solution according to 3) or (14). (16) The reaction indicator aqueous solution according to any one of the above (13) to (15), wherein the amount of the dispensed reaction indicator aqueous solution is equivalent to that of serum or plasma by containing a protein. (17) By including a protein, a calibration coefficient equivalent to that obtained by calculating the calibration coefficient from serum or plasma containing a reaction indicator can be obtained.
3) The aqueous solution of the reaction indicator according to any one of the above items (16) to (16). (18) The above (13) to (17), which is an aqueous solution of a reaction indicator when the measurement of a substance to be measured is performed by an analyzer.
The reaction indicator aqueous solution according to any one of the above. (19) A method for calculating a calibration coefficient, wherein the calculation of the calibration coefficient includes the step of calculating the calibration coefficient with water or an aqueous solution of a reaction indicator containing the protein together with the reaction indicator. (20) The method for calculating a calibration coefficient according to (19), wherein the concentration of the protein contained in the aqueous solution of the reaction indicator is 4 to 8%. (21) The method for calculating a calibration coefficient according to (19) or (20), wherein the protein contained in the reaction indicator aqueous solution is albumin. (22) By allowing the protein to be contained in the aqueous solution of the reaction indicator, the dispensed amount of the aqueous solution of the reaction indicator can be increased with serum,
Alternatively, the method of calculating a calibration coefficient according to any one of (19) to (21), which is equivalent to plasma. (23) By including a protein in the reaction indicator aqueous solution, a calibration coefficient equivalent to that obtained by calculating the calibration coefficient using serum or plasma containing the reaction indicator can be obtained. 22) The method for calculating a calibration coefficient according to any one of the above items 22). (24) The above-mentioned (19) to (23), which is a method of calculating a calibration coefficient when measuring a substance to be measured by an analyzer.
The method for calculating a calibration coefficient according to any one of the above.

[0047]

BEST MODE FOR CARRYING OUT THE INVENTION (1) Standard Solution The standard solution of the present invention is a standard solution characterized by containing a protein together with a substance to be measured in water or an aqueous solution.

The protein to be contained in the standard solution together with the substance to be measured includes albumin, casein, globulin, gelatin and the like. As this protein, albumin is preferred. Examples of albumin include human serum albumin (HSA), bovine serum albumin (BSA), horse serum albumin, and ovalbumin.

The protein to be contained in the standard solution may be one type of protein alone or a plurality of types of protein.

The concentration of the protein contained in the standard solution is as follows:
1 to 12% is preferable, 3 to 10% is more preferable, and 4
-8% is particularly preferred.

The substance to be measured contained in the standard solution is not particularly limited as long as it is intended to measure the concentration or activity value of the substance to be measured contained in the sample.

Examples of the substance to be measured are described below. First, among substances to be measured which can be measured by a reaction between an enzyme and a substance serving as a substrate of the enzyme, substances serving as an enzyme substrate include, for example, creatinine, creatine, uric acid (UA), urea nitrogen (BUN) , UN), glucose, total cholesterol, free cholesterol, ester cholesterol, HDL-cholesterol, LD
L-cholesterol, β-lipoprotein, triglyceride (TG, neutral fat), phospholipid (PL), free fatty acid (NEFA, FFA), glucose, lactic acid, pyruvate, galactose, sialic acid, citric acid, fructosamine, 1 , 5-anhydro-D-glucitol, glycogen, fucose, total bilirubin, direct bilirubin, indirect bilirubin, conjugated bilirubin, unconjugated bilirubin, sodium, chlor, potassium, calcium, inorganic phosphorus, magnesium, bile acid, etc. Can be mentioned.

Among the substances to be measured which can be measured by reacting the enzyme with a substance serving as a substrate of the enzyme, examples of the enzyme include α-amylase, acid phosphatase, creatine kinase (CK), and asparaquine. Acid aminotransferase (AST, GOT), alanine aminotransferase (ALT, GPT), lactate dehydrogenase (LDH, LD), alkaline phosphatase (ALP), leucine aminopeptidase (LA
P), γ-glutamyl transpeptidase (γ-GT
P), cholinesterase (Ch-E), lipase, or lipoprotein lipase (LPL).

