JP2003161733A - Specific binding analysis method and specific binding analysis device - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a specific binding analysis method for accurately performing the qualitative analysis and the quantitative analysis of an analyte in a sample without being influenced by a prozone phenomenon. <P>SOLUTION: A sample containing an analyte is brought into contact with a retention section for retaining a first specific binding substance that is labeled by a labeling material, the analyte is made to bined with the first specific binding substance, the sample is brought into contact with the first detection section where the second specific binding substance is immobilized to bind the excessive analyte to the second specific binding substance, the sample is brought into contact with the second detection section where a substance that is the same as or similar to the analyte is fixed to bind the first specific binding substance to the substance, signals 1 and 2 originating from the labeled substance obtained at the first and second detection sections are measured respectively, and the concentration of the analyte in the sample is obtained based on the strength. <P>COPYRIGHT: (C)2003,JPO

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a specific binding analysis method for qualitative or quantitative analysis of an analyte in a sample.

[0002]

2. Description of the Related Art In recent years, with the improvement of medical treatment in homes and areas, and the increase of highly urgent clinical examinations,
There has been a wealth of demand for the development of a specific binding assay method that enables quick, simple, and accurate measurement even if not a clinical laboratory expert. As such a specific binding analysis method, many methods are known, such as an immunoassay applying an antigen-antibody reaction, a receptor assay using a receptor, and a nucleic acid probe assay using hybridization of complementary nucleic acid sequences. Because of its high specificity, it is widely used in a wide range of fields including clinical tests.

Further, in a chromatographic analysis method, which is a type of immunoassay, for example, a liquid sample is brought into contact with a matrix composed of a porous carrier or a fine particle-filled carrier in which a specific binding substance is insolubilized or immobilized. The presence or absence of the target substance in the sample is analyzed by utilizing the phenomenon in which the sample flows out by the osmotic force due to the capillary phenomenon along the matrix (for example, Japanese Patent No. 2504).
923, Japanese Patent No. 2667793, Japanese Patent Publication No. 7-
78503, JP-A-10-73592, and JP-A-8-240591).

Specifically, a specific binding substance labeled with a labeling material that can be arbitrarily detected by the naked eye or an optical method is allowed to undergo a specific binding reaction with an analyte. Then, the specific binding substance that has undergone a specific binding reaction with the analyte is bound to the binding material immobilized on the matrix, and the presence or absence of the analyte in the sample is finally determined according to the amount of the label immobilized on the matrix. To analyze. In this chromatographic analysis method, since the surface area of the carrier in the matrix is large, it is possible to insolubilize a large amount of specific binding substances, and the frequency of collisions between reactive molecules that can cause specific binding reactions is higher than that in the case of reactions in liquid phase. Since it is large, it is advantageous in terms of measurement sensitivity and measurement time.

[0005]

However, the conventional chromatographic analysis method has a problem called prozone phenomenon. The prozone phenomenon is a phenomenon in which the concentration of the analyte cannot be uniquely determined with respect to the signal intensity derived from the specific binding reaction. Specifically, when the analyte is excessively present in the sample, it is labeled with the analyte (specifically binding substance-specific binding substance-analyte binding substance) that specifically binds to the labeled specific binding substance. Thus, the specific binding substance and the simple substance of the analyte not specifically bound are present on the matrix.

[0006] Then, the analyte specifically bound to the labeled specific binding substance and the simple substance of the analyte compete with the binding material immobilized on the matrix to cause a specific binding reaction. Therefore, the analyte alone may be bound to the binding material, and the analyte specifically bound to the labeled specific binding substance may flow out due to the osmotic force due to the capillary phenomenon. As a result, the amount of the labeled specific binding substance bound to the binding material decreases, and the signal intensity based on the specific binding reaction of the specific binding substance does not correspond to the amount of the analyte contained in the sample.

Therefore, when the amount of the analyte in the sample is excessively large, the amount of the analyte specified by the labeled amount is measured to be abnormally small, and the presence or absence of the analyte in the sample is detected. There are cases in which the On the other hand, in order to accurately measure the analyte without being affected by the prozone phenomenon, by measuring the signal intensity multiple times using diluted samples of different concentrations, the analyte can be highly concentrated. A method of confirming its existence has been proposed. However, it is necessary to use a plurality of reaction vessels, which complicates the measurement process and causes an increase in size and complexity of the analyzer.

Therefore, the present invention provides a specific binding analysis method that is not affected by the prozone phenomenon and can accurately and quantitatively measure an analyte in a sample even with a small and simple measuring device. An object is to provide a specific binding analyzer. Among them, in the present invention, the labeling substance-first specific binding substance-analyte binding substance is immobilized on the first detection unit on which the second specific binding substance is immobilized, and the first detection unit is used. Since the quantification is performed based on the signal intensity of, the measurement accuracy is high, especially in the low concentration range.

[0009]

According to the present invention, (A) a sample containing an analyte is brought into contact with a first specific binding substance labeled with a detectable labeling material, and the first specific binding substance is added to the sample. A step of binding an analyte to obtain a conjugate of a labeling material, a first specific binding substance, and an analyte; (B) contacting the sample with a first detection unit on which a second specific binding substance is immobilized. ,
An excess of the analyte that did not bind to the first specific binding substance, and a step of binding the binder to the second specific binding substance and immobilizing it on the first detection unit, (C) the analyte The sample is brought into contact with a second detection unit on which the same or similar substance is immobilized, and the first specific binding substance in the conjugate is bound to the substance to immobilize the conjugate on the second detection unit. And (D) measuring the intensities of the signal 1 and the signal 2 derived from the labeling material obtained in the first detection unit and the second detection unit, respectively, and (E) the signal 1 and the signal 2 The present invention relates to a specific binding analysis method, which comprises the step of qualitatively or quantitatively determining the concentration of an analyte in the sample based on the intensity.

