JP4556605B2 - Target substance measurement method and reagent - Google Patents

Description

  The present invention relates to a measurement method and a measurement reagent for detecting a target substance contained in a test sample using a detection substance that specifically binds to the target substance.

  In hospitals and testing centers, with the development of automated analyzers, clinical tests in the immune serum department, which have been tested by the method of use, are rapidly being automated. There are many kinds of measurement methods used for such clinical examinations. For example, there are measurement methods such as RIA (radioimmunoassay), EIA (enzyme immunoassay), TIA (immunoturbidimetry), LIA (latex immunoturbidimetry) and the like. It is used. Among these methods, RIA and EIA require a dedicated model suitable for each measurement method because they use radioactive substances and enzymes. On the other hand, aggregation methods such as TIA and LIA can be carried out very simply because the change in turbidity of the reaction solution accompanying the aggregation caused by the antigen-antibody reaction is measured. Therefore, TIA and LIA can be applied to general-purpose automatic analyzers, and a system is employed in which an immune serum test using these is performed on the same line as a biochemical test.

Each of these measurement methods has a detection concentration suitable for the measurement method, and is applied according to the abundance of the antigen in the sample solution (test sample). For example, TIA is used for measuring a relatively high concentration of antigen. For example, TIA is usually used for measuring an immunoglobulin present in a relatively large amount in serum. Moreover, since LIA has higher sensitivity than TIA, it is preferably used for a lower concentration of antigen.
However, with these measurement methods, it is difficult to accurately detect a target substance in a test sample in a wide measurement range from a low concentration to a high concentration.
For example, when the antigen concentration in the test sample is low, TIA may have insufficient sensitivity.
There is also a problem of zone phenomenon. That is, in the aggregation methods such as TIA and LIA, generally, a calibration curve is prepared from the absorbance increase value, and the concentration is determined, but this is usually in proportion to the antigen concentration. This is because of the rise. However, if there is an excessive amount of antigen in the test sample, on the contrary, there is a possibility that the absorbance decreases in proportion to the antigen concentration (zone phenomenon), and the antigen concentration in the test sample may be erroneously determined. There is. For example, immunoglobulins are present in human serum components next to albumin even under normal conditions, but in samples such as myeloma serum, there are tens of thousands of mg / dl, several tens of times the normal value. There is a possibility that misjudgment due to antigen excess may occur.

For example, in order to realize high-sensitivity detection at a low concentration, countermeasures such as using a large amount of a detection reagent such as an antibody or a specimen, or adding an additive such as a sensitizer, etc. are taken for such a problem. (For example, refer to Patent Document 1).
In addition, a method of diluting a specimen expected to contain a high concentration of antigen and measuring the absorbance is used. Furthermore, for the purpose of suppressing the zone phenomenon, an immunoassay reagent containing a specific polymer substance in addition to an insoluble carrier carrying an antibody or the like, a test sample whose measured value is equal to or higher than a predetermined reference value, and then a specific concentration There are proposed a method of measuring with the above reagent, a method of allowing an antibody to exist separately from an antibody fixed on a carrier, and the like (see, for example, Patent Documents 2 to 4).
JP-A-9-304388 JP-A-9-304389 Japanese Patent Laid-Open No. 10-239317 Patent No. 3005400

However, a sensitizer non-specifically promotes an increase in absorbance and can also cause aggregation of immunocomplexes and the like contained in a sample, which is likely to be a factor of erroneous determination.
Further, when diluting the sample solution, especially when there are a large number of samples, a great amount of time and labor is spent on the dilution operation, and there is a possibility of an error in the concentration measurement result accompanying the dilution. Furthermore, even if a measure for suppressing the zone phenomenon is performed, the zone phenomenon is derived from the measurement principle and cannot be completely avoided, and the possibility of erroneous determination remains.
In addition, in order to deal with high-concentration samples that may be encountered at low frequency during testing, uniformly use a large amount of antibodies or add additives to the samples handled in the testing. Is not economical and unnecessarily increases non-specific reactions, reducing test accuracy. On the other hand, performing the above-described treatment only on a specimen containing a high concentration of antigen particularly hinders application to an automatic analyzer.
As described above, it has been difficult to easily and accurately quantify the target substance such as an antigen contained in the test sample from a low concentration to a high concentration.

