JPH10332694A - Immunological measurement method - Google Patents

Abstract

PROBLEM TO BE SOLVED: To achieve an accurate measurement without diluting an object to be measured and without repeated measurement by setting pH in a liquid and sodium chloride to values within a specific range on immunological reaction. SOLUTION: In an immunological measurement method for immunologically reacting an object A to be measured and a substance B that is specifically connected to the object to be measured, pH in a liquid on immunological reaction is set to approximately 3.5-5.5 and sodium chloride concentration is set to approximately 10-250 g/l. By setting pH to the specific range, an accuracy can be improved also for the high-concentration substance A to be measured. The sandwich method is used for the immunological measurement method. The object A to be measured is related to, for example, protein, hormone, chemicals, and infectious disease. For example, an antibody and an antigen that are specifically connected to the object A to be measured are used as the substance B to be specifically connected to the object A to be measured. For example, isotope, enzyme, fluorescent substance, and luminous substance are used for a labeled substance, thus improving the accuracy in measurement greatly.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to an immunoassay. More specifically, the substance to be measured is accurately measured,
The present invention relates to an immunoassay method capable of measuring a substance to be measured without diluting the substance or without repeatedly measuring the substance.

[0002]

2. Description of the Related Art In an immunoassay, a method for measuring a substance to be measured without a complicated operation of diluting the substance even when the concentration of the substance is high has been conventionally known as a method which is specific to a substance to be measured and a substance to be measured. The pH of the reaction solution when reacting a substance that is chemically bound is 6.0 to 8.0, the sodium chloride concentration is 20 to 250 g / L, the immune reaction is suppressed, and the substance to be measured is not diluted. A method for direct measurement has been proposed [JP-A-09-089894].

[0003]

However, if the substance to be measured is directly measured without dilution by this measuring method, the accuracy of reproducibility of the measured value is insufficient depending on the kind of the substance to be measured, so that a sufficiently accurate measurement is performed. When a value was required, the substance to be measured was repeatedly measured.

[0004]

Means for Solving the Problems The present inventors have made intensive studies to solve the above-mentioned problems, and as a result, have improved the conventional method,
By replacing H with a more acidic or alkaline specific region, the present inventors have found that the accuracy of a substance to be measured is unexpectedly greatly improved, and the present invention has been achieved. That is, the present invention relates to the following immunological assay methods. In an immunoassay for immunoreacting a substance to be measured (A) and a substance (B) which specifically binds to the substance to be measured, the pH of the liquid at the time of the immune reaction is adjusted to 3.5 to 5.5,
An immunoassay wherein the sodium chloride concentration is 10 to 250 g / L. In an immunoassay for immunoreacting the substance to be measured (A) and the substance (B) which specifically binds to the substance to be measured, the pH in the liquid during the immune reaction is adjusted to 9.0 to 12.0, An immunoassay wherein the sodium chloride concentration is 10 to 250 g / L.

[0005]

BEST MODE FOR CARRYING OUT THE INVENTION In the method of the present invention, the pH of the solution used for the immunoreaction of (A) and (B) is usually 3.5 to 5.5, preferably 4.5 to 5.5 in the above method. In the above method, it is usually 9.0 to 12.0, preferably 1
0.0 to 11.0.

[0006] In the method of the present invention, the concentration of sodium chloride in the liquid used for the immunoreaction of (A) and (B) is usually 10 to 250 g in any of the methods.
/ L. In the case of the method of (1), the upper limit of the sodium chloride concentration in the liquid is preferably set to 250 g / L, and the following formula (1)
Is a concentration that satisfies Y ≧ 5X−7.5 (1) [wherein X represents the pH of the solution during the immune reaction, and Y
Indicates the concentration of sodium chloride (g / L) in the liquid. In the case of the above method, the upper limit is preferably set to 250 g /
L is a density satisfying the following expression (2). Y ≧ −3.33X + 50 (2) [wherein X represents the pH of the solution during the immune reaction, and Y
Indicates the concentration of sodium chloride (g / L) in the liquid. ], The sodium chloride concentration in the liquid is more preferably 20 to 250 g / L, particularly preferably 40 to 250 g / L.
２５０250 g / L.

Although the immunoassay of the present invention can be used for all immunoassays such as competitive assays and non-competitive assays, accurate measurements cannot be obtained if the concentration of (A) is too high. It is preferably used in non-competitive assays, which are often immunological assays. The non-competitive assay is an immunoassay in which (A) having at least two homologous or heterologous epitopes is measured. As long as (A) in the sample increases, unless a prozone phenomenon occurs, This is a method of increasing the number of specific reactants such as antibodies and antigens that bind to (A). Measurement signals such as turbidity, scattered light, transmitted light, absorbance, radioactivity, luminescence, and fluorescence accompanying the increase of the specific reactants This is a method of measuring the fluctuation of the value as an index.

