JP2001033454A - Method and apparatus for determining substance to be detected on porous solid-phase ligand measuring test piece - Google Patents

Abstract

PROBLEM TO BE SOLVED: To accurately determine with high specificity by utilizing a combination of a ligand pair substantially not effected by an influence of a substance coexisting in a sample to be detected to an internal control installed in an apparatus. SOLUTION: A sample to be detected is reacted with standard particles, and moved by a capillary flow in a matrix. A reaction composite is captured by an antigen held in a detecting region, and its signal is detected. Meanwhile, an internal control holds a gold colloid or fluorescence labeled latex particles in which an avidin is adsorbed on a test piece as a substance of reaction pair not substantially affected by an influence of the substance coexisted in the sample. Then, a biotin is previously held in the internal control area to capture moved avidin particles by an avidin-biotin bonding reaction. This signal is detected to obtain an internal control signal. A ratio of both the signals is obtained, compared with a working curve line data obtained from the sample of known concentration and the substance to be detected is determined.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to an immunoassay method and apparatus used for clinical examination.

[0002]

2. Description of the Related Art Ligand measuring methods and devices utilizing capillary flow are widely used in clinical tests as immunochromatographic measuring methods. In this measurement method, a test substance is generally qualitatively or semi-quantitatively detected by visual detection. On the other hand, attempts have been made to quantitatively measure a test substance by this type of immunoassay. For example, JP-A-8-24
According to 0591, a substance which is the same or similar to the test substance is held in one area of the test piece, and a signal which has reacted with the label by immunological reaction is detected using this area as an internal control. A method is disclosed in which a ratio of a signal generated in an area for detecting a complex of a label and an analyte to form an immune binding pair with a signal generated in the internal control area is determined to quantify the test substance. This method is characterized in that the amount of the test substance is determined by utilizing the fact that the signal from the test substance and the signal in the internal control area have an inverse proportional relationship. Further, US Pat. No. 5,753,517 discloses a method in which an immune reaction different from a test substance is used as an internal control. In this method, the signal in the internal control area is constant without depending on the concentration of the test substance, and the test substance is quantified by obtaining the ratio between the signal in the internal control area and the signal of the test substance. All of these methods utilize an immune reaction by an antigen-antibody reaction in the step of generating an internal control signal. According to the method of US Pat. No. 5,753,517, internal control particles to which an antibody is adsorbed are prepared, and a binding pair that immunoreacts with the antibody adsorbed to the particles is retained in the internal control region. -Designed such that the antibody reaction generates an internal control signal. As described above, in the internal control using the antigen-antibody reaction, a commonly used biological sample (for example, blood,
Serum, etc.), there are many samples containing antibodies against various animals and rheumatoid factors, and non-specific immune reactions other than the specific immunological reaction of interest occur, making accurate measurement impossible. there were.

[0003]

SUMMARY OF THE INVENTION In a method and an apparatus for measuring a ligand utilizing capillary flow, a method and an apparatus for quantifying a test substance are provided.

[0004]

[Means for Solving] As a result of intensive studies, the present inventors have found that
Accurate and highly specific by utilizing a combination of ligand pairs that are substantially unaffected by substances coexisting in the test sample for the ligand measurement method using capillary flow and the internal control installed in the device They have found that a quantitative method can be provided, and have completed the present invention.

[0005] For example, when a test substance (antigen) is quantified by immunochromatographic measurement, labeled particles obtained by sensitizing and adsorbing an antibody against an antigen to colloidal gold or colored or fluorescently labeled latex particles are held on a matrix test piece. The test sample is reacted with the labeled particles, moved by capillary flow in the matrix, the reaction complex is captured by the antibody retained in the detection area, and the signal is detected. On the other hand, as an internal control, for example, a gold colloid or a colored or fluorescently labeled latex particle adsorbed with avidin is held on a test piece as a substance of a reaction pair substantially unaffected by a coexisting substance in a test sample, The sample is moved in the matrix by the addition of the test sample. Avidin particles that have been moved by holding biotin in advance in the internal control area are captured by the avidin-biotin binding reaction. By detecting this signal, an internal control signal is obtained. Then, the ratio of both signals is obtained by a known method, and the analyte is quantified by comparing with a calibration curve data obtained by similarly operating a sample of a known concentration.