In response to such a problem, the present inventors are the same as the target substance or the target substance as a measurement method capable of accurately quantifying the target substance in the test sample with a simple operation in a wide measurement range from a low concentration to a high concentration. When a specific substance consisting of a substance having specificity is added to a detection solution containing a detection substance, a reference concentration at which the absorbance is maximized is obtained, and a concentration region above the reference concentration, that is, a decrease in absorbance (probe) A method for measuring a target substance concentration in a test sample by creating a calibration curve in (Zone) has been proposed (Japanese Patent Application No. 2003-282675).
Although the present invention is excellent, it is required to express even higher reproducibility.
The present invention has been made in order to solve the above-described problem, and can measure a target substance in a test sample with high accuracy and reproducibility with a simple operation in a wide measurement range from a low concentration to a high concentration. And a measuring reagent suitably used for the measuring method.

As a result of further studies, the present inventor has found that the above problem can be solved by blending a polyhydric alcohol into the detection solution used in the above measurement method.
That is, the method for measuring a target substance of the present invention is a method for measuring a target substance concentration in a test sample using a detection substance that specifically binds to the target substance.
When the absorbance of the mixture obtained by adding a specific substance solution containing a specific substance consisting of a target substance or a substance having the same specificity as the target substance to the detection solution containing the detection substance is maximized A first step of determining a specific substance concentration in the specific substance solution to be a reference concentration;
A second step of adding a specific substance solution of the reference concentration or higher and a standard solution containing a target substance having a known concentration to a second detection solution of the same type as the detection solution, and measuring the absorbance thereof;
Adding a specific substance solution having the same concentration as the second step to a third detection solution of the same type as the detection solution, and a test sample, and measuring the absorbance thereof,
The detection solution contains glycerin .
The target substance measurement reagent of the present invention is a target substance measurement reagent in a test sample,
A detection solution containing a detection substance that specifically binds to a target substance and glycerin, and a specific substance solution containing a specific substance consisting of the target substance or a substance having the same specificity as the target substance, The specific substance concentration in the specific substance solution is not less than a reference concentration at which the absorbance of the mixture obtained by adding the specific substance solution to the detection solution is maximum.

  According to the measurement method of the present invention, the target substance in the test sample can be quantified with a simple operation with high accuracy and reproducibility in a wide measurement range from a low concentration to a high concentration. Moreover, the measuring reagent of this invention is used suitably for the measuring method of this invention.

First, the method for measuring a target substance of the present invention will be described.
The target substance measurement method of the present invention measures a target substance concentration in a test sample using a detection substance that specifically binds to the target substance.

The test sample only needs to be liquid, and examples thereof include blood, serum, body fluid, urine, and a supernatant of a liquid in which a solid specimen is dispersed.
The target substance is not particularly limited, and examples thereof include causative substances such as various diseases and allergies, pathogenic bacteria, physiologically active substances, and antibodies thereof. Specifically, immunoglobulins (eg, human and animal immunoglobulins, modified immunoglobulins), various viral antigens (eg, hepatitis virus-related antigens, rubella HA antigens, etc.), microbial antigens such as various bacteria, fungi, toxins ( For example, toxoplasma, syphilis treponema, etc.), various plasma protein components (eg, α-fetoprotein, C-reactive protein (CRP), albumin, complement component, etc.), various hormones (eg, estrogen, human chorionic gonadotropin, (HCG) ), Etc.), antibodies (eg, anti-CEA (carcinoembryonic antigen) antibodies, anti-reactive protein antibodies, anti-fibrinogen antibodies, anti-γ-globulin antibodies, etc.), hormone receptors (eg, thyroid stimulating hormone receptor, HCG receptor, etc.), An enzyme (eg Phosphatase, cholinesterase, etc.), a substrate (e.g., cholesterol, etc. uric acid), hemoglobin derivatives, sugar derivatives (such as sugar compound, glycoproteins, etc.).