[0008] The immunological assay of the present invention is preferably used in the sandwich method among non-competitive assays, because the overall measurement accuracy is improved. In the sandwich method, for example, the amount of a labeled substance in a complex obtained by reacting (A), a substance in which (B) is bound to an insolubilized carrier, and a substance in which (B) is bound to a labeled substance is measured. Thereby, (A) is measured. The complex formation reaction may be performed in a one-step reaction or a two-step reaction. In the case of a two-step reaction, for example, a substance in which (A) and (B) are bound to an insolubilizing carrier is first reacted, and then a substance in which (B) is bound to a label is reacted to form a complex. . In the case of this two-stage reaction, the method according to the present invention is used for the first-stage immune reaction.
It is more preferable to use the method according to the present invention also for the second stage immune reaction.

The analyte (A) that can be used in the method of the present invention includes (1) protein-related items (AFP, CEA, CA19-9, CA125, CA
15-3, CA72-4, CA50, troponin I, troponin T, pepsinogen I, pepsinogen II, PA,
PAP, SCC, NSE, IgE, specific IgE, β2-
Microglobulin (abbreviated as β2m), ferritin, IA
P, C3, C4, C5, CRP, α2-MG, IgA,
IgM, IgG, IgE, IgD, CK-MB, CK-
MM, myoglobin, myosin, transferrin, apolipoprotein, glycoprotein, albumin, microalbumin, hemoglobulin, glycohemoglobin, fructosamine, HDL, LDL, RF, lymphocyte subset, LE cell, anti-thyroglobulin antibody, anti-microsomal antibody, ASO ), (2) hormone-related items (insulin, HCG, βHCG, growth hormone, TS
H, LH, FSH, prolactin, T3, T4, FT
3, FT4, TBG, C-peptide, T-Uptak
e, estrogen, HPL, E2, cortisol, progesterone, testosterone, somatostatin, etc.),
(3) drug-related items (digoxin, phenytoin, phenobarbital, theophylline, etc.), and (4)
Numerous infectious disease related items (fungi, streptococci, Escherichia coli, Mycobacterium tuberculosis, hepatitis virus, herpes virus, AIDS virus, Candida, mycoplasma, toxoplasma, syphilis, malaria parasite, dysentery ameber, etc.) Metabolites (such as verotoxin) and antibodies. Of these, items requiring dilution (β2m, TBG, IAP, RF, LE cells, anti-thyroglobulin antibody, anti-microsomal antibody, C3, C4,
C5, CRP, α2-MG, IgA, IgM, IgG, I
gE, specific IgE, IgD, estrogen, HPL, transferrin, albumin, microalbumin, hemoglobulin, glycated hemoglobin, fructosamine,
HDL, LDL, ASO, infectious disease-related items themselves and their metabolites and antibodies).

Substance (B) that specifically binds to the substance to be measured
As an antibody that specifically reacts with the analyte (A),
An antigen or the like can be used. For example, (A) is AFP
For antigen, (B) is anti-AFP antibody, (A) is anti-HB
For the s antibody, (B) is an HBs antigen or the like. In addition, the substance (B) in the case of binding to the insolubilized carrier and the substance (B) in the case of binding to the labeling substance are both substances as long as they are substances that specifically bind to the substance to be measured. They can be used even if they are not the same. For example, a combination of a monoclonal antibody and a polyclonal antibody, a combination of a rabbit-derived antibody and a goat-derived antibody, and the like.

Examples of the insolubilizing carrier include (1) a siliceous inorganic carrier [glass (porous, frosted glass, etc.), silica gel, benite, etc.], (2) a magnetic substance (magnetic fine particles, etc.), and (3) an organic substance. Known materials are used for all carriers (polyethylene, polypropylene, polystyrene, methylpentene resin, polycarbonate, vinyl chloride resin, methacrylic resin, ABS resin, epoxy resin, fluororesin, nylon, dextran, paper, etc.).
In addition, these insolubilized carriers are sometimes used in the form of fine particles in suspension.

As a method for bonding (B) to the insolubilized carrier, (1) a method for chemically bonding (B) to glass (for example, US Pat. Nos. 4,280,992 and 365,2761) and , (2) a method of physically adsorbing (B) to a plastic (for example, A. Engbal et al .; Biosim Biofiz Acta, Vol. 251, 4)
27 Tribute, 1971). Furthermore, (3) (B)
A method in which (B) is bound to an insolubilized carrier by deriving from a product in which biotin is bound to a biotin and a product in which streptavidin is bound to an insolubilized carrier; Thus, there is a method of indirectly binding (B) to the insolubilized carrier, such as a method of binding (B) to the insolubilized carrier (for example, Japanese Patent Application No. 04-174911).

