JP3654591B2 - Specific binding analysis method and specific binding analysis device - Google Patents

Description

[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a specific binding analysis method for performing qualitative analysis or quantitative analysis of an analyte in a sample.
[0002]
[Prior art]
In recent years, with the expansion of medical care at home and in the community, and the increase in urgent clinical tests, development of specific binding analysis methods that enable quick, simple and accurate measurement even if you are not a clinical laboratory specialist Has come to be desired in abundance.
As such a specific binding analysis method, many methods 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 are known. Because of its high specificity, it is widely used in a wide range of fields including clinical tests.
[0003]
Furthermore, in a chromatographic analysis method which is a kind of immunoassay, for example, a liquid sample is brought into contact with a matrix comprising a porous carrier or a fine particle-packed carrier in which a specific binding substance is insolubilized or immobilized, and the liquid sample is brought into contact with the matrix. The presence or absence of the analyte in the sample is analyzed by utilizing the phenomenon of flowing out due to the osmotic force due to the capillary phenomenon (for example, Japanese Patent No. 2504923, Japanese Patent No. 2667793, Japanese Patent Publication No. 7-78503) JP-A-10-73592 and JP-A-8-240591).
[0004]
Specifically, a specific binding substance labeled with a labeling material that can be arbitrarily detected by the naked eye or an optical method is subjected to a specific binding reaction between the analyte. Then, the specific binding substance that has undergone specific binding reaction with the analyte is bound to the binding material immobilized on the matrix, and finally the presence or absence of the analyte in the sample is determined according to the amount of label immobilized on the matrix. Analyze.
In this chromatographic analysis method, since the surface area of the support in the matrix is large, a large amount of specific binding substances can be insolubilized, and the collision frequency between reaction molecules that can cause specific binding reactions is higher than that in the case of reactions in the liquid phase. Therefore, it is advantageous in terms of measurement sensitivity and measurement time.
[0005]
[Problems to be solved by the invention]
However, the conventional chromatographic analysis method has a problem called a 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 an analyte is excessively present in a sample, it is labeled with an analyte that is specifically bound to the labeled specific binding substance (labeled material-specific binding substance-analyte conjugate). The specific binding substance and the single analyte that did not specifically bind exist on the matrix.
[0006]
Then, the analyte specifically bound to the labeled specific binding substance and the analyte alone compete with the binding material immobilized on the matrix and undergo a specific binding reaction. For this reason, a single analyte may be bound to the binding material, and the analyte specifically bound to the labeled specific binding substance may flow out due to osmotic force due to capillary action. 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.
[0007]
Therefore, when the amount of the analyte in the sample is excessive, the amount of the analyte specified from the labeled amount is measured to be abnormally small, and the presence or absence of the analyte in the sample is accurately determined. There are cases that cannot be grasped.
On the other hand, in order to accurately measure the analyte without being affected by the prozone phenomenon, the analyte is increased in concentration by measuring the intensity of the signal multiple times using different diluted samples. A method for confirming the existence has been proposed. However, it is necessary to use a plurality of reaction vessels, resulting in a complicated measurement process and an increase in size and complexity of the analyzer.
[0008]
Therefore, the present invention provides a specific binding analysis method and a specific binding analysis that are not affected by the prozone phenomenon and can accurately and qualitatively and quantitatively measure an analyte in a sample even with a small and simple measuring device. An object is to provide an apparatus. In particular, the present invention fixes the conjugate of the labeling material-first specific binding substance-analyte to the first detection unit to which the second specific binding substance is fixed, and the first detection unit uses the first detection unit. Since the quantification is performed based on the intensity of the signal, the measurement accuracy is particularly high in the low concentration range.
[0009]
[Means for Solving the Problems]
In the present invention, (A) a sample containing an analyte is brought into contact with a first specific binding substance labeled with a detectable labeling substance, and the analyte is bound to the first specific binding substance to thereby label the substance. -First specific binding substance-a step of obtaining a conjugate of the analyte, (B) the sample is brought into contact with the first detection unit to which the second specific binding substance is immobilized, and bound to the first specific binding substance An excess analyte that was not, and a step of binding the conjugate to the second specific binding substance and immobilizing it on the first detection unit, (C) immobilizing the same or similar substance as the analyte A step of bringing the sample into contact with a second detection unit, binding the first specific binding substance in the conjugate to the substance, and fixing the conjugate to the second detection unit; The signal 1 derived from the labeling material obtained in the detection unit and the second detection unit And (E) qualitatively or quantitatively determining the concentration of the analyte in the sample based on the intensity of the signal 1 and the signal 2, respectively. To a specific binding analysis method.