The detection substance only needs to be a substance that specifically binds to the target substance, that is, a substance having a site that binds to the target substance, and can be appropriately set according to the target substance to be measured. For example, when an anti-immunoglobulin antibody is used as the detection substance, immunoglobulin can be detected as the target substance.
Examples of the substance for detection include a substance that binds to a target substance by an antigen-antibody reaction, an enzyme-substrate reaction, a ligand-receptor reaction, or the like. As the detection substance that specifically binds to the target substance by the antigen-antibody reaction, for example, an antibody can be used when the target substance is an antigen, and an antigen can be used when the target substance is an antibody.
Among these, when the detection substance specifically binds to the target substance by the antigen-antibody reaction, a wide variety of antibodies or antigens that bind to the target substance antigen or antibody are available. This is preferable because the target substance can be measured.

When an antibody is used as the detection substance, the origin of the antibody is not limited. For example, those derived from serum of any animal species such as rabbit antibody, rat antibody, mouse antibody, goat antibody can be used. In terms of cost, antibodies purified from goat serum are preferred.
The antibody may be a monoclonal antibody or a polyclonal antibody.
The antibody may be the antibody itself or an antibody fragment (having a site that binds to the target substance) such as F (ab ′) ₂ obtained by fragmenting the antibody.
The purification method and purity of the antibody are not particularly limited, and for example, an antibody purified by ammonium sulfate salting out can be used.

  In the measurement method of the present invention, first, a specific substance solution containing a specific substance consisting of a target substance or a substance having the same specificity as the target substance is added to a detection solution containing a detection substance. A first process is performed in which the specific substance concentration in the specific substance solution at the time when the absorbance of the mixed solution is maximized is obtained as a reference concentration.

The specific substance used for obtaining the reference concentration may be a target substance or a substance having the same specificity as the target substance, but the target substance is particularly preferable.
The dispersion medium for the specific substance solution is not particularly limited, and known ones can be used. Examples of such a dispersion medium include phosphate buffer, glycine buffer, Tris buffer, Good's buffer, and the like. The pH of the medium is preferably 5.5 to 8.5.
Specific substance solutions include bovine serum albumin and sucrose as stabilizers; water-soluble polysaccharides such as polyethylene glycol and dextran as measurement sensitivity improvers; sodium azide as preservatives; sodium chloride as a salt concentration regulator. Such additives can be added as appropriate.
Further, the specific substance solution may contain a polyhydric alcohol as in the detection solution described later.

The detection solution contains a detection substance and a polyhydric alcohol as essential components. Thereby, the reproducibility of the result measured by the measuring method of the present invention is excellent.
The polyhydric alcohol is a relatively low molecular weight alcohol having a plurality of hydroxyl groups in the same molecule, such as glycerin, ethylene glycol, trimethylolpropane, pentaerythritol, 1,2,6-hexanetriol, sorbitol, 1, Conventional industrially produced alcohols such as 1,4,4-tetrakishydroxymethylsiloxane, tris (2-hydroxyethyl) isocyanurate and dipentaerythritol are used. Since these are used in large quantities as immunodiagnostic agents, the grade is preferably of a high purity, that is, reagent grade 1 or reagent grade level.
The polyhydric alcohol concentration in the detection solution is preferably 1 to 50% by volume and more preferably 1 to 30% by volume with respect to the total volume of the detection solution.

In the detection solution, the detection substance may be used as it is dispersed in the detection solution, or may be used by being supported on an insoluble carrier, but it is preferably used by being supported on an insoluble carrier. Thereby, since the change rate of the light absorbency with respect to the amount of change in the target substance concentration becomes higher, highly sensitive detection can be performed.
When a detection substance is supported on an insoluble carrier, it is preferable to use a substance that does not prevent aggregation of the insoluble carrier.