In the step (E), the step (D)
When two different concentrations 1 and 2 are introduced in specifying the concentration of the analyte based on the intensity of the signal 1 obtained in step 1, the concentration 1 and the concentration based on the intensity of the signal 2 It is preferable to determine which of the two is true to determine the concentration of the analyte. It is preferable to determine the prozone phenomenon based on the intensity of the signal 2 obtained in the step (D).

The sample after step (A) is brought into contact with the first detection part on which the second specific binding substance is immobilized, and an excessive amount of the analyte not bound to the first specific binding substance, and the binding. The step (B) of binding the body to the second specific binding substance and immobilizing it on the first detection unit is performed, and then the step (B) is performed on the second detection unit on which the same or similar substance as the analyte is immobilized. It is preferable to perform the step (C) of contacting the sample having passed through B), binding the first specific binding substance in the binding substance to the substance, and fixing the binding substance to the second detection unit.

Further, the sample after the step (A) is brought into contact with a second detection part on which a substance which is the same as or similar to the substance to be analyzed is fixed, and the first specific binding substance in the conjugate is treated as described above. The step (C) of binding the substance to the substance to fix the conjugate to the second detection unit is performed, and then the first detection unit having the second specific binding substance fixed thereto is contacted with the sample subjected to the process (C). And the step (B) of binding the excess analyte not bound to the first specific binding substance and the binding substance to the second specific binding substance and immobilizing the bound substance on the first detection unit. it can.

In addition, the step (A) is performed in advance, and the first
And the second detection unit are provided on a sheet chromatograph matrix, and the sample after step (A) is dropped on the upstream side of the first detection unit and the second detection unit to obtain the matrix. It is also preferable to perform the steps (B) to (E) by moving the sample to the first detection section and the second detection section by the osmotic force due to the capillary phenomenon inside the sample.

Further, a holding unit for holding the first specific binding substance labeled with a detectable labeling material, a first detection unit and a second detection unit are provided on a sheet chromatograph matrix, and the sample is provided. Is dropped onto the holding part or the upstream side thereof, and the sample is moved to the holding part, the first detection part and the second detection part by the osmotic force of the capillary phenomenon in the matrix, and the steps (A) to ( It is also preferable to carry out E).

It is preferable that the signal derived from the labeling material is a signal showing coloration, fluorescence or luminescence. At least one of the first specific binding substance and the second specific binding substance is preferably an antibody. The labeling material is preferably metal sol, dye sol or particles containing a fluorescent substance, or colored latex particles.

In the step (D), it is preferable that the signal 1 and the signal 2 are continuously measured in this order along the moving direction of the analyte. Step (D)
In step (E), a corresponding curve showing the relationship between the continuously measured signals 1 and 2 and the position in the moving direction of the analyte is created, and in step (E), a qualitative or quantitative determination is made based on the corresponding curve. It is preferable to carry out. In the step (E), the quantification of the analyte is performed based on the quantitative information obtained from the sample of which the concentration of the analyte is known and the signal 1 and the signal 2 obtained in the step (D). Is preferred.

Furthermore, the present invention provides a sheet-like chromatograph matrix, a first detection unit provided on the matrix and having a specific binding substance immobilized thereon, and a substance provided on the matrix which is the same as or similar to the analyte. And a second detection unit having immobilized thereon, the sample containing the analyte and the specific binding substance is moved to the first detection unit and the second detection unit by the osmotic force due to the capillary phenomenon to perform the specific binding reaction. Provided is a specific binding analysis device characterized by qualitatively or quantifying the analyte by detecting a signal generated and caused by the specific binding reaction.

Further, the device comprises a holding portion provided on the upstream side of the first detection portion on the matrix, which holds a specific binding substance labeled with a detectable labeling material. However, it is preferable to move the sample from the holding unit to the first detection unit and the second detection unit by the osmotic force due to the capillary phenomenon.

In addition to the holding part, the device further comprises a sample spotting part provided on the matrix on the upstream side of the holding part, wherein the sample is spotted by the osmotic force of the capillary phenomenon. It is preferable to move from the attachment part to the holding part, the first detection part and the second detection part.

The device does not have the holding unit, but has a sample spotting unit provided on the upstream side of the first detection unit on the matrix, and the sample is applied to the sample by an osmotic force by a capillary phenomenon. It is also preferable to move from the spotting section to the first detection section and the second detection section.

[0021]

BEST MODE FOR CARRYING OUT THE INVENTION First, a specific binding analysis method according to the present invention will be described with reference to the drawings. FIG. 1 is a schematic perspective view of a test strip that is a specific binding analysis device that can be used in the specific binding analysis method of the present invention. The test strip 1 is a sheet-shaped test strip, and is, for example, a matrix 2 which is a region on the surface of a chromatographic test strip into which a sample containing an analyte can permeate and develop.
Have. The strip 1 is made of, for example, a porous carrier or a fine particle-filled carrier that allows a sample to be permeated and developed by a capillary phenomenon.

A sample spotting section 3, a holding section 4, a first detecting section 5 and a second detecting section 6 are arranged on the matrix 2. The sample spotting part 3 is an area provided in the matrix 2, and the sample is spotted on the area. Holding part 4
Is the sample spotting section 3, the first detecting section 5 and the second detecting section 6
The first specific binding substance labeled with a labeling material is provided in the region where the spotted sample flows in and is provided between the first and second binding substances. The first detection unit 5 is a region where the sample flows in via the holding unit 4, and the second specific binding substance is immobilized as a binding material. The second detection unit 6 is a region into which the sample that has passed through the holding unit 4 and then the first detection unit 5 flows, and the analyte or its analogue is immobilized.