The substances to be measured by the complex formation reaction include, for example, iron, unsaturated iron binding ability (UIBC), total iron binding ability (TIBC), copper,
Examples include aluminum, zinc, calcium, and magnesium.

Further, as a substance to be measured which can be measured by an antigen-antibody reaction (immunological reaction), for example, HBs antigen, anti-HBs antibody, HBe antigen, anti-HBe
A virus-related antigen or antibody such as an antibody, an anti-HBc antibody, an anti-HCV antibody, an anti-HIV antibody, an anti-ATLV antibody; ) Or immunoglobulin G (IgG), immunoglobulin A (IgA), immunoglobulin M (IgM), or immunoglobulin E (I
gE) and other immunoglobulins; C-reactive protein (CR
P), inflammatory markers such as α1-acid glycoprotein, haptoglobin, complement C3, complement C4, rheumatoid factor;
tumor markers such as α-fetoprotein, CEA, CA19-9; hormones such as human placental chorionic gonadotropin; allergens or allergen-related antigens or antibodies such as allergen-specific IgE antibodies; antithrombin III (ATI
Blood coagulation-related substances such as II); fibrinolytic substances (FDP), fibrinolysis-related substances such as D-dimer; blood group-related antigens or antibodies such as ABO blood group antibodies and irregular antibodies; lipoprotein (a ), Substances related to other diseases such as ferritin; drugs or toxic substances.

The substance to be measured which can be measured by a binding reaction of a nucleic acid with a nucleic acid complementary thereto includes, for example, DNA or RNA of a virus such as a pathogenic virus and bacteria such as a pathogenic bacterium. Examples include DNA or RNA, or DNA or RNA of animals such as humans or plants.

As the substance to be measured contained in this standard solution, only one kind of substance to be measured may be contained in the standard solution, or a plurality of kinds of substances to be measured may be contained.

The standard solution of the present invention contains a protein together with a substance to be measured in water or an aqueous solution. The aqueous solution may be any solution containing water as a solvent. The aqueous solution may contain, if necessary, a buffer, an organic solvent, a surfactant, an inorganic substance such as a saccharide, an amino acid, a peptide, a lipid, a coenzyme, or a metal ion, a stabilizer, a preservative, an activator, or a chelate. Agents or other high molecular substances may be included.

As the buffer, a buffer having a buffering capacity in a target pH range may be appropriately used. Examples of such a buffer include tris [hydroxymethyl] aminomethane (Tris), phosphate buffer, imidazole, glycylglycine, PIPES, ACES, B
ES, MOPS, TES, HEPES, DIPSO, T
APSO, POPSO, HEPPSO, EPPS, HE
PPS, Tricine, Bicine, TAPS, C
HES, CAPSO, CAPS, MES, Bis-Tr
is, Bis-Tris propane, ADA, MOPS
O, and the like.

The standard solution of the present invention comprises a protein and water or an aqueous solution together with a substance to be measured.
The dispensed volume of this standard solution is serum, plasma, a standard serum having a composition (matrix) similar or similar to the composition (matrix) of serum, or a composition (matrix) similar or similar to the composition (matrix) of plasma. This is equivalent to the standard plasma of the present invention.

In addition, the standard solution of the present invention has a composition (matrix) similar to or similar to the composition (matrix) of serum, plasma, and serum by including the protein together with the substance to be measured in water or an aqueous solution. ) Or a standard plasma having a composition (matrix) similar to or similar to the composition (matrix) of plasma, and a measurement value of the substance to be measured is obtained which is equivalent to that obtained by calibration.

The standard solution of the present invention is suitable as a standard solution when the measurement of a substance to be measured is performed by an analyzer.

The standard solution of the present invention can be prepared by dissolving a substance to be measured and a protein in water such as distilled water or purified water, or the above-mentioned aqueous solution.