Examples of the insoluble carrier include organic polymer particles, inorganic substance particles, latex particles, cell membrane pieces, blood cells, microorganisms and the like. Among these, latex particles are preferred because they can be mass-produced with industrially constant quality performance.
The material of the latex particles is not particularly limited as long as the particle size is relatively constant and industrially constant quality and performance can be mass-produced. For example, styrene, vinyl chloride, Single polymers and copolymers of vinyl monomers such as acrylonitrile, vinyl acetate, acrylic acid esters, and methacrylic acid esters; butadiene copolymers such as styrene-butadiene copolymers and methyl methacrylate-butadiene copolymers It is done. Of these, polystyrene-based polymer particles are more preferable from the viewpoints of being excellent in the adsorptivity of substances such as antibodies and being able to retain biological activity for a long period of time.
The particle size of the latex particles is preferably from 0.01 to 10 μm, particularly preferably from 0.03 to 1 μm.
In addition, the concentration of the insoluble carrier in the mixed solution of the specific substance solution and the detection solution can be appropriately determined in consideration of the nature of the target substance, the expected abundance, the obtained scattered light intensity, etc. Generally, 0.01 to 10% by mass is preferable, and 0.1 to 1% by mass is particularly preferable.

  When a detection substance is supported on an insoluble carrier, a physical adsorption method, a chemical bonding method, or the like can be used. In the case of physical adsorption, an insoluble carrier and a detection substance are simply mixed. In the case of chemical bonding, the bonding can be performed using a functional group present on the surface of the insoluble carrier or the detection substance, or via a spacer having an appropriate functional group. Examples of the spacer include carbodiimide and N-hydroxysuccinimide ester.

The dispersion medium for the detection solution is not particularly limited, and known ones can be used. Examples of such a dispersion medium include those exemplified as the dispersion medium for the specific substance solution.
In addition, an additive can be optionally added to the detection solution as in the case of the specific substance solution.

In the present invention, the reference concentration can be determined as follows, for example.
First, a specific substance solution containing a specific substance is added to a detection solution while changing the concentration of the specific substance. A graph is prepared with the specific substance concentration in the specific substance solution on the axis and the measured absorbance on the vertical axis. In the graph, the absorbance increases in proportion to the concentration until reaching a specific concentration, and decreases in proportion to the concentration when the specific concentration is exceeded. Then, from the graph, the specific substance concentration of the specific substance solution when the absorbance reaches the maximum is obtained, and the concentration is set as the reference concentration.
The instrument used for measuring the absorbance is not particularly limited as long as it can measure the absorbance of the mixed solution over time, but a general-purpose biochemical automatic analyzer is preferable.
The reference concentration can be estimated in advance from the affinity between the detection substance and the target substance, the concentration of the detection substance, and the like.

Next, a second process in which a specific substance solution having a reference concentration or higher and a standard solution containing a target substance with a known concentration are added to a second detection solution of the same type as the detection solution, and the absorbance is measured. I do.
Here, the “same species as the detection solution” indicates that the detection solution used in the first process has the same conditions such as composition, concentration, temperature, and pH. Further, the “specific substance solution having a reference concentration or higher” means a specific substance solution containing a specific substance at a concentration higher than the reference concentration.
The order of adding the specific substance solution and the standard solution to the second detection solution is not particularly limited. For example, the standard solution may be added after adding the specific substance solution, or the specific substance solution and the standard solution may be added. And may be added simultaneously.

In the second process, for example, when adding a standard solution after adding a specific substance solution, the absorbance is measured after a certain time from the addition of the standard solution. Moreover, when adding a specific substance solution and a standard solution simultaneously, it measures after progress for a fixed time after adding these.
Absorbance measurement conditions are the same as in the first step.
A calibration curve can be created by plotting the absorbance measured in this way against the concentration of the standard substance in the standard solution. Here, a calibration curve is obtained in which the absorbance decreases in proportion to the target substance concentration in the standard solution.

In addition, by selecting which concentration of the specific substance solution of the specific substance solution to be added to the detection solution should be set in the region above the reference concentration, a target in a concentration range from a minute concentration to a high concentration can be selected. Substances can be more accurately quantified. For example, when measuring a target substance at a very low concentration, it is possible to obtain a calibration curve with a greater difference in absorbance with respect to a minute difference in the concentration of the target substance by making the specific substance concentration larger than the reference concentration. it can.
Moreover, when measuring a high concentration target substance, it is preferable that the specific substance concentration is not less than the reference concentration and near the reference concentration. This requires less detection material and is more economical.