[0010]
In the step (E), when specifying the concentration of the analyte based on the intensity of the signal 1 obtained in the step (D), when two different concentrations 1 and 2 are derived, It is preferable to determine which one of the concentration 1 and the concentration 2 is true based on the intensity of the signal 2 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).
[0011]
The first detection unit to which the second specific binding substance is immobilized is brought into contact with the sample that has undergone the step (A), and the excess analyte that has not bound to the first specific binding substance and the conjugate are converted into the first detection part. The step (B) of binding to the first specific binding substance by binding to the two specific binding substances is performed, and then the step (B) is applied to the second detection part to which the same or similar substance as the analyte is fixed. It is preferable to perform the step (C) of contacting the passed sample, binding the first specific binding substance in the conjugate to the substance, and fixing the conjugate to the second detection unit.
[0012]
In addition, the sample that has undergone the step (A) is brought into contact with a second detection unit in which a substance that is the same as or similar to the analyte is fixed, and the first specific binding substance in the conjugate is bound to the substance. Then, the step (C) of fixing the conjugate to the second detection unit is performed, and then the sample having undergone the step (C) is brought into contact with the first detection unit to which the second specific binding substance has been immobilized. It is also possible to perform the step (B) of binding the excess analyte to be bound to one specific binding substance and the conjugate to the second specific binding substance and immobilizing it on the first detection unit.
[0013]
In addition, the step (A) is performed in advance, the first detection unit and the second detection unit are provided on a sheet-like chromatographic matrix, and the sample that has undergone the step (A) is provided with the first detection unit and the second detection unit. The sample is dropped on the upstream side of the detection unit, and the sample is moved to the first detection unit and the second detection unit by osmotic force due to capillary action in the matrix, and steps (B) to (E) are performed. preferable.
[0014]
In addition, 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-like chromatographic matrix, and the sample is held by the holding unit. And the sample is moved to the holding unit, the first detection unit, and the second detection unit by osmotic force due to capillary action in the matrix, and steps (A) to (E) are performed. It is also preferable to do this.
[0015]
The signal derived from the labeling material is preferably a signal indicating coloration, fluorescence or luminescence.
It is preferable that at least one of the first specific binding substance and the second specific binding substance is an antibody.
The labeling material is preferably a metal sol, a dye sol or particles containing a fluorescent substance, or colored latex particles.
[0016]
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 analysis object.
In the step (D), a corresponding curve indicating the relationship between the signal 1 and the signal 2 continuously measured and the position in the moving direction of the analysis object is created, and in the step (E), based on the corresponding curve. Qualitative or quantitative determination is preferred.
In the step (E), the analyte is quantified based on the quantitative information obtained from the sample whose concentration of the analyte is known and the signals 1 and 2 obtained in the step (D). Is preferred.
[0017]
Furthermore, the present invention provides a sheet-like chromatographic matrix, a first detection unit provided on the matrix and fixed with a specific binding substance, and a substance provided on the matrix and the same or similar to the analyte. A second detection unit, and a sample containing an analyte and a specific binding substance is moved to the first detection unit and the second detection unit by osmotic force by capillary action to cause a specific binding reaction, Provided is a specific binding analysis device characterized in that the analyte is qualitatively or quantitatively detected by detecting a signal derived from the specific binding reaction.
[0018]
Further, the device includes a holding unit provided on the upstream side of the first detection unit on the matrix, the holding unit holding a specific binding substance labeled with a detectable labeling material, It is preferable to move the sample from the holding unit to the first detection unit and the second detection unit by osmotic force due to capillary action.
[0019]
In addition to the holding unit, the device includes a sample spotting unit provided on the upstream side of the holding unit on the matrix, and the sample is separated from the sample spotting unit by osmotic force due to capillary action. It is preferable to move to the holding unit, the first detection unit, and the second detection unit.