Here, the sample used in the specific binding analysis method of the present invention is a liquid sample which is predicted to contain an analyte. For example, urine, serum, plasma, whole blood, saliva,
Tear fluid, cerebrospinal fluid, and secretions from the nipple and the like are included. Further, it may be a sample obtained by suspending or dissolving a solid, gel or sol such as mucus, body tissue or cells in a liquid such as a buffer, an extract or a solution.

Further, the analyte in the present invention may be any substance as long as it has a specific binding substance having a property of specifically binding to the analyte, for example, various proteins or poly-proteins functioning as an antibody or an antigen. Examples include peptides, glycoproteins, polysaccharides, complex glycolipids, nucleic acids, effector molecules, receptor molecules, enzymes and inhibitors. More specifically, α-fetoprotein, carcinoembryonic antigen (CEA), tumor markers such as CA125 and CA19-9, various proteins such as β2-microglobulin (β2m) and ferritin, glycoproteins or complex glycolipids, estradiol. (E2), estriol (E3), human chorionic sex stimulating hormone (hCG), luteinizing hormone (LH), human placental lactogen (hPL) and other hormones, HBs antigen, HBs antibody, HBc antigen, HBc
Antibodies, HCV antibodies, HIV-related various virus-related antigens and virus-related antibodies, allergens and corresponding IgE antibodies, narcotic drugs, medicinal drugs and their metabolites, nucleic acids of virus- and tumor-related polynucleotide sequences And so on.

The specific binding substance in the present invention may be any substance as long as it specifically binds to the analyte.
Examples include antibodies, antigens, glycoproteins, polysaccharides, complex glycolipids, nucleic acids, effector molecules, receptor molecules, enzymes, inhibitors and the like. In the present invention, the first specific binding substance labeled with a labeling material and the second specific binding substance are used, and the labeled first specific binding substance and the second specific binding substance are analyzed. It is preferable that they can be bound via an entity. From the viewpoint of high specificity, it is desirable that at least one of the first specific binding substance and the second specific binding substance is an antibody. Further, a monoclonal antibody is preferable.

The first specific binding substance and the second specific binding substance do not necessarily have to be the same. Further, when the analyte does not have a plurality of identical epitopes, it is preferable that the first specific binding substance and the second specific binding substance have specificities for different epitopes. However, if the analyte has a plurality of identical epitopes, the same specific binding substance may be used.

The substance to be immobilized on the second detection section 6 may be the analyte itself or a similar substance that behaves similarly to the analyte. That is, any substance that binds to the first specific binding substance labeled with the labeling material may be used. For example, a substance having the same epitope as the epitope of the analyte that binds to the first specific binding substance may be used.

The labeling material used in the present invention is a substance whose presence can be arbitrarily detected. For example, not only a direct label that is visible to the naked eye in the natural state, or a direct label that is easily visible by using an optical filter or a method that stimulates fluorescence by applying a stimulus such as ultraviolet light, is used. It also includes an indirect label capable of detecting a visible signal by adding a different developing reagent.

Direct labels include dye sol, metal sol,
Examples include fine colored particles such as colored latex particles, and particles containing a fluorescent substance. Indirect labels include enzymes such as alkaline phosphatase and horseradish peroxidase. The direct label is preferable to use because the analytical result can be obtained instantaneously because a detectable signal is generated without addition of another reagent, and since it is robust and stable.

In the following, urine is used as a sample, hCG is used as an analyte contained in the sample, and an anti-hCG monoclonal antibody capable of participating in a sandwich reaction with hCG for the first specific binding substance and the second specific binding substance. An embodiment of the present invention using gold colloid as a labeling material will be described. When colored particles such as gold colloid are used here, since the colored particles are fine, it is possible to concentrate the label in a small area or volume, and the first detection unit 5 and the second detection unit 6 are included. In, the signal derived from the reaction in which the colloidal gold as the labeling agent of the first specific binding substance contributes can be used to accurately perform the qualitative and / or quantitative determination of hCG.

First, a sample for quantitative analysis or qualitative analysis is spotted on the sample spotting section 3. The spotted sample reaches the holding section 4 while permeating and developing the matrix 2 from the sample spotting section 3 by the capillary phenomenon. That is, a sample containing an analyte is brought into contact with a holding unit that holds the first specific binding substance labeled with a detectable labeling material, and the analyte is bound to the first specific binding substance for labeling. A material-first specific binding substance-analyte binding substance is obtained (step (A)). As described above, the matrix 2 may be any place where the analyte and the specific binding substance are developed, and examples thereof include a porous carrier and a gel carrier. Here, the case of using nitrocellulose will be described.

Nitrocellulose is superior to other matrix materials, such as paper, because it has the ability to inherently bind proteins without prior sensitization. By directly applying a specific binding substance such as an antibody to nitrocellulose, the specific binding substance can be surely immobilized, and no chemical treatment that hinders the specific binding ability of the specific binding substance is required. For example, when the matrix material is paper, CNBr, carbonyldiimidazole,
It is necessary to carry out chemical bonding using tresyl chloride or the like.

Further, since nitrocellulose having pores of various sizes is available, it becomes easy to select a matrix material according to requirements such as sample flow rate. When a nitrocellulose sheet is used, it is desirable to increase the handling strength by laminating (lining) a sheet-like reinforcing material made of a material such as plastic on the back of this sheet. It can be easily manufactured by forming a thin film of nitrocellulose on the reinforcement.

After immobilizing the antibody on the detection part, it is preferable to block the matrix to reduce non-specific adsorption to the matrix. Blocking can be achieved by treatment with a protein (such as bovine serum albumin or milk protein), polyvinyl alcohol, ethanolamine, or a combination thereof.

The holding section 4 is provided with an anti-hCG monoclonal antibody which is a first specific binding substance having an immunological binding property to hCG which is an analyte. The anti-hCG monoclonal antibody is labeled with colloidal gold as a labeling material. Analyte h contained in the sample urine
The CG flows into the holding unit 4 and specifically binds to the first specific binding substance to obtain a conjugate of the labeling material-first specific binding substance-analyte. That is, the colloidal gold is attached to the hCG that is the analysis target through the anti-hCG monoclonal antibody.