Next, a specific substance solution having the same concentration as the second process and a test sample are added to a third detection solution of the same type as the detection solution, and a third process of measuring the absorbance is performed.
That is, first, a detection solution of the same type as the detection solution used in the first process and the second process is prepared separately, and the specific substance solution added to the second detection solution in the second process is added to the detection solution. A specific substance solution having the same concentration and a test sample are added. At this time, the ratio (volume ratio) of the third detection solution, the specific substance solution, and the test sample is the same as the ratio of the second detection solution, the specific substance solution, and the standard sample in the second process. . The order of addition of the specific substance solution and the test sample to the third detection solution is the same as the order of addition of the specific substance solution and the standard solution to the second detection solution in the second process.
After the addition of the specific substance solution and the test sample, the absorbance is measured after the same time has elapsed as the measurement of the absorbance in the second step. The absorbance measurement conditions are the same as those in the first process and the second process.
Subsequently, the concentration of the target substance contained in the test sample is determined by referring to the concentration of the target substance corresponding to the point showing the same value as the absorbance obtained in the third process on the calibration curve created in the second process. Can be sought.

In the measurement method of the present invention, a target substance ranging from a very small amount to a large amount can be easily and accurately quantified. Therefore, since dilution operation is not required even for a high concentration target substance, it is easy to handle a large number of samples at once. For example, automatic analysis can be performed with a simple mechanism regardless of individual differences, normality / abnormality, and type of target substance of a test sample derived from a living body, so that cost reduction and time required for analysis can be realized. In addition, the measurement method of the present invention is excellent in reproducibility and can perform highly reliable measurement.
Furthermore, when the detection substance is supported on an insoluble carrier in the detection solution, the target substance can be detected with higher sensitivity, and the amount of the detection substance used can be reduced.
In addition, when an anti-immunoglobulin antibody is used as a detection substance, an immunoglobulin (immunoglobulin) can be detected as a target substance, and in particular, immunoglobulin G ( IgG), immunoglobulin A (IgA), immunoglobulin M (IgM), and other immunoglobulins present in large amounts in normal specimens can be accurately quantified without dilution.
Further, in the measurement method of the present invention, target substances that are present in large amounts in normal specimens such as complement components other than immunoglobulins, such as C3 and C4, can be accurately quantified without diluting the test sample. It can be carried out.
The measurement method of the present invention can be suitably used for automatic analysis using a general-purpose automatic analyzer in hospitals, examination centers, and the like.

Next, the reagent for measuring a target substance of the present invention will be described.
The reagent for measuring a target substance of the present invention is a specific substance comprising a detection solution containing a detection substance and a polyhydric alcohol that specifically binds to the target substance, and the target substance or a substance having the same specificity as the target substance. The specific substance concentration in the specific substance solution is equal to or higher than a reference concentration that maximizes the absorbance of the mixture obtained by adding the specific substance solution to the detection solution. It is characterized by being.
The target substance measurement reagent of the present invention is suitably used in the above-described measurement method of the present invention. That is, a calibration curve is created by adding a standard solution containing a target substance with a known concentration to such a measurement reagent, and measuring the absorbance after a lapse of a certain time. Separately, a test sample is placed on the same type of measurement reagent. The absorbance after the same time has elapsed after the addition. And the target substance concentration in a test sample can be calculated | required from the measurement result using the calibration curve created above.

[Example 1]
(1) Preparation of Detection Solution and Specific Substance Solution As a detection solution, a latex sample solution (anti-IgG antibody-supported latex) in which a detection substance composed of an anti-human IgG goat polyclonal antibody is supported on an insoluble carrier composed of latex particles by physical adsorption. Sample solution) was prepared by the following procedure.
That is, a detection substance consisting of an anti-human IgG goat polyclonal antibody (International Immunology Corp .: hereinafter referred to as “anti-IgG antibody”) at a concentration of 2.0 mg / ml and a 50 mM phosphate buffer (pH 7.2: Hereinafter, 10.0 ml of polystyrene particles having an average particle diameter of 110 nm (manufactured by Fujikura Kasei Co., Ltd .: solid content 1 mass%) are added to 10.0 ml of a solution dissolved in “PBS”, and the mixture is added at 4 ° C. for 60 minutes. Stir. Next, 50 mM PBS (pH 7.2) containing 1% by mass of bovine serum albumin (hereinafter referred to as “BSA”) was added to this solution, stirred at 4 ° C. for 60 minutes, and then centrifuged at 20000 × g for 60 minutes. Separated and washed. The washing operation was performed 3 times. To the obtained precipitate, 9.5 ml of 50 mM PBS containing 0.1% by mass of BSA and 0.5 ml of glycerin were added, and the polystyrene particles were suspended, followed by dispersion treatment with an ultrasonic crusher. An anti-IgG antibody-supported latex test solution (glycerin concentration 5% (V / V)) having a solid content of 1% (W / V) was obtained.

