JP2005049264A - Measuring method and measuring reagent of target material - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a measuring method capable of quantitating a target material in a test sample accurately by a simple operation in a wide measuring range from a low concentration to a high concentration. <P>SOLUTION: Concerning a method for measuring the target material concentration in the test sample by using a detection material bonding specifically with the target material, this measuring method of the target material is characterized by having the first process for determining the reference concentration wherein the absorbance becomes maximum when adding the target material or a specific material comprising a material having the same specificity as the target material to detection solution including the detection material, the second process for adding the specific material having the standard or higher concentration and the reference solution including the target material having the known concentration to the second detection solution of the same kind as the detection solution, and measuring its absorbance, and the third process for adding the specific material having the same concentration as that in the second process and the test sample to the third detection solution of the same kind as the detection solution, and measuring its absorbance. <P>COPYRIGHT: (C)2005,JPO&NCIPI

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 specific for the target substance.

With the development of automatic analyzers in hospitals and laboratories, clinical tests in the immune serum department, which have been tested using methods, are rapidly being automated. There are many types of measurement methods used in such clinical tests. For example, RIA (radioimmunoassay), EIA (enzyme immunoassay), TIA (immunoturbidimetry), insoluble carriers are supported with antibodies, etc. An aggregation method such as LIA (latex aggregation method) utilizing the aggregation of the accompanying carrier is used. Among these methods, RIA and EIA require dedicated models that match the measurement method, whereas TIA and LIA can be applied to general-purpose automatic analyzers. A system is adopted in which the inspection is performed on the same line.
In the test, in addition to the need to detect with high sensitivity an antigen with a small amount present in a sample solution (test sample), it may be necessary to detect and quantify an antigen with a very large amount. For example, immunoglobulins are present in human serum components next to albumin even when normal, and in samples such as myeloma serum, there are tens of thousands of mg / dl, several tens of times the normal value.
In contrast, for example, TIA may have insufficient sensitivity to detect low concentrations of antigen.
On the other hand, LIA has higher sensitivity than TIA and is preferably used for low concentrations of antigen. In LIA, a calibration curve is usually created using an increase in absorbance proportional to the concentration of antigen contained in a test sample. However, when the amount of antigen is excessive with respect to the antibody for detection, a phenomenon (zone phenomenon) in which the absorbance decreases is generated. Therefore, when the antigen concentration in the test sample is very high, the concentration may be erroneously determined.
Therefore, for example, for the purpose of realizing high-sensitivity detection at a low concentration, measures such as using a large amount of a detection reagent or sample such as an antibody or adding an additive such as a sensitizer have been taken (for example, , See 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 aggregate immunocomplexes and the like contained in a specimen, which is likely to cause 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.
On the other hand, even if a device for suppressing the zone phenomenon is used, the zone phenomenon is derived from the measurement principle and cannot be completely avoided, and the possibility of erroneous determination remains. Furthermore, in order to deal with low-frequency, high-concentration specimens that may be encountered during testing, it is economical to uniformly use a large amount of antibodies or add additives to the specimens handled in the testing. And unnecessarily increase non-specific reactions to reduce 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.

  The present invention has been made to solve the above-described problems, and an object of the present invention is to provide a measurement method capable of accurately quantifying a target substance in a test sample with a simple operation in a wide measurement range from a low concentration to a high concentration. And

The inventor of the present invention pays attention to the fact that the decrease in absorbance due to the zone phenomenon still persists even when the amount of added antigen is further increased. I found a method that could not be judged.
That is, the target substance measurement method 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.
A first step of obtaining a reference concentration that maximizes the absorbance when 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 second step of adding the specific substance equal to or higher than the reference concentration to a second detection solution of the same type as the detection solution and a standard solution containing a target substance having a known concentration and measuring the absorbance thereof;
A third step of adding the specific substance having the same concentration as the second step and a test sample to a third detection solution of the same type as the detection solution and measuring the absorbance thereof. And
The target substance measurement reagent of the present invention is a target substance measurement reagent in a test sample,
It contains a detection substance that specifically binds to a target substance and a specific substance composed of the target substance or a substance having the same specificity as the target substance, and the concentration of the specific substance is the detection of the specific substance. It is characterized in that it is above the reference concentration at which the absorbance is maximized when added to the substance for use.