Here, it is preferable that the first specific binding substance is provided in the holding portion 4 in a dry state. For example, a reagent containing the first specific binding substance is applied to the matrix after blocking and freeze-dried to support the first specific binding substance in a dry state on the holding unit 4. As a result, when the holding unit 4 is in a dry state, the first specific binding substance is held by the holding unit 4, and the liquid sample permeates and develops the matrix 2 to wet the matrix 2. The specific binding substance of can freely move on the matrix 2.

Therefore, the sample containing the above-mentioned bound substance in which the analyte and the first specific binding substance are bound to each other is permeated and developed in the matrix 2 while the first detection portion 5 and the first 2 into the detector 6. An anti-hCG monoclonal antibody, which is a second specific binding substance capable of binding to the analyte hCG, is substantially immobilized on the first detection unit 5. For example, when the matrix is made of nitrocellulose, the antibody can be physically and chemically adsorbed and immobilized on the matrix by applying a reagent containing the antibody to the matrix. This second specific binding substance, anti-hCG monoclonal antibody, does not move even in a wet state.
In the second detection unit 6, anti-hC which is the first specific binding substance
Analyte (hCG) that binds to G monoclonal antibody
Or the analogue thereof is substantially immobilized. This too does not move in the wet state.

The sample may be spotted on the sample spotting part after being reacted with the first specific binding substance outside the test reagent strip in advance. The first specific binding substance to be reacted with the sample outside the strip may be one which is not bound to the labeling material, and further, the labeled first primary binding substance in the matrix is used.
It may be bound to a labeling material that generates a signal different from that of the specific binding substance. When the labeled first specific binding substance and the sample are preliminarily reacted outside the strip, the holding unit 4 provided with the first specific binding substance may not be arranged in the matrix 2.

The analyte in the sample that has reached the first detection unit 5 specifically binds to the second specific binding substance. As a result, the analyte is fixed to the first detection unit 5 via the second specific binding substance. That is, the anti-hCG monoclonal antibody, which is the first specific binding substance labeled with colloidal gold, is the anti-hC which is the second specific binding substance immobilized on the first detection unit 5 via the analyte hCG.
It binds to the G monoclonal antibody. This makes the first detection part, to which the second specific binding substance is immobilized, contact the sample subjected to the step (A), and removes the excess analyte not bound to the first specific binding substance to the second detection part. It is a step (B) of binding to a specific binding substance and immobilizing it on the first detection unit.

Here, when hCG is present in excess in the sample, hCG not specifically bound to the anti-hCG monoclonal antibody labeled with gold colloid and anti-h with gold colloid.
The hCG specifically bound to the CG monoclonal antibody competes with the second specific binding substance in the first detection unit 5 in the specific binding reaction. As a result, the single hCG preferentially reacts with and binds to the specific binding substance immobilized on the first detection unit 5. Then, the amount of the anti-hCG monoclonal antibody labeled with the colloidal gold bound to the first detection unit 5 decreases, the signal intensity contributed by the colloidal gold decreases, and the signal detected by the first detection unit 5 decreases. The strength is hCG in the sample
No longer reflects the amount of. The phenomenon in which the signal intensity does not increase according to the amount of the analysis target due to the excess analysis target is thus referred to as the prozone phenomenon.

Among the anti-hCG monoclonal antibodies, which are the first specific binding substance labeled with the colloidal gold and have reached the second detection section 6, the first anti-hCG monoclonal antibody capable of further binding to the analyte hCG.
Anti-hCG monoclonal antibody which is a specific binding substance of
It will be specifically bound to the immobilized analyte hCG and immobilized on the second detection unit 6. That is, the anti-hCG monoclonal antibody that is the first specific binding substance labeled with gold colloid is immobilized on the second detection unit 6 through the analyte hCG. This is the step (B) in which the second detection part having the same or similar substance as the analyte is immobilized.
It is a step (C) of bringing the sample that has passed through the step of contacting, and binding the first specific binding substance in the binding substance to the substance to fix the binding substance to the second detection unit.

Here, the sample is the first detector 5 and the second detector.
It is preferable that the sample be developed so that it continues to flow even if it passes over the detection section 6 of. For that purpose, a sufficient amount of the sample is spotted on the sample spotting unit 3, and in the first detection unit 5 and the second detection unit 6, an extra labeled first specific binding substance that does not participate in the binding reaction is detected. , And is washed away from the first detection unit 5 and the second detection unit 6 by the flow of the sample that continues beyond the detection unit. For this purpose, an absorption part may be provided at the end of the matrix 2 where the sample is developed. Any material may be used as the material of the absorption part as long as it has sufficient absorption capacity to wash away the sample other than the analysis target, and examples thereof include glass fiber filter paper GA200 (manufactured by Toyo Corporation).

When the absorption section is provided in this way, unreacted substances are washed away with the flow of the sample, so that after the specific binding reaction,
The signal derived from the specific binding reaction can be detected by the first detection unit 5 and the second detection unit 6 without performing the separation operation of the unreacted material. Therefore, in the specific binding analysis method of the present invention, the measurement of the intensity of the signal obtained from the specific binding reaction between the analyte and the specific binding substance is performed at any place in the place where the specific binding reaction is performed. Is possible, but
The distribution state of the signal intensity is detected in the first detection unit 5 and the second detection unit 6, or in the region including the first detection unit 5 and the second detection unit 6, and the distribution state of the signal intensity is detected in the sample. It is preferable to qualitatively or quantitatively analyze the analyte.