Further, human IgG (International Immunology Corp .: hereinafter referred to as “IgG”) was used as a specific substance, and a specific substance solution (specific substance solution) was prepared by the following procedure.
Specifically, glycine 50 mM and sodium chloride 70 mM were dissolved in pure water, adjusted to pH 7.2, and IgG concentrations 10 mg / dl, 15 mg / dl, 20 mg / dl, 25 mg / dl, 50 mg / dl, 75 mg / 75 were added to this solution. Human IgG was mixed so that it might become dl, 200 mg / dl, and 500 mg / dl, and the specific substance solution was obtained.

(2) Determination of Reference Concentration First, the specific substance solution of each IgG concentration prepared in (1) above was prepared as a separate detection solution (glycerin concentration 5% (V / V)), and the absorbance after 5 minutes from the time of addition was measured. Hereinafter, the absorbance was measured using an automatic general-purpose measuring instrument Hitachi 7020 (manufactured by Hitachi, Ltd.) at a measurement wavelength of 800 nm and a measurement temperature of 37 ° C.
As a result, when the IgG concentration was 25 mg / dl, the absorbance was maximized as compared with other concentrations. That is, the reference concentration for IgG was 25 mg / dl.

(3) Preparation of calibration curve Next, 300 μl of a specific substance solution having an IgG concentration of 25 mg / dl prepared by the same method as in (1) above, and 3 μl of serum containing IgG of known concentration as a target substance as a standard solution Mixed.
The standard solution was prepared by diluting serum with known IgG concentration so that the IgG concentrations were 0 mg / dl, 400 mg / dl, 1600 mg / dl, 3200 mg / dl, 4800 mg / dl, and 8000 mg / dl.
To this mixed solution, 100 μl of a detection solution separately prepared by the method of (1) above was added, and the absorbance was measured 5 minutes after the addition. The absorbance was plotted against the IgG concentration of the standard solution, and a calibration curve for IgG measurement was prepared. The results are shown in FIG. The horizontal axis in FIG. 1 is the IgG concentration of the standard solution, and the vertical axis is the absorbance.

(4) Measurement of test sample Subsequently, 300 μl of a specific substance solution with an IgG concentration of 25 mg / dl prepared by the same method as in (1) above, and 3 μl of human serum with an IgG concentration of 1000 mg / dl as a test sample Mixed.
To this mixed solution, 100 μl of a detection solution (glycerin concentration 5%) separately prepared by the method (1) was added, and the absorbance after 5 minutes from the addition was measured. Using the obtained absorbance and the calibration curve prepared in (3) above, the IgG concentration in the test sample was determined. As a result, the value of the IgG concentration in the test sample obtained from the calibration curve was 1143 mg / dl, which almost coincided with the value of 1142 mg / dl of the IgG concentration of the test sample used.
The same measurement was further performed nine times, and the average value and standard deviation of these results were determined. The average value of the IgG concentration in the test sample was 1143 mg / dl, and the standard deviation was 17.86.

[Example 2]
A calibration curve was prepared in the same manner as in Example 1 except that (1) the detection solution and the specific substance solution in Example 1 were prepared and the glycerol concentration of the detection solution was changed to 10% (V / V). The result is shown in FIG.
Moreover, when the average value of IgG concentration in a test sample and a standard deviation (10 times measurement) were calculated | required similarly to the above using the created calibration curve, the average value of IgG concentration in a test sample was 1144 mg / dl. Yes, the standard deviation was 15.40.