  According to the measurement method of the present invention, a target substance in a test sample can be accurately quantified by a simple operation in a wide measurement range from a low concentration to a high concentration.

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 used in the measurement method of the present invention only needs to be liquid, and examples thereof include blood, serum, body fluid, urine, and supernatant of a liquid in which a solid specimen is dispersed.

  In the present invention, the target substance to be measured is not particularly limited, but includes causative substances such as various diseases and allergies, pathogenic bacteria, physiologically active substances, antibodies thereof and the like. Specifically, immunoglobulins (for example, human and animal immunoglobulins, modified immunoglobulins), various viral antigens (for example, 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 components, etc.), various hormones (eg, estrogen, human chorionic gonadotropin, (HCG) )), Antibodies (eg, anti-CEA (carcinoembryonic antigen) antibody, anti-reactive protein antibody, anti-fibrinogen antibody, anti-γ-globulin antibody, etc.), hormone receptors (eg, thyroid stimulating hormone receptor, HCG receptor, etc.), Enzymes (eg alkaline phosphatase, cholinester) , Etc.), substrates (eg, cholesterol, uric acid, etc.), hemoglobin derivatives, sugar derivatives (eg, sugar compounds, glycoproteins, etc.).

  The detection substance used in the measurement method of the present invention may be any substance that specifically binds to the target substance. Examples of such a detection substance 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. Among these, when a substance that binds to a target substance by an antigen-antibody reaction is used, a wide variety of antibodies or antigens that bind to the target substance antigen or antibody can be obtained, so that various kinds of target substances can be measured. .

First, a first process is performed for obtaining a reference concentration that maximizes absorbance when a target substance or a specific substance having the same specificity as the target substance is added to a detection solution containing a detection substance.
In the present invention, the substance having the same specificity as the target substance used for the specific substance refers to a substance that specifically binds to a site on the detection substance that binds to the target substance.
A specific substance can be used in determining the reference concentration at which the absorbance is maximized, but the same substance as the target substance is particularly 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.

In the detection solution, the detection substance may be used dispersed in the solution or supported on an insoluble carrier, but is preferably used supported on an insoluble carrier. As a result, the rate of change in absorbance with respect to the amount of change in the target substance concentration becomes higher, so that highly sensitive detection can be performed.
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.

  When the 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, the insoluble carrier and the 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.

When an antibody is used as the detection substance, the origin of the antibody is not limited. For example, antibodies derived from sera of any animal species such as rabbit antibodies, rat antibodies, mouse antibodies, goat antibodies can be used. The antibody may be an antibody or an antibody fragment such as F (ab ′) ₂ , but an antibody purified from goat serum is preferable in terms of cost. The purification method and purity of the antibody are not particularly limited, and for example, an antibody purified by ammonium sulfate salting out or the like can be used. When an antibody is used as the detection substance and supported on the insoluble carrier, the antibody may be any antibody that does not prevent aggregation of the insoluble carrier.
Furthermore, when an anti-immunoglobulin antibody is used as an antibody, immunoglobulin can be detected as a target substance.

  Examples of the method for measuring absorbance include an immunoturbidimetric method and an immunonephrolytic method, and the immunoturbidimetric method is preferably used.

Next, a second process of adding the specific substance having 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. I do.
At this time, the standard solution may be added after the specific substance is added, or the specific substance and the standard solution may be added simultaneously.
Here, the detection solution of the same type 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.