Examples of the label include known substances such as isotope [I125 etc.], enzymes [Peroxidase, alkaline phosphatase, β-galactosidase etc.], fluorescent substances [Europium derivatives etc.], luminescent substances [Acridium derivatives etc.]. used.

As a method of binding (B) to a label, there is a method of binding (B) to a label (for example, Biochemical Experiment Method 15, Tokyo Kagaku Dojin, pp. 308-330 (19)
93)). Furthermore, a method of binding (B) to a label by binding streptavidin to (B) and binding to biotin, or a method of binding an antibody against (B) to a label beforehand (B) ) Is bound to a label (for example, Japanese Patent Application No. Sho 63).
-262479) and the method of indirectly binding (B) to a label.

In carrying out the method of the present invention, in order to further improve the accuracy of the measurement, the substance to be measured and the buffer for the immunoreaction during the measurement are automatically dispensed by a machine instead of manually. Is more preferred.

[0016]

EXAMPLES The present invention will be further described with reference to the following examples, but the present invention is not limited to these examples.

Preparation of a buffer for an immunoreaction; 5.15 g / L of sodium hydrogen phosphate, 0.764 g / L of potassium dihydrogen phosphate and 10% of bovine serum albumin in ion-exchanged water.
g / L, and the sodium chloride concentration was further increased to 10 g / L, 20 g / L, 40 g / L,
80 g / L, 160 g / L and 250 g / L were added to prepare a total of six kinds of buffer solutions before pH adjustment. Next, in order of the buffer solution before pH adjustment of
By the operation of adding an appropriate amount of Cl or NaOH to prepare six samples of the same pH in numerical order, buffer solutions for the following comparative examples and examples were prepared. Buffers for comparative examples (pH is out of range): immune reaction buffers a1 to a6 at pH 2.5, immune reaction buffers a7 to a12 at pH 6.0, and immune reaction buffer a13 at pH 7.2
To a18, pH 8.5 buffer solution for immune reaction a19 to a2
4, buffer solution for immune reaction a25 to a30 at pH 13.0, buffer solution for Examples (pH within range): buffer solution for immune reaction b1 to b6 at pH 3.5, buffer solution for immune reaction at pH 4.5 b7 to b12, an immune reaction buffer b13 having a pH of 5.5
To b18, pH 9.0 buffer for immune reaction b19 to b2
4, an immune reaction buffer b25 to b30 at pH 10.0,
Immune reaction buffers b31 to b36 at pH 11.0, pH
12.0 buffer buffer for immune reaction b37-b42

Comparative Examples 1 to 30 and Examples 1 to 42 β2
m Immunological Assay Using a fully automatic enzyme immunoassay analyzer OLYDAS-120 (manufactured by Olympus Optical Co., Ltd.), a1 was used as the first reaction.
0.3 mL of each of ~ a30, b1 ~ b42 and standard β2
0.005 mL of m-solution or β2m-containing specimen and anti-β2
m antibody-bound glass beads (hereinafter referred to as G1)
G1 + β2 was reacted in a reaction tube at 37 ° C. for 16 minutes.
m complex was formed. After the reaction, the mixture was washed with a B / F separation liquid dedicated to the device, and B / F separation was performed.

As a second reaction, G1 + β2m in a test tube was used.
0.02 M phosphate buffer containing 0.2 mg / L of peroxidase-labeled anti-β2m antibody (hereinafter referred to as P1), 10 g / L of bovine serum albumin, and 8.5 g / L of sodium chloride per complex. (PH 7.2) to 0.
3 mL was dispensed and immunoreacted at 37 ° C. for 16 minutes in a reaction tube to form a G1 + β2m + P1 complex. After the reaction,
Washing was performed with a B / F separation liquid dedicated to the apparatus, and B / F separation was performed.

As a third reaction, G1 + β2m in a test tube
+ P1 complex was dispensed with 0.3 mL of an acetate buffer containing 0.1 g / L of hydrogen peroxide and 0.17 g / L of tetramethylbenzidine, and the mixture was placed in a reaction tube at 37 ° C., 1
After a color development reaction for 5.5 minutes, 0.7 ml of deionized water was mixed after the reaction, and photometry was performed at 380 nm.

Finally, the concentration value and photometric value of each standard β2m solution were automatically regressed with a spline curve, and β was determined from the calibration curve.
The concentration value for the photometric value of the 2m-containing specimen was calculated.

Comparative Examples 31 to 60 and Examples 43 to 84
TBG immunoassay Using a fully automatic enzyme immunoassay analyzer OLYDAS-120 (manufactured by Olympus Optical Co., Ltd.), a
0.3 mL of each of 1 to a30, b1 to b42, and standard T
0.005 mL of BG solution or TBG-containing sample and anti-T
A single BGm antibody-bound glass bead (hereinafter referred to as G2) was immunoreacted in a reaction tube at 37 ° C. for 16 minutes, and G2 + T
A BG complex was formed. After the reaction, the mixture was washed with a B / F separation liquid dedicated to the device, and B / F separation was performed.