[Example 3]
A calibration curve was prepared in the same manner as in Example 1 except that (1) the detection solution and the specific substance solution in Example 1 were prepared with a glycerol concentration of 20% (V / V). The result is shown in FIG.
Moreover, when the average value of IgG concentration in a test sample and a standard deviation (10 times measurement) were calculated | required similarly to the above using the created calibration curve, the average value of IgG concentration in a test sample was 1140 mg / dl. Yes, the standard deviation was 11.66.

[Example 4]
A calibration curve was prepared in the same manner as in Example 1 except that in the preparation of the detection solution and the specific substance solution in Example 1, the glycerol concentration of the detection solution was changed to 30% (V / V). The result is shown in FIG.
Moreover, when the average value and standard deviation (10 times measurement) of IgG concentration in a test sample were calculated | required similarly to the above using the created calibration curve, the average value of IgG concentration in a test sample was 1148 mg / dl. Yes, the standard deviation was 11.65.

[Comparative Example 1]
A calibration curve was prepared in the same manner as in Example 1 except that glycerin was not added to the detection solution in the preparation of (1) the detection solution and the specific substance solution in Example 1. The result is shown in FIG.
Moreover, when the average value of IgG concentration in a test sample and a standard deviation (10 times measurement) were calculated | required similarly to the above using the created calibration curve, the average value of IgG concentration in a test sample was 1151 mg / dl. Yes, the standard deviation was 33.94.

  As is clear from the above results, in Examples 1 to 4 in which glycerin was added to the detection solution, a calibration curve in which the IgG concentration and the absorbance uniquely corresponded until the IgG concentration of the standard solution changed from a low concentration to a high concentration. I was able to get it. Moreover, the results measured using the obtained calibration curve were excellent in accuracy and reproducibility.

It is a figure which shows the light absorbency with respect to IgG density | concentration of a standard solution in Example 1. FIG.

Claims (12)

In a method of measuring a target substance concentration in a test sample using a detection substance that specifically binds to the target substance,
When the absorbance of the mixture obtained by adding a specific substance solution containing a specific substance consisting of a target substance or a substance having the same specificity as the target substance to the detection solution containing the detection substance is maximized A first step of determining a specific substance concentration in the specific substance solution to be a reference concentration;
A second step of adding a specific substance solution of the reference concentration or higher and a standard solution containing a target substance having a known concentration to a second detection solution of the same type as the detection solution, and measuring the absorbance thereof;
Adding a specific substance solution having the same concentration as the second step to a third detection solution of the same type as the detection solution, and a test sample, and measuring the absorbance thereof,
The method for measuring a target substance, wherein the detection solution contains glycerin . The method for measuring a target substance according to claim 1, wherein the concentration of glycerin in the detection solution is in the range of 1 to 50% by volume. The method for measuring a target substance according to claim 1 or 2 , wherein the detection substance specifically binds to the target substance by an antigen-antibody reaction. The detection material, method of measuring a target substance according to any one of claims 1 is carried on the insoluble carrier in the detection solution 3. The method for measuring a target substance according to claim 4 , wherein the insoluble carrier is latex particles. The detection material is an anti-immunoglobulin antibody, method for measuring a target substance according to any one of the target substance claims 1 immunoglobulin 5. A reagent for measuring a target substance in a test sample,
A detection solution containing a detection substance that specifically binds to a target substance and glycerin, and a specific substance solution containing a specific substance consisting of the target substance or a substance having the same specificity as the target substance, The target substance measurement reagent, wherein the specific substance concentration in the specific substance solution is equal to or higher than a reference concentration at which the absorbance of the mixture obtained by adding the specific substance solution to the detection solution is maximum. . The reagent for measuring a target substance according to claim 7, wherein the concentration of glycerin in the detection solution is in the range of 1 to 50% by volume. The reagent for measuring a target substance according to claim 7 or 8 , wherein the detection substance specifically binds to the target substance by an antigen-antibody reaction. Reagent for measuring a target substance according to any one of the detection substance, the preceding claims 7 carried on the insoluble carrier detection solution 9. The reagent for measuring a target substance according to claim 10 , wherein the insoluble carrier is latex particles. The reagent for measuring a target substance according to any one of claims 7 to 11 , wherein the detection substance is an anti-immunoglobulin antibody, and the target substance is an immunoglobulin.

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