Then, the step (D) of measuring the intensities of the signal 1 and the signal 2 derived from the labeling material obtained in the first detector and the second detector, respectively, and the signal 1 and the signal 2 The step (E) of performing the qualitative or quantitative determination by obtaining the concentration of the analyte in the sample based on the intensity of. Quantitative information indicating the correspondence relationship between the concentration of the signal intensity distribution obtained by measurement using a sample containing the analyte of known concentration and the concentration is created in advance,
The concentration may be determined from the quantitative information and the distribution status of the signal intensity obtained by the analysis.

FIG. 2 is a schematic diagram showing the structure of an analyzer used together with the strip 1 in the specific binding analysis method according to the present invention. This analyzer includes, for example, a first electrode 11, a second electrode 12, a measurement start detector 10, an analyzer 9, a light source 7 and a photodetector 8. First electrode 11
The second electrode 12 and the second electrode 12 are electrodes for measuring the conductivity of the sample spotting part 3. The measurement start detector 10 is a device that detects that the sample has been spotted on the sample spotting unit 3 based on a change in conductivity, and supplies a detection signal to the analyzer 9. After receiving the detection signal, the analyzer 9 controls the stage 13 to scan the entire test reagent strip 1 in the Z direction when a predetermined time has elapsed. Furthermore, the light source 7 is controlled and the output signal of the photodetector 8 is analyzed. The light source 7 is a device that irradiates the first detection unit 5 and the second detection unit 6 with light. The photodetector 8 is a device that detects the reflected light from the first detection unit 5 and the second detection unit 6.

The operation of this analyzer will be described. First, before the sample spotting, the conductivity of the sample spotting part 3 in the dry state and the conductivity of the sample spotting part 3 in the wet state at the time of spotting the sample are measured. The information obtained thereby is input to the measurement start detector 10 as measurement start information. Then, a sample containing hCG is spotted on the sample spotting part 3, and when the sample spotting part 3 changes from a dry state to a wet state, the sample points monitored by the first electrode 11 and the second electrode 12 The conductivity of the attachment changes. The measurement start detector 10 can detect that the sample has been spotted on the sample spotting portion 3 by referring to the change in conductivity and the measurement start information.

It is preferable that the spotting of the sample on the sample spotting portion 3 is carried out after mounting the test reagent strip 1 in the analyzer, but the test reagent strip 1 spotted with the sample is mounted on the analyzer. May be. When the measurement start detector 10 detects spotting of the sample, the analyzer 9 controls the stage 13 to scan the test reagent strip 1 in the Z direction. At the same time, the analyzer 9 controls the light source 7 and the photodetector 8 to measure the change in the signal intensity accompanying the scanning, and obtains the chromatograms shown in FIGS. 3 and 4. The light source 7 irradiates a region including the first detection unit 5 and the second detection unit 6 with light having a predetermined wavelength (for example, 650 nm), and the photodetector 8 detects the reflected light. As the measurement wavelength, a wavelength suitable for coloring the sample or the labeling material in the first detection unit 5 and the second detection unit 6 may be selected.

Here, the signal may be any signal that can be generated and detected by a reaction in which the labeling material contributes, and for example, fluorescence that can be measured by a fluorophotometer, luminescence that can be measured by a luminescence photometer, or 1 detection unit 5 and second detection unit 6
It is also possible to use a color that can be visually determined and measured with a color difference meter. In this case, the light reflected by the first detection unit 5 and the second detection unit 6, the first detection unit 5 and the second reflected light
The intensity of fluorescence or luminescence generated in the detection unit 6 will be detected. The detection of this signal is continuously performed while the positional relationship between the test reagent strip 1 and the light source 7 and the photodetector 8 is relatively changed in parallel to the penetration direction of the sample, that is, in the Z direction. Either the test reagent strip 1 or the light source 7 and the photodetector 8 may be moved in parallel with the penetration direction of the sample, or both may be moved.

The photodetector 8 sends this signal to the analyzer 9. The analyzer 9 measures the signal intensity by analyzing the signal from the photodetector 8. The analyzer 9 creates a corresponding curve consisting of the intensity of the measured signal and the scanning distance, that is, a chromatogram, and analyzes it. 3 and 4
[Fig. 4] is a schematic diagram of corresponding curves (chromatograms) drawn by analyzing distribution states of signal intensities near the first detection unit 5 and the second detection unit 6, respectively. This chromatogram is
It is drawn by irradiating the matrix 2 with light from the light source 7 while scanning the test reagent strip 1 or the light source 7, and analyzing the reflected light detected by the photodetector 8 with the analyzer 9.
In this chromatogram, the vertical axis represents the signal intensity in the vicinity of the first detection unit 5 and the second detection unit 6, and the horizontal axis represents the region of the detection unit (scanning distance from the sample spotting unit 3). .

The height H of this chromatogram is measured as the intensity of the signal based on the specific binding reaction. Here, the area S of this chromatogram may be used as the intensity of the signal based on the specific binding reaction. The heights of the chromatograms of the first detection unit 5 and the second detection unit 6 are set to Hs and Hr, respectively.
And the areas of the chromatograms of the first detection unit 5 and the second detection unit 6 are shown as Ss and Sr, respectively.
In addition, here, the test reagent strip 1 or the light source 7 is scanned to continuously measure the reflected light in the region including the first detection unit 5 and the second detection unit 6 to obtain a chromatogram. For example, the first detection unit 5 and the second detection unit 6 are sandwiched between the upstream and near the spotting unit 3 and the downstream signal intensity on the opposite side is connected by a straight line to form the first detection unit 5 and the second detection unit. The height obtained by subtracting the height of the straight line from the height of the maximum value of the chromatogram in Part 6 shall be the height H of the chromatogram, and the area of the area enclosed by the chromatogram and the straight line should be the chromatogram. By setting the area S of the gram, it is possible to obtain the signal intensity based on the specific binding reaction in which the signal intensity derived from the background is subtracted from the signal intensity in the first detection unit 5 and the second detection unit 6. Even if the sample is colored such as whole blood, the analyte in the sample can be qualitatively or quantified without being affected by the background and contaminants.