[0006] The combination of a reaction pair that generates an internal control signal and is substantially unaffected by the coexisting substance in the test sample is a combination of avidin-biotin, biotin-streptavidin, and a biotin analog. It is selected from a combination of avidin or streptavidin. Further, in a known method, a combination of animal IgG and an antibody thereto is shown. In this case, it is as described above that the presence of the antibody against animal IgG in human serum affects the measurement. However, in the method of the present invention, a combination of an artificial compound (hapten) sensitized and adsorbed particles and an antibody against the compound is also suitable. For example, 2,4-dinitrophenol (DNP)
Can be adsorbed on the particles, and the anti-DNP antibody can be retained in the internal control signal detection area. As other examples, fluorescein, rhodamine and the like and antibodies thereto are also useful. However, anti-DN, which is the same method as that for adsorbing a known antibody to internal control particles, is used.
The method in which DNP is retained in the internal control detection region by the particles to which the P antibody is adsorbed is not suitable because it is affected by anti-animal antibodies and rheumatoid factors coexisting in the test sample. Which ligand pair should be selected may be selected in consideration of a substance coexisting in the test sample.

When blood or serum is used as a test sample,
It is advantageous to select a combination of avidin or streptavidin and biotin or an enzyme and substrate that are not present in human serum, such as a combination of mannase and mannan. On the other hand, when the test sample measures environmental hormones or the like in soil, a combination of haptoglobin and hemoglobin is also advantageous.

Therefore, one of the features of the present invention is that an optimum internal control can be selected according to the properties of a test sample.

FIG. 1 shows an example of a specific embodiment of the present invention. The member 1 is a matrix test piece such as a nitrocellulose membrane, an acetylcellulose membrane, a cellulose filter paper, a glass filter paper, and a polyethylene sintered body, and the portion 2 is a sample addition portion provided on the test piece.
A separation filter or separation pad capable of removing solids such as blood cells, and a member for removing solids using a nonwoven fabric gauze or the like can be provided at the sample addition site. The site 3 is a site for holding labeled particles. When measuring an antigen, labeled particles obtained by sensitizing and adsorbing an antibody against the antigen to colloidal gold or colored or fluorescently labeled latex particles are held. Generally, the labeled particles are kept dry. This site also holds particles for internal control. As the particles for internal control, particles giving a signal different from the particles for the test substance are appropriate. For example, in contrast to gold colloid, metal colloids having different wavelengths are used, colored particles are colored with dyes having different absorption wavelengths, and fluorescent dyes are two types of particles having different fluorescence wavelengths. It is appropriate that the particle size of the internal control particles is substantially the same as the particle size of the test substance particles. If the difference between the particle diameters is large, the mobility of the particles for the test substance and the particles for the internal control due to the capillary flow may be different due to the influence of the viscosity of the test sample and the like, and accurate analysis may not be performed. As the substance to be adsorbed to the internal control particles, a combination suitable for analysis can be selected from the above-mentioned ligand pairs. Avidin-
When biotin is selected, either avidin or biotin is adsorbed on particles and used. Site 4 is a detection area, and in the case of antigen measurement, generally, an antibody against the test substance is retained. If a test substance is present in the test sample, the test substance and the labeled particles form a complex, move by capillary flow, and are captured at the site 4. The signal of the captured labeled particles is detected. Site 5 is located close to site 4 and is an internal control area. Internal control particles that have migrated by capillary flow are captured at one of the ligand pairs at this site. For example, when avidin-adsorbed particles are used as internal control particles, biotin is retained at this site, and the particles are captured by the biotin-avidin bond. Detect the signal of the captured particles. Determine the ratio of the signal detected by the test substance and the signal detected by the internal control, compared with a calibration curve obtained by previously operating a sample containing a substance of known concentration,
The test substance is quantified. As a signal to be detected, dye absorption, reflection, fluorescence and the like can be applied.

[0010]

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

[0011]

Example 1 (1) Preparation of anti-HBs monoclonal antibody-labeled latex Anti-HBs monoclonal antibody (clone HB0011)
Was adjusted to 1 mg / mL in a 20 mM phosphate buffer (p
H7.0). 1 mL of a suspension of blue latex particles having a particle size of 0.25 μm (10% solid content) was added to 10 mL of the diluted solution, mixed, and allowed to stand at room temperature for 2 hours. 1
The latex particles were collected by centrifugation at 5,000 × g for 20 minutes, and 20 μm containing 10 mg / mL bovine serum albumin.
M phosphate buffer (BSA-phosphate buffer)
Washed twice. Suspended in a final 10 mL of BSA-phosphate buffer.