As a second reaction, G2 + TBG in a test tube was used.
0.02M phosphate buffer containing 0.4 mg / L of peroxidase-labeled anti-TBG antibody (hereinafter referred to as P2), 10 g / L of bovine serum albumin and 8.5 g / L of sodium chloride per complex. (PH 7.2) to 0.
3 mL was dispensed and immunoreacted at 37 ° C. for 16 minutes in a reaction tube to form a G2 + TBG + P2 complex. After the reaction,
Washing was performed with a B / F separation liquid dedicated to the apparatus, and B / F separation was performed.

As a third reaction, G2 + TBG in a test tube
+ P2 complex was dispensed with 0.3 mL of an acetate buffer containing 0.1 g / L of hydrogen peroxide and 0.17 g / L of tetramethylbenzidine in a reaction tube at 37.degree.
After a color development reaction for 5.5 minutes, 0.7 ml of deionized water was mixed after the reaction, and photometry was performed at 380 nm.

Finally, the concentration value and photometric value of each standard TBG solution are automatically regressed with a spline curve, and T
The concentration value for the photometric value of the BG-containing sample was calculated.

Evaluation of Comparative Examples 1 to 30 and Examples 1 to 42 In order to confirm that the immunoassay method can accurately measure a high concentration of an analyte, Comparative Examples 1 to 3
0 and the β2m immunoassay of Examples 1-42
Each sample containing 0 mg / L β2m was measured at n = 20, and the simultaneous reproducibility [standard deviation of the measured value / mean value × 100]
(%)]. Table 1 shows the results.

[0027]

[Table 1]

Further, Comparative Examples 31 to 60 and Example 4
A TBG immunoassay of 3-84 with 50 mg / L T
The BG-containing samples were measured at n = 20, and the simultaneous reproducibility [standard deviation of the measured values / mean value × 100 (%)] was determined. Table 2 shows the results.

[0029]

[Table 2]

Consideration of evaluation results: From the results of Tables 1 and 2, it is not possible to accurately obtain measured values of a high concentration of a substance to be measured by the conventional method, but the accuracy of the measured values obtained by the present invention is greatly improved. It can be seen that it has improved. That is, in this comparative example, the average of the simultaneous reproducibility near neutral pH 6.0 to 8.5 was 20 to 25% in the β2m immunoassay and 15 to 16% in the TBG immunoassay. However, the average reproducibility of the pH range in this example is 3-5% for the β2m immunoassay and 2-4% for the TBG immunoassay.
In addition, pH 5.5 of the acidic upper limit and pH 6 and pH out of the range.
It is confirmed that a slight difference between pH 8.5 and pH 9, which is the lower limit of alkalinity, has a critical significance. further,
When the relationship between X and Y is out of the range of Expressions (1) and (2), it is also confirmed that the simultaneous reproducibility in the range is worse.

[0031]

According to the present invention, the measured value of a substance to be measured at a high concentration could not be obtained sufficiently accurately. Therefore, it is necessary to increase the accuracy of the measured value by diluting or measuring the substance to be measured or repeating the measurement. However, according to the present invention, the accuracy of the obtained measurement value is dramatically improved, and the measurement can be performed without diluting the measurement target substance or measuring it repeatedly, and the immunological measurement method The simplicity of the measurement operation is greatly improved.

Claims (4)

[Claims] In an immunoassay for immunoreacting a substance to be measured (A) and a substance (B) which specifically binds to the substance to be measured, the pH of the solution during the immunoreaction is adjusted to 3.5 to 3.5.
5.5 and the sodium chloride concentration is 10-250
g / L. 2. The method according to claim 1, wherein the concentration of sodium chloride in the solution at the time of the immune reaction satisfies the following formula (1). Y ≧ 5X−7.5 (1) [wherein X represents the pH of the solution during the immune reaction, and Y
Indicates the concentration of sodium chloride (g / L) in the liquid. ] 3. An immunological assay for immunoreacting a substance to be measured (A) and a substance (B) which specifically binds to the substance to be measured, wherein the pH of the solution during the immunoreaction is 9.0 to 9.0. 1
2.0 and the sodium chloride concentration is 10-250.
g / L. 4. The immunological assay method according to claim 3, wherein the concentration of sodium chloride in the solution at the time of the immune reaction satisfies the following formula (2). Y ≧ −3.33X + 50 (2) [wherein X represents the pH of the solution during the immune reaction, and Y
Indicates the concentration of sodium chloride (g / L) in the liquid. ]