At this time, the signal intensity at any place on the matrix 2 other than the first detection unit 5 and the second detection unit 6 may be intensity derived from the background,
For example, the minimum value of the signal intensities in the vicinity of the first detection unit 5 and the second detection unit 6 may be selected. Background is the behavior exhibited by a sample that does not contain the analyte. For example, the first detection unit 5 and the second
Non-specific adsorption of a specific binding substance or the like in the detection unit 6 or coloration of the sample itself when measuring a signal due to coloration influences the background, thus lowering the measurement accuracy. And, the contaminant is a substance other than the analyte contained in the sample, but the contaminant that is a problem in the specific binding analysis method is
In the binding reaction with the specific binding substance, a substance that behaves similarly to the analyte (for example, a structurally related substance of the analyte), or by binding with the analyte, the analyte and the specific binding substance become Substances that interfere with the specific binding reaction of. The analyzer 9 has quantitative information in a memory (not shown) and has analysis values Hs and Hr or Ss and S.
By referring to r and this quantitative information, the concentration of the analyte is specified. Hereinafter, the specific binding analysis method according to the present invention will be described based on Examples performed using the strip shown in FIG. 1 and the analyzer shown in FIG. 2, but the present invention is not limited to these. .

[0052]

EXAMPLES << Examples >> The specific binding analysis method of the present invention was performed using the strip 1 shown in FIG. 1 and the analyzer shown in FIG. Here, the analyzer 9 specified the concentration of the analysis target from Hs and Hr. 650n in the first detector 5
A graph showing the relationship between the signal intensity Hs and the hCG concentration when light of m wavelengths is irradiated is shown in FIG. hCG was added to the control urine for quality control, where the hCG concentration was virtually zero.
Was added as a sample. The hCG concentration of each sample was 0 (IU / L), 10 (IU / L), 30 (IU / L).
/ L), 50 (IU / L), 100 (IU / L), 30
0 (IU / L) 500 (IU / L), 1000 (IU / L
L), 1500 (IU / L), 2000 (IU / L),
3000 (IU / L), 5000 (IU / L), 750
It was set to 0 (IU / L) or 10000 (IU / L), and measurement was performed 2 or 3 times at each concentration.

As can be seen from FIG. 5, when the hCG concentration in the sample is low, most of the analytes bind to the labeled first specific binding substance and are immobilized on the first detection part 5. The second specific binding substance was bound. Therefore,
The signal intensity Hs derived from the reaction contributed by the labeling material detected by the first detection unit 5 increased as the concentration increased. If the hCG concentration in the sample is high, the labeled first
Since the analyte that is not specifically bound to the specific binding substance is present in the reaction system in excess, the analyte that specifically binds to the labeled first specific binding substance and the labeled first specific binding substance The labeling material detected by the first detection unit 5 as a result of the competition with the analyte that is not specifically bound with and the specific binding reaction with the second specific binding substance immobilized on the first detection unit 5. The signal intensity Hs derived from the reaction contributed by H.sub.2 decreased as the concentration increased. By using the dotted line in FIG. 5 as a calibration curve, the concentration of the analyte could be specified. That is, when the signal intensity Hs on the vertical axis shows a certain value, the horizontal axis of the dotted line showing this value corresponds to the density. For example, when the signal strength Hs is 5.0, it was possible to specify about 1200 (IU / L).

However, here, there are cases where the analyzer 9 has a plurality of concentrations specified when the dotted line in FIG. 5 is referred to as a calibration curve, and the concentration of the analyte with respect to the signal intensity cannot be uniquely determined. It was hCG concentration is 6000 (IU /
L) or more, the signal strength Hs is 10
At that time, the hCG concentration is about 3500 (IU / L) or about 7
Either 600 (IU / L) was indistinguishable. When the concentration of the analyte with respect to such signal intensity Hs is not uniquely determined, the second detection unit 6 is operated as follows.
It was possible to simultaneously determine the signal intensity Hr from the chromatogram and analyze the Hr and Hs simultaneously to specify the concentration.

FIG. 6 is a graph showing the relationship between the signal intensity Hr and the hCG concentration when the second detector 6 is irradiated with light having a wavelength of 650 nm. As a sample, hCG was added to control urine for quality control in which the hCG concentration was substantially zero. HCG concentration of each sample is 0
(IU / L), 10 (IU / L), 30 (IU / L),
50 (IU / L), 100 (IU / L), 300 (IU
/ L) 500 (IU / L), 1000 (IU / L), 1
500 (IU / L), 2000 (IU / L), 3000
(IU / L), 5000 (IU / L), 7500 (IU
/ L) or 10000 (IU / L), 2 at each concentration
Or, it was measured three times.

As can be seen from FIG. 6, since the labeled first specific binding substance which is not bound to the analyte binds to the analyte or the analogue immobilized on the second detection section 6, Signal intensity Hr derived from the reaction contributed by the labeling material
Decreased with increasing concentration. That is, when the labeled first specific binding substance that is not bound to the analyte is present due to a decrease in concentration, these are identified as the analyte or the analogue immobilized on the second detection unit 6. Due to the binding, a signal intensity Hr derived from the reaction contributed by the labeling material was generated. However, when there is a high concentration and a large excess of the analyte, substantially all of the labeled first specific binding substance is bound to the analyte, and therefore reaches the second detection unit 6. However, since it could not bind to the immobilized analyte or the like and was washed away, the signal intensity Hr derived from the reaction contributed by the labeling material was not substantially generated.