The standard solution of the present invention may be in any form such as a liquid form, a lyophilized form, a powder form, or a solid form.

(2) Method of Measuring Substance to be Measured In the method of measuring a substance to be measured according to the present invention, in the measurement of the substance to be measured, a protein and water to be measured are calibrated (standardized) in a water or aqueous solution together with the substance to be measured. ) Is performed.

A protein is added together with the substance to be measured.
The standard solution contained in water or the aqueous solution is as described in the above (1).

In the present invention, the measurement of the substance to be measured is not limited as long as the substance to be measured contained in the sample is measured by using a chemical reaction. For example, an enzyme and a substance serving as a substrate of the enzyme may be used. Of a target substance using a reaction with a target, measurement of a target substance using a complex formation reaction, measurement of a target substance using an antigen-antibody reaction (immunological reaction), nucleic acid complementary to the nucleic acid Measurement of a substance to be measured using a binding reaction with, measurement of a substance to be measured using a hydrophobic binding reaction, measurement of a substance to be measured using other chemical reactions, and the like.

The method for measuring a substance to be measured according to the present invention comprises the steps of:
Calibration may be performed according to a commonly used calibration method (for example, the method described in the section of “Prior Art”), and the concentration or activity value of the measurement target substance contained in the sample may be measured.

In the method for measuring a substance to be measured according to the present invention, by calibrating with a standard solution containing a protein together with the substance to be measured, the dispensed amount of this standard solution can be adjusted to the composition of serum, plasma, and serum ( This is equivalent to a standard serum having a composition (matrix) similar or similar to that of the (matrix), or a standard plasma having a composition (matrix) similar or similar to the composition (matrix) of the plasma.

Further, in the method of measuring a substance to be measured according to the present invention, the calibration is performed using a standard solution containing a protein together with the substance to be measured, so that the composition (matrix) of serum, plasma and serum can be obtained. Alternatively, the same measured value of the substance to be measured can be obtained as when calibration is performed using a standard serum having a similar composition (matrix) or a standard plasma having a composition (matrix) similar or similar to that of plasma (matrix). Things.

In the method for measuring a substance to be measured according to the present invention, the measurement of the substance to be measured is preferably performed by an analyzer.

(3) Aqueous solution of reaction indicator The aqueous solution of reaction indicator of the present invention is an aqueous solution of a reaction indicator, wherein a protein is contained in water or an aqueous solution together with the reaction indicator.

Examples of proteins to be contained in the reaction indicator aqueous solution together with the reaction indicator include albumin, casein, globulin, gelatin and the like. As this protein, albumin is preferred. Examples of albumin include human serum albumin (HSA), bovine serum albumin (BSA), horse serum albumin, and ovalbumin.

The protein to be contained in the reaction indicator aqueous solution may be one kind of protein alone or a plurality of proteins.

The concentration of the protein contained in the reaction indicator aqueous solution is preferably 1 to 12%, more preferably 3 to 10%, and particularly preferably 4 to 8%.

The reaction indicator contained in the reaction indicator aqueous solution is not particularly limited as long as it is used for calculating the calibration coefficient used for the measurement of the substance to be measured contained in the sample. .

Examples of the reaction indicator are described below. For example, aspartate aminotransferase (AS
T, GOT) activity, alanine aminotransferase (ALT, GPT) activity, lactate dehydrogenase (LD
H, LD) activity, creatine kinase (CK) activity, or cholinesterase (Ch-E) activity. Glucose, α-amylase activity, or alkaline phosphatase (ALP) used to calculate a calibration coefficient used for measurement. 4-nitrophenol or γ-glutamyl transpeptidase (γ-GTP) activity value used for calculating a calibration coefficient used for measuring an activity value or the like, or a calibration coefficient used for measuring a leucine aminopeptidase (LAP) activity value or the like. Examples thereof include 4-nitroaniline used for the calculation.

Although the reaction indicator is contained in the reaction indicator aqueous solution, only one kind of reaction indicator may be contained in the reaction indicator solution, or a plurality of types of reaction indicator may be contained. .