(2) Preparation of biotin-labeled latex Sulfo-NHS-LC-biotin (manufactured by Sigma) was dissolved in a 20 mM phosphate buffer (pH 7.0) to a concentration of 0.1 mg / mL, and the solution was dissolved in 10 mL of the solution. 1 mL of a suspension of red latex particles having a particle size of 0.28 μm (10 solids) was added to the mixture, and the mixture was stirred and left at room temperature for 2 hours. Thereafter, the mixture was centrifuged at 15,000 × g for 20 minutes to collect latex particles, and 2 mg of 10 mg / mL bovine serum albumin was added.
0 mM phosphate buffer (BSA-phosphate buffer) 10 mL
And washed three times. Suspended in a final 10 mL of BSA-phosphate buffer.

(3) Each of the above latex suspensions is 1: 1
A 0.5% polyvinyl alcohol was added to the suspension mixed with the above, and a non-woven fabric pad (1 cm × 10 cm, thickness 2 m) was added.
m) was immersed in the solution and freeze-dried. The pad was cut to a length of 0.8 cm and used for one test.

(4) Nitrocellulose membrane (1 cm × 10
cm) was used as a test piece and processed as shown in FIG. At site 4 in the figure, anti-HBs monoclonal antibody (clone HB0022) was added at 100 μg / mL for 10 μL.
L was applied to provide a detection site. At the site 5 in the figure, 10 μL of 100 μg / mL of avidin was applied to provide a control site. After coating and drying each, the entire nitrocellulose membrane was blocked with a BSA-phosphate buffer and dried. The test piece prepared in the above (3) was placed on the site 3 of the test piece prepared in this manner, and the test piece was connected to the sample addition site 2 with a nonwoven fabric gauze so as to be connected by a capillary.

(5) Measurement of HBs antigen A known concentration of HBs antigen solution (0, 10, 20, 50, 1)
(00 ng / mL) was added dropwise to the sample addition section, and after 8 minutes, the reflectance was determined at 410 nm and 530 nm using a densitometer for thin layer chromatography (manufactured by Shimadzu Corporation). Table 1 shows the results.

[0016]

[Table 1]

As a result, a ratio depending on the HBs antigen concentration was obtained, and it became clear that the HBs antigen could be quantified.

[0018]

The present invention provides a highly specific assay system which avoids the influence of coexisting substances when quantifying a test sample by the signal ratio between the internal control and the detection section of the test sample.

[0019]

[Brief description of the drawings]

FIG. 1 is a schematic example of an immunochromatography method.

[Explanation of symbols]

 DESCRIPTION OF SYMBOLS 1 ... Immunochromatography support 2 ... Sample addition site 3 ... Labeled latex site 4 ... Detection site 5 ... Control site

Claims (4)

[Claims] 1. A method for quantifying the concentration of a test substance in a fluid sample, the method comprising: a) a test piece containing a matrix in which the fluid sample can move by capillary action, which can be detected according to the concentration of the test substance. B) detecting a signal (internal control signal) from the labeling substance which can be detected without depending on the concentration of the test substance; c) detecting the signal from the step a); Determining the ratio of the signals in step b), d) measuring the ratio of the signals determined in a similar manner for the fluid sample containing the analyte at a known concentration, and measuring the ratio of the signals determined in step c). The method for quantifying the concentration of a test substance in a fluid sample comprising the step of measuring the concentration of the test substance in the fluid sample by comparing Wherein the use of a combination of receiving no ligand to substantially the effects of the coexisting materials in the postal. 2. A combination of a ligand pair which is substantially unaffected by a substance coexisting in a test sample in a step of generating and detecting an internal control signal is at least a combination of biotin or a biotin analog and avidin or streptavidin. Alternatively, the method according to claim 1, which is selected from a combination of a hapten and an antibody. 3. An apparatus for quantifying the concentration of a test substance in a fluid sample, comprising: a) a test strip containing a matrix in which the fluid sample can move by capillary action, which can be detected in accordance with the concentration of the test substance. B) detecting a signal (internal control signal) from the labeling substance which can be detected without depending on the concentration of the test substance; c) detecting the signal from the step a); Determining the ratio of the signals in step b), d) measuring the ratio of the signals determined in a similar manner for the fluid sample containing the analyte at a known concentration, and measuring the ratio of the signals determined in step c). In an apparatus for quantifying the concentration of a test substance in a fluid sample comprising a step of measuring the concentration of the test substance in a fluid sample by comparing And wherein the use of combinations of ligand pair which does not substantially affected by the coexisting materials in the postal. 4. A combination of a ligand pair which is substantially unaffected by a substance coexisting in a test sample in a step of generating and detecting an internal control signal is a combination of biotin or a biotin analog and avidin or streptavidin, or 4. The device of claim 3, wherein the device is selected from a combination of a hapten and an antibody.