More precisely, when the first specific binding substance has a plurality of binding sites as in the case of an antibody (two sites in the case of an IgG antibody) as in the present embodiment, each of these binding sites is more strictly defined. The binding state of was reflected in the signal intensity. That is, the first binding site where all binding sites are filled with analyte.
Since the specific binding substance (1) has a very low probability of being newly bound to the analyte, the probability of binding to the analyte immobilized on the second detection unit 6 was also extremely low. However, if at least one binding site capable of binding to the analyte remains, the probability of binding to the analyte immobilized on the second detection unit 6 depends on the ratio of the remaining binding sites. Increased. In any case, the signal increase Hr shown by the dotted line in FIG. 6 decreased as the analyte concentration increased.

By using the dotted line in FIG. 6 and the dotted line in FIG. 5 as quantitative information, the concentration of the analyte can be specified as follows. As described above, there are cases where there are a plurality of concentrations specified when only the dotted line in FIG. 5 is referred to, and the concentration of the analyte with respect to the signal intensity cannot be uniquely determined. For example, when the hCG concentration may be 6000 (IU / L) or more, when the signal strength Hs is 10, the hCG concentration is about 3500 (IU / L) or about 7600 (IU / L).
It was not possible to distinguish which one was. However, the concentration could be specified by further referring to the dotted line in FIG. That is, if the signal strength Hr of FIG. 6 is 1.2, the density is about 3500 (IU / L) from the dotted line, and if the signal strength Hr is 0.45, the density is about 76 from the dotted line.
It was confirmed to be 00 (IU / L). As described above, even if the concentration of the analyte is not uniquely determined only from the signal intensity Hs in FIG. 5, the signal intensity Hr is obtained from the chromatogram of the second detection unit 6, and both Hr and Hs are obtained. It was possible to specify the concentration by analyzing in combination with.

Here, it was possible to specify the concentration only from Hr in FIG. 6, but since the slope of the dotted line with respect to the concentration in the low concentration region is small, the accuracy in specifying the concentration from the measured Hr is high. Was lower than when Hs in FIG. 5 was specified. Therefore, it was preferable to utilize Hr only to determine which of the two concentrations derived from Hs is true, and to utilize the concentration only from Hs. Also, for simplicity, H in FIG.
It was also possible to specify the concentration from Hs in FIG. 5 only when r was a predetermined value or more. For example, in the case of this embodiment,
Only when Hr is 0.6 or more, the concentration is specified from Hs in FIG. 5, and when Hr is 0.6 or less, it is determined that the prozone phenomenon is occurring, and the concentration is 6000 (IU / L) or more. However, it was easy if the concentration was not specified. That is, Hr was effective for simplification if it was used only for pro zone determination.

Further, in the present embodiment, the second detection section 6 is arranged on the downstream side of the first detection section 5 in the direction (Z direction) in which the liquid sample develops. Thus, the accuracy of quantification was improved. Because when a liquid passes through a matrix in which proteins such as antibodies are immobilized,
The flow was turbulent, and the phenomenon that the liquid sample flowed only to a specific part of the matrix was apt to occur, resulting in uneven coupling degree at the downstream detection unit. This reduced reproducibility. The obstruction of the flow of the liquid sample by the protein immobilized on the matrix was caused by spatial obstruction and uneven hydrophilicity due to uneven immobilization of the density. Since it was possible to improve the quantification accuracy by ensuring the reproducibility of the signal generated in the first detection unit 5 that specifies the concentration, that is, quantified, it is preferable to arrange the quantification detection unit on the upstream side. won.

As described above, by analyzing both Hs and Hr as quantitative information, the concentration could be specified without being affected by the so-called prozone phenomenon.
In addition, in the present embodiment, an example in which Hs and Hr are utilized is shown, but the same effect is obtained even when Ss and Sr shown in FIGS. 3 and 4 are utilized in the same manner. That is, it was possible to quantify with Ss and to carry out prozone determination with Sr. As described above, according to the specific binding analysis method according to the present example, even when the prozone phenomenon occurs, the qualitative or quantitative analysis of the analyte in the sample can be performed easily and quickly. It was

[0062]

As described above, according to the present invention, the correspondence between the analysis value of the signal intensity based on the specific binding reaction of the specific binding substances generated at the two detection portions and the concentration of the analyte is shown. Since the amount of the analyte in the sample is specified with reference to the quantitative information, the accurate qualitative or quantitative analysis of the analyte in the sample can be performed easily and quickly without being affected by the prozone phenomenon. You can

[Brief description of drawings]

FIG. 1 is a schematic perspective view of an embodiment of a strip for a test reagent used in the specific binding analysis method according to the present invention.

FIG. 2 is a schematic diagram showing the configuration of an embodiment of an analyzer used in the specific binding analysis method according to the present invention.

FIG. 3 is a schematic diagram showing a distribution state of signal intensities of the first detection unit 5 of the strip 1 detected by the analyzer shown in FIG.

FIG. 4 is a schematic diagram showing the distribution of signal intensities of the second detector 6 of the strip 1 detected by the analyzer shown in FIG.

FIG. 5 is a graph showing the relationship between the hCG concentration and the signal intensity Hs when the first detector 5 is irradiated with light having a wavelength of 650 nm.

FIG. 6 is a graph showing the relationship between the hCG concentration and the signal intensity Hr when the second detector 6 is irradiated with light having a wavelength of 650 nm.

[Explanation of symbols]

1 strip 2 matrix 3 Sample spotting part 4 holding part 5 First detector 6 Second detector 7 light source 8 Photodetector 9 analyzer 10 Measurement start detector 11 First electrode 12 Second electrode

   ─────────────────────────────────────────────────── ─── Continued front page    (72) Inventor Fumihisa Kitawaki             1006 Kadoma, Kadoma-shi, Osaka Matsushita Electric             Sangyo Co., Ltd. (72) Inventor Noriko Gonjo             1006 Kadoma, Kadoma-shi, Osaka Matsushita Electric             Sangyo Co., Ltd.