The aqueous solution of the reaction indicator of the present invention contains the protein together with the reaction indicator in water or an aqueous solution. The aqueous solution may be any one containing water as a solvent.

The aqueous solution may contain a buffer, if necessary.
Contains organic substances, surfactants, saccharides, amino acids, peptides, lipids, coenzymes, inorganic substances such as metal ions, stabilizers, preservatives, activators, chelating agents, and other high molecular substances. You may.

As the buffer, a buffer having a buffering capacity in a target pH range may be used as appropriate. Examples of such a buffer include tris [hydroxymethyl] aminomethane (Tris), phosphate buffer, imidazole, glycylglycine, PIPES, ACES, B
ES, MOPS, TES, HEPES, DIPSO, T
APSO, POPSO, HEPPSO, EPPS, HE
PPS, Tricine, Bicine, TAPS, C
HES, CAPSO, CAPS, MES, Bis-Tr
is, Bis-Tris propane, ADA, MOPS
O, and the like.

The reaction indicator aqueous solution of the present invention is such that the protein is contained in water or an aqueous solution together with the reaction indicator so that the dispensed amount of the reaction indicator aqueous solution becomes equivalent to serum or plasma. is there.

In addition, the aqueous solution of the reaction indicator of the present invention contains the protein together with the reaction indicator in water or an aqueous solution, so that the calibration coefficient can be calculated from serum or plasma containing the reaction indicator. In this case, the same calibration coefficient as in the case of performing the above is obtained.

The reaction indicator aqueous solution of the present invention is suitable as a reaction indicator aqueous solution when the measurement of a substance to be measured is performed by an analyzer.

The reaction indicator aqueous solution of the present invention can be prepared by dissolving the reaction indicator and the protein in water such as distilled water or purified water, or the above-mentioned aqueous solution.

The reaction indicator aqueous solution of the present invention may be in any form such as a liquid form, a lyophilized form, a powder form, or a solid form.

(4) Method for Calculating the Calibration Coefficient The method for calculating the calibration coefficient according to the present invention provides a method for calculating a calibration coefficient used for measuring a substance to be measured contained in a sample. It is characterized in that a calibration coefficient is calculated using the contained reaction indicator aqueous solution.

The protein together with this reaction indicator is
The reaction indicator aqueous solution contained in water or the aqueous solution is as described in the above (3).

The method of calculating the calibration coefficient of the present invention is based on the method of calculating the calibration coefficient or the like (for example, the method described in the “Prior Art” section) which is usually performed by using the above-mentioned aqueous solution of the reaction indicator. The concentration may be calculated, and the concentration or activity value of the target substance contained in the sample may be measured using the calculated calibration coefficient.

In the present invention, the measurement of the substance to be measured is not limited as long as the substance to be measured contained in the sample is measured by using a chemical reaction. For example, an enzyme and a substance serving as a substrate of the enzyme may be used. Of a target substance using a reaction with a target, measurement of a target substance using a complex formation reaction, measurement of a target substance using an antigen-antibody reaction (immunological reaction), nucleic acid complementary to the nucleic acid Measurement of a substance to be measured using a binding reaction with, measurement of a substance to be measured using a hydrophobic binding reaction, measurement of a substance to be measured using other chemical reactions, and the like.

In the method for calculating a calibration coefficient according to the present invention,
By including the protein together with the reaction indicator, the dispensed amount of the reaction indicator aqueous solution is equivalent to that of serum or plasma.

[0092] Thus, in the method for calculating the calibration coefficient of the present invention, the protein was contained in the reaction indicator aqueous solution, and the calibration coefficient was calculated from the serum or plasma containing the reaction indicator. A calibration coefficient equivalent to that in the case is obtained.

The method for calculating a calibration coefficient according to the present invention is suitable as a method for calculating a calibration coefficient when a substance to be measured is measured by an analyzer.