[0020]
The device does not have the holding unit, and includes a sample spotting unit provided on the upstream side of the first detection unit on the matrix, and the sample spotting unit is caused to penetrate the sample by osmotic force due to capillary action. It is also preferable to move the first detector to the first detector.
[0021]
DETAILED DESCRIPTION OF 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 which 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-like test piece, and has, for example, a matrix 2 on the surface of a chromatographic test piece, which is a region in which a sample containing an analyte can permeate and develop. The strip 1 is made of a porous carrier or a fine particle-packed carrier that can permeate and develop a sample by capillary action, for example.
[0022]
On the matrix 2, a sample spotting unit 3, a holding unit 4, a first detection unit 5 and a second detection unit 6 are arranged. The sample spotting unit 3 is an area provided in the matrix 2, and a sample is spotted thereon. The holding unit 4 is disposed between the sample spotting unit 3 and the first detection unit 5 and the second detection unit 6, and is an area into which the spotted sample flows, and is labeled with a labeling material. One specific binding substance is provided. The 1st detection part 5 is an area | region into which a sample flows in via the holding | maintenance part 4, and the 2nd specific binding substance is fix | immobilized as a binding material. The second detection unit 6 is a region into which the sample flows through the first detection unit 5 following the holding unit 4, and the analysis object or the like is immobilized thereon.
[0023]
Here, the sample used in the specific binding analysis method of the present invention is a liquid sample that is predicted to contain the analyte. For example, urine, serum, plasma, whole blood, saliva, tears, cerebrospinal fluid, and secretions from the nipple are included. Alternatively, 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, extract or lysate.
[0024]
In addition, the analyte in the present invention may be any substance as long as a specific binding substance having the property of specifically binding to the analyte is present, for example, various proteins, polypeptides, sugars that function as antibodies or antigens. Examples include proteins, 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), various hormones such as human chorionic gonadotropin (hCG), luteinizing hormone (LH), human placental lactogen (hPL), HBs antigen, HBs antibody, HBc antigen, HBc antibody Various virus-related antigens and virus-related antibodies such as HCV antibody and HIV antibody, various allergens and corresponding IgE antibodies, narcotic drugs, medical drugs and metabolites thereof, nucleic acids of virus and tumor-related polynucleotide sequences, etc. Is mentioned.
[0025]
The specific binding substance in the present invention may be any substance that specifically binds to the analyte, such as an antibody, an antigen, a glycoprotein, a polysaccharide, a complex glycolipid, a nucleic acid, an effector molecule, a receptor molecule, an enzyme, And inhibitors.
In the present invention, the first specific binding substance labeled with the labeling material and the second specific binding substance are used, and the first specific binding substance and the second specific binding substance labeled are analyzed. It is preferable that it can couple | bond together through a thing. In view of high specificity, at least one of the first specific binding substance and the second specific binding substance is preferably an antibody. Furthermore, a monoclonal antibody is preferable.
[0026]
Note that the first specific binding substance and the second specific binding substance are not necessarily required to be the same. Moreover, when the analysis object does not have two or more same epitopes, it is preferable to use the 1st specific binding substance and the 2nd specific binding substance which have specificity with respect to a different epitope, respectively. However, when the analyte has a plurality of the same epitope, the same specific binding substance may be used.
[0027]
In addition, the substance immobilized on the second detection unit 6 may be the analysis object itself, or may be a similar substance that exhibits the same behavior as the analysis object. 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 to be bound to the first specific binding substance may be used.
[0028]
The labeling material used in the present invention is a substance whose presence can be arbitrarily detected. For example, a direct label that is visible with the naked eye when in the natural state, or a direct label that is easily visible by using an optical filter or a method that promotes fluorescence by applying a stimulus such as ultraviolet light, as well as a substrate Indirect labels that can detect visible signals by adding a simple developing reagent are also included.
[0029]
Direct labels include finely colored particles such as dye sols, metal sols, colored latex particles, and particles containing fluorescent materials. Indirect labels include enzymes such as alkaline phosphatase and horseradish peroxidase. Since direct labeling generates a detectable signal without adding another reagent, it is possible to obtain an analysis result instantaneously, and it is better to use direct labeling because it is robust and stable.
[0030]
In the following, urine is labeled as a sample, hCG is labeled as an analyte contained in the