When the standard solution is added after the specific substance is added, the absorbance is measured after a lapse of a certain time from the addition of the standard solution. When a specific substance and a standard solution are added at the same time, the absorbance is measured after a lapse of a certain time after adding them. The absorbance measurement conditions are the same as in the first step.
By plotting the absorbance against the absorbance and the concentration of the target substance in the standard solution, a calibration curve can be created. Here, a calibration curve in which the absorbance decreases as the concentration of the standard solution increases is obtained.
In addition, by selecting which concentration of the specific substance to be added to the detection solution is set above the reference concentration, the target substance in a concentration range from a minute concentration to a high concentration can be more accurately determined. It can be quantified. For example, when measuring a target substance at a very low concentration, a calibration curve having a greater difference in absorbance than a minute difference in the concentration of the target substance is obtained by increasing the concentration of the specific substance to be added above the reference concentration. be able to. When measuring a high concentration of the target substance, it is sufficient that the target concentration is equal to or higher than the reference concentration and in the vicinity of the reference concentration. In this case, less detection substances are used, which is more economical.

Further, the third step of adding the specific substance having the same concentration as the second step and the test sample to the third detection solution of the same type as the detection solution and measuring the absorbance thereof is performed. That is, 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 having the same concentration as the second process and the test sample are added to the detection solution. . After the addition, the absorbance is measured after the same time has elapsed as the measurement of 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.

The reagent for measuring a target substance of the present invention contains a detection substance that specifically binds to the target substance, and a specific substance composed of the target substance or a substance having the same specificity as the target substance. The concentration is characterized by being equal to or higher than a reference concentration at which the absorbance becomes maximum when the specific substance is added to the detection solution containing the detection substance.
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 certain period of time. Separately, add a test sample to the same type of measurement reagent. By measuring the absorbance after the lapse of the same time, the target substance concentration in the test sample can be obtained using the calibration curve.

In the measurement method of the present invention, by adding a specific substance to the detection solution at the reference concentration or higher, a target substance that is in a small amount to a large amount can be quantified easily and accurately. Since a 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.
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, immunoglobulin (immunoglobulin) can be detected as a target substance, and in particular, immunoglobulin G (which has conventionally been difficult to quantify without dilution) IgG), immunoglobulin A (IgA), immunoglobulin M (IgM), and other immunoglobulins present in large amounts in normal specimens can be accurately quantified without dilution.

  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. Here, for example, a target substance that is present in a large amount in a normal sample, such as a complement component containing C3, C4, etc., can be accurately quantified without dilution.

[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. Test solution) was prepared.
A detection substance consisting of an anti-human IgG goat polyclonal antibody (International Immunology Corp .: hereinafter referred to as “anti-IgG antibody”) was dissolved in a 50 mM phosphate buffer (pH 7.2) at a concentration of 2.0 mg / ml. 10.0 ml of polystyrene particles having an average particle diameter of 110 nm (manufactured by Fujikura Kasei Co., Ltd .: solid content 1 mass%) were added to 10.0 ml of the liquid, and the mixture was stirred at 4 ° C. for 60 minutes. Next, a 50 mM phosphate buffer solution (pH 7.2: hereinafter referred to as “PBS”) containing 1% by mass of bovine serum albumin (hereinafter referred to as “BSA”) was added to this solution, followed by stirring at 4 ° C. for 60 minutes. Then, it was washed by centrifugation at 20000 × g for 60 minutes. The washing operation was performed 3 times. After adding 10 ml of PBS containing 0.1% by mass of BSA to the resulting precipitate and suspending the polystyrene particles, dispersion treatment was performed with an ultrasonic crusher to obtain a solid content of 0.1% (W / V). Anti-IgG antibody-supporting latex test solution was obtained.
Further, a human IgG (International Immunology Corp .: hereinafter referred to as “IgG”) was used as a specific substance to prepare a specific substance solution (specific substance solution). Glycine 50 mM and sodium chloride 70 mM were dissolved in pure water and adjusted to pH 7.2. To this solution, an IgG concentration of 10 mg / dl, 15 mg / dl, 20 mg / dl, 25 mg / dl, 50 mg / dl, 75 mg / dl, Human IgG was mixed so that it might become 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 the above (1) is added to each of the different detection solutions prepared by the same method as in the above (1). Absorbance after minutes 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. The results are shown in FIG. FIG. 1 is a semilogarithmic graph with the horizontal axis as a logarithmic scale.
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 to obtain IgG concentrations of 10 mg / dl, 25 mg / dl, 100 mg / dl, 500 mg / dl, 1000 mg / dl, 2000 mg / dl, 5000 mg / dl, 8000 mg / dl. It prepared so that it might become.
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. FIG. 2 is a semilogarithmic graph with a logarithmic scale on the horizontal axis.