[0094]

EXAMPLES Hereinafter, the present invention will be described in more detail with reference to examples, but the present invention is not limited to these examples.

[Example] (Confirmation of Dispensed Amount of Standard Solution Containing Bovine Serum Albumin) An analyzer for a standard solution containing bovine serum albumin (BSA) in water together with the dye Orange G as a substance to be measured. Was confirmed to be equivalent to the amount of serum dispensed.

(1) Preparation of Standard Solutions The following various standard solutions containing the dye Orange G as a substance to be measured were prepared. The volumetric flasks and whole pipettes used for the preparation of these standard solutions were all used with their volumes corrected according to the regulations.

Preparation of BSA / Orange G-containing standard solution (a) Bovine serum albumin (BS
A) [Armor Co.] at a concentration of 2%, 3
%, 4%, 5%, 6%, and 10% in purified water to obtain a 2% aqueous BSA solution, a 3% aqueous BSA solution,
% BSA aqueous solution, 5% BSA aqueous solution, 6% BSA aqueous solution, and 10% BSA aqueous solution were prepared.

(B) 2% BSA aqueous solution, 3% B
SA aqueous solution, 4% BSA aqueous solution, 5% BSA aqueous solution, 6
To each of the 10% BSA aqueous solution and the 10% BSA aqueous solution, 15 mg of the dye Orange G (Tokyo Kasei Kogyo Co., Ltd.), which is the substance to be measured in this measurement, was added and dissolved. 0.
2% BSA aqueous solution containing 075% orange G, 3
% Aqueous BSA, 4% aqueous BSA, 5% aqueous BSA, 6% aqueous BSA, and 10% aqueous BSA were prepared.

These were respectively subjected to 2% BSA / orange G standard solution, 3% BSA / orange G standard solution, 4% BSA / orange G standard solution, 5% BSA / orange G standard solution, 6%
A BSA / orange G standard solution and a 10% BSA / orange G standard solution were used.

Preparation of Orange G-Containing Standard Serum To human serum, 15 mg of the dye Orange G (Tokyo Kasei Kogyo Co., Ltd.), which is a substance to be measured in this measurement, was added and dissolved, and the volume was adjusted to 20 mL with a volumetric flask. .07
It was human serum containing 5% orange G.

This was used as a standard serum containing orange G.

Preparation of a Standard Solution Containing Orange G To purified water, 15 mg of a dye Orange G (Tokyo Kasei Kogyo Co., Ltd.), which is a substance to be measured in this measurement, was added and dissolved. 0.075
% Orange G.

This was used as an orange G-containing standard solution.

(2) Measurement of dispensed amount of each standard solution in the analyzer

The amount of each standard solution prepared in the above (1) was measured using a Hitachi 7150 type automatic analyzer.

Measurement by Analyzer (a) Hitachi 7150 Automatic Analyzer [Hitachi, Ltd.]
The 2% BS prepared in (1) above
Standard solution containing A and orange G, standard solution containing 3% BSA and orange G, standard solution containing 4% BSA and orange G, 5% B
Standard solution containing SA / Orange G, 6% BSA / Orange G
The standard solution, the standard solution containing 10% BSA / orange G, the standard serum containing orange G, and the standard solution containing orange G were used as samples. The dispensed volume of each standard solution used as this sample was 3 μL, 5 μL, 7 μL, and 10 μL, respectively.
Specified in the analyzer.

(B) In the analyzer, after each standard solution as the sample is dispensed into a cuvette, 300 μL of purified water as a measuring reagent is dispensed and placed at 37 ° C.

(C) About 10 minutes after the measurement reagent was dispensed, the absorbance of the solution [standard solution (sample) and purified water (measurement reagent)] in the cuvette was measured at a wavelength of 480 nm.

Calculation of Dispensed Amount of Standard Solution From the absorbance of the solution in the cuvette measured in the above, the amount of each standard solution actually dispensed in the analyzer was calculated.