Claims (17)

[Claims] 1. (A) A sample containing an analyte is brought into contact with a first specific binding substance labeled with a detectable labeling material, and the analyte is bound to the first specific binding substance for labeling. A step of obtaining a material-first specific binding substance-analyte binding substance, (B) contacting the sample with a first detection part on which a second specific binding substance is immobilized, and Excessive unbound analyte and the step of binding the conjugate to a second specific binding substance and immobilizing it on the first detection part, (C) immobilizing the same or similar substance as the analyte Contacting the sample to the second detector, and binding the first specific binding substance in the binder to the substance to fix the binder to the second detector (D) Signal 1 derived from the labeling material obtained in the first detection section and the second detection section And (2) measuring the intensities of the signals 2 and (E) qualitatively or quantitatively determining the concentration of the analyte in the sample based on the intensities of the signals 1 and 2. Specific binding analysis method. 2. In the step (E), when the concentration of the analyte is specified based on the intensity of the signal 1 obtained in the step (D), two different concentrations 1 and 2 are detected. 2. The specific binding analysis method according to claim 1, wherein when guided, the concentration of the analyte is determined by determining which of concentration 1 and concentration 2 is true based on the intensity of the signal 2. . 3. The specific binding analysis method according to claim 1, wherein the prozone phenomenon is determined based on the intensity of the signal 2 obtained in the step (D). 4. An excess analyte that has not bound to the first specific binding substance, by bringing the sample after step (A) into contact with the first detection part on which the second specific binding substance is immobilized, and The step (B) of binding the conjugate to a second specific binding substance and immobilizing it on the first detection part is performed, and then, on the second detection part on which the same or similar substance as the analyte is immobilized,
Performing the step (C) of bringing the sample subjected to the step (B) into contact, binding the first specific binding substance in the binder to the substance, and fixing the binder to the second detection unit. The method for analyzing specific binding according to claim 1, which is characterized in that: 5. The sample which has undergone step (A) is contacted with a second detection part to which a substance that is the same as or similar to the analyte is immobilized, and the first specific binding substance in the conjugate is added to the second detection part. The step (C) of binding the substance to the substance to fix the conjugate to the second detection unit is performed, and then the first detection unit having the second specific binding substance fixed thereto is contacted with the sample subjected to the process (C). And performing the step (B) of binding the excess analyte not bound to the first specific binding substance and the binding substance to the second specific binding substance and immobilizing it on the first detection unit. The method for analyzing specific binding according to claim 1, which is characterized in that: 6. The step (A) is carried out in advance, the first detection part and the second detection part are provided on a sheet chromatograph matrix, and the sample which has undergone the step (A) is treated as the first detection part and the second detection part. Dropping on the upstream side of the second detection unit, the sample is moved to the first detection unit and the second detection unit by the osmotic force due to the capillary phenomenon in the matrix,
The method for analyzing specific binding according to claim 1, wherein steps (B) to (E) are performed. 7. A first labeled with a detectable label.
Of the specific binding substance, the first detection unit and the second detection unit are provided on the matrix for sheet chromatograph, and the sample is dropped on the holding unit or on the upstream side thereof, and in the matrix. The specific binding according to claim 1, wherein the sample is moved to the holding unit, the first detection unit and the second detection unit by an osmotic force due to a capillary phenomenon, and steps (A) to (E) are performed. Analysis method. 8. The specific binding analysis method according to claim 1, wherein the signal derived from the labeling material is a signal showing coloration, fluorescence, or luminescence. 9. The specific binding analysis method according to claim 1, wherein at least one of the first specific binding substance and the second specific binding substance is an antibody. 10. The specific binding analysis method according to claim 1, wherein the labeling material is a metal sol, particles containing a dye sol or a fluorescent substance, or colored latex particles. 11. The signal 1 in the step (D).
2. The specific binding analysis method according to claim 1, wherein the signal 2 and the signal 2 are continuously measured in this order along the moving direction of the analyte. 12. In the step (D), a corresponding curve showing the relationship between the continuously measured signals 1 and 2 and the position of the analysis target in the moving direction is created, and in the step (E), The specific binding analysis method according to claim 11, wherein qualitative or quantitative determination is performed based on the corresponding curve. 13. The analysis target in the step (E) is based on the quantitative information obtained from a sample having a known concentration of the analysis target and the signal 1 and the signal 2 obtained in the step (D). The method for quantifying specific binding according to claim 1, wherein the amount of the substance is quantified. 14. A sheet-like chromatograph matrix, a first detection unit provided on the matrix and having a specific binding substance immobilized thereon, and a substance provided on the matrix, which is the same as or similar to an analyte, is immobilized. A second detection unit, wherein a sample containing an analyte and a specific binding substance is moved to the first detection unit and the second detection unit by an osmotic force due to a capillary phenomenon to cause a specific binding reaction, A specific binding analysis device, characterized in that the analyte is qualitatively or quantified by detecting a signal derived from the specific binding reaction. 15. A holding part, which is provided on the upstream side of the first detection part on the matrix and holds a specific binding substance labeled with a detectable labeling material, 15. The specific binding analysis device according to claim 14, wherein is moved from the holding part to the first detection part and the second detection part by an osmotic force due to a capillary phenomenon. 16. A sample spotting unit provided on the upstream side of the holding unit on the matrix, wherein the sample is spotted from the holding unit to the first detection by an osmotic force of the capillary phenomenon of the sample. 16. The specific binding analysis device according to claim 15, which is moved to the second section and the second detection section. 17. A sample spotting unit provided on the upstream side of the first detection unit on the matrix, wherein the sample is spotted from the sample spotting unit by the osmotic force of the capillary phenomenon. The specific binding analysis device according to claim 14, which is moved to the second detection unit.