(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 separately prepared by the method of (1) above was added, and the absorbance was measured 5 minutes after the addition. 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 1012 mg / dl, which was consistent with the IgG concentration of the test sample used.

[Comparative Example 1]
Measurement was performed in the same manner as in Example 1 except that IgG was not added in the preparation of (1) the detection solution and the specific substance solution in Example 1. The results are shown in FIG. FIG. 3 is a semi-logarithmic graph with a logarithmic scale on the horizontal axis.

As is clear from FIG. 2, in Example 1 in which the reference concentration of IgG was added to the latex test solution, the calibration curve in which the IgG concentration and the absorbance uniquely corresponded until the IgG concentration of the standard solution changed from low to high. And the possibility of misjudging the IgG concentration from the absorbance measured for the sample with unknown concentration could be eliminated. This allowed the IgG concentration to be accurately determined in the test sample without dilution.
In Comparative Example 1 in which the latex test solution did not contain a specific substance consisting of IgG, when the IgG concentration in the standard solution was high, two or more IgG concentrations corresponded to the same absorbance value. In other words, a calibration curve in which the IgG concentration and absorbance uniquely correspond could not be obtained.

In Example 1, it is a figure which shows the light absorbency with respect to the density | concentration of a specific substance when IgG is added to a detection solution as a specific substance. It is a figure which shows the light absorbency with respect to IgG density | concentration of a standard solution in Example 1. FIG. It is a figure which shows the light absorbency with respect to IgG density | concentration of a standard solution in the comparative example 1.

Claims (9)

In a method of measuring a target substance concentration in a test sample using a detection substance that specifically binds to the target substance,
A first step of obtaining a reference concentration that maximizes the absorbance when 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 second step of adding the specific substance equal to or higher than the reference concentration to a second detection solution of the same type as the detection solution and a standard solution containing a target substance having a known concentration and measuring the absorbance thereof;
A third step of adding the specific substance having the same concentration as the second step and a test sample to a third detection solution of the same type as the detection solution and measuring the absorbance thereof. A method for measuring a target substance. The method for measuring a target substance according to claim 1, wherein the detection substance specifically binds to the target substance by an antibody-antigen reaction. The method for measuring a target substance according to claim 1, wherein the detection substance is supported on an insoluble carrier in a detection solution. The method for measuring a target substance according to claim 3, wherein the insoluble carrier is latex particles. 5. The method for measuring a target substance according to claim 2, wherein the detection substance is an anti-immunoglobulin antibody, and the target substance is an immunoglobulin. A reagent for measuring a target substance in a test sample,
It contains a detection substance that specifically binds to a target substance and a specific substance composed of the target substance or a substance having the same specificity as the target substance, and the concentration of the specific substance is the detection of the specific substance. A reagent for measuring a target substance, wherein the reagent is equal to or higher than a reference concentration at which absorbance is maximized when added to a substance for use. The reagent for measuring a target substance according to claim 7, wherein the detection substance is supported on an insoluble carrier. 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 antibody-antigen reaction. The reagent for measuring a target substance according to claim 8, wherein the detection substance is an anti-immunoglobulin antibody, and the target substance is an immunoglobulin.

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