(A) “Amount of standard solution actually dispensed in analyzer” [actual dispensed amount], “dispensed amount of measurement reagent”
The following relationship exists between [reagent amount], "absorbance of standard solution" [absorbance of standard solution], and "absorbance of solution in cuvette measured by analyzer" [absorbance of measurement].

The “absorbance of standard solution” [absorbance of standard solution] refers to the absorbance at 480 nm of each of the standard solutions prepared in the above (1), which was diluted exactly 50-fold with purified water.
It is measured by a UVIDEC-430B spectrophotometer (JASCO Corporation), and the absorbance obtained by this measurement is multiplied by 50.

Further, “amount of reagent to be measured” [reagent amount]
Is 300 μL in the above measurement.

[Measurement absorbance] = [actual dispensed amount] × [standard solution absorbance] ÷ ([actual dispensed amount] + [reagent amount])

When this equation is expanded, the following equation is obtained.
By substituting [reagent amount], [measured absorbance], and [standard solution absorbance] into this equation, the “amount of standard solution actually dispensed in the analyzer” [actual dispensed volume] is calculated. can do.

[Actual dispensed amount] = [reagent amount] × [measured absorbance] ÷ ([standard solution absorbance] − [measured absorbance])

Table 1 shows the "amount of the standard solution actually dispensed in the analyzer" [actual dispensed amount] calculated by substituting the respective values into this equation. The unit of the numerical values shown in this table is “μL”.

[0117]

[Table 1]

(B) The ratio of the “amount of standard solution actually dispensed in the analyzer” [actual dispensed amount] to the “dispensed amount specified in the analyzer” (“the actual amount dispensed in the analyzer”). Table 2 shows “amount of standard solution dispensed” ÷ “dispensed amount specified in analyzer” × 100). The unit of the numerical values shown in this table is “percent (%)”.

[0119]

[Table 2]

(3) Discussion From the results shown in Tables 1 and 2, the amount of the standard solution actually dispensed in the analyzer (actual dispensed amount) was orange G
The contained standard solution was 4.2 times better than the orange G-containing standard serum.
% To 7.7%, indicating that an error has occurred.

In particular, it can be seen that the smaller the dispensed volume specified in the analyzer, the more noticeable this difference is and the more the error increases.

For example, when the dispensed volume specified in the analyzer is 3 μL, the difference is 7.7%.

In contrast, bovine serum albumin (BS
It can be seen that in each of the orange G standard solutions containing A), the difference from the dispensed amount of the orange G-containing standard serum is reduced.

For example, in the standard solution containing 2% BSA / orange G, when the dispensed volume specified in the analyzer is 3 μL, the difference between the standard solution containing orange G and the dispensed volume of the standard serum containing orange G is different. Decreased by 1.4%.

Particularly, a standard solution containing 4% BSA / orange G, a standard solution containing 5% BSA / orange G, 6% BS
In the standard solution containing A and orange G and the standard solution containing 10% BSA and orange G, the difference from the dispensed volume of the standard serum containing orange G at any given dispensed volume is at most 4%.
It can be seen that the effect of eliminating errors is remarkable.

From the above results, it can be seen that the standard solution containing bovine serum albumin (BSA), which is a protein, in water together with orange G, which is a substance to be measured, has the same volume of the standard serum as the standard serum. Was confirmed.

That is, orange G, which is a substance to be measured,
Along with bovine serum albumin (BS
When calibration is performed with a standard solution containing A) in water,
It was confirmed that the same measured value of the substance to be measured as that obtained by calibration with the standard serum could be obtained.

[0128]

According to the method of the present invention for measuring a standard solution and a substance to be measured, the amount of the standard solution dispensed in the analyzer is equivalent to that of a sample such as serum or plasma, and the serum, plasma, standard serum, or standard It is possible to obtain the same measured value of the substance to be measured as when calibration is performed using plasma, and to perform accurate calibration and accurate measurement.

Further, according to the method of calculating the reaction indicator aqueous solution and the calibration coefficient of the present invention, the dispensed amount of the reaction indicator aqueous solution in the analyzer is the same as the original dispensed amount, and the correct calibration coefficient containing no error is determined. It can be calculated, and as a result, an accurate measured value of the substance to be measured can be obtained.

Claims (24)

[Claims] 1. A standard solution wherein a protein is contained in water or an aqueous solution together with a substance to be measured. 2. The standard solution according to claim 1, wherein the concentration of the protein is 4 to 8%. 3. The standard solution according to claim 1, wherein the protein is albumin. 4. The method according to claim 1, wherein the amount of the standard solution to be dispensed is equivalent to serum, plasma, standard serum, or standard plasma by including the protein. Standard solution. 5. The method according to claim 1, wherein a measurement value equivalent to that obtained by calibration with serum, plasma, standard serum, or standard plasma is obtained by including the protein. Standard solution as described. 6. The standard solution according to claim 1, which is a standard solution when the measurement of the substance to be measured is performed by an analyzer.
Standard solution as described in item. 7. A method for measuring a substance to be measured, characterized in that in the measurement of the substance to be measured, calibration is performed using a standard solution containing water or an aqueous solution together with the protein together with the substance to be measured. 8. The method according to claim 7, wherein the concentration of the protein contained in the standard solution is 4 to 8%. 9. The method according to claim 7, wherein the protein contained in the standard solution is albumin. 10. The method according to claim 7, wherein the calibration is performed using a standard solution containing a protein together with the substance to be measured, whereby the dispensed amount of the standard solution is equivalent to serum, plasma, standard serum, or standard plasma. The method for measuring a substance to be measured according to claim 9. 11. Calibration using a standard solution containing a protein together with a substance to be measured provides a measurement value equivalent to that obtained by calibration using serum, plasma, standard serum, or standard plasma. The method for measuring a substance to be measured according to any one of claims 7 to 10. 12. The method for measuring a substance to be measured according to claim 7, wherein the measurement of the substance to be measured is performed by an analyzer. 13. A protein together with a reaction indicator,
A reaction indicator aqueous solution characterized by being contained in water or an aqueous solution. 14. The method according to claim 14, wherein the concentration of the protein is 4 to 8%.
The reaction indicator aqueous solution according to claim 13. 15. The reaction indicator aqueous solution according to claim 13, wherein the protein is albumin. 16. Inclusion of a protein,
The reaction indicator aqueous solution according to any one of claims 13 to 15, wherein a dispensed amount of the reaction indicator aqueous solution is equivalent to serum or plasma. 17. Inclusion of a protein,
The reaction indicator aqueous solution according to any one of claims 13 to 16, wherein a calibration coefficient equivalent to the case where the calibration coefficient is calculated using serum or plasma containing the reaction indicator is obtained. 18. The reaction indicator aqueous solution according to any one of claims 13 to 17, which is an aqueous solution of a reaction indicator when the measurement of a substance to be measured is performed by an analyzer. 19. A method for calculating a calibration coefficient, wherein the calculation of the calibration coefficient includes the step of calculating the calibration coefficient using water or an aqueous solution of a reaction indicator containing the protein together with the reaction indicator. 20. The method according to claim 19, wherein the concentration of the protein contained in the reaction indicator aqueous solution is 4 to 8%. 21. The protein according to claim 19, wherein the protein contained in the aqueous solution of the reaction indicator is albumin.
Calculation method of the calibration coefficient described. 22. Inclusion of the protein in the aqueous solution of the reaction indicator, the dispensed amount of the aqueous solution of the reaction indicator is reduced.
22. It is equivalent to serum or plasma.
The method for calculating a calibration coefficient according to any one of the above. 23. A serum containing a reaction indicator by adding a protein to an aqueous solution of the reaction indicator,
The method of calculating a calibration coefficient according to any one of claims 19 to 22, wherein a calibration coefficient equivalent to a case where the calibration coefficient is calculated using plasma is obtained. 24. The method for calculating a calibration coefficient according to any one of claims 19 to 23, wherein the method is a method for calculating a calibration coefficient when measuring a substance to be measured by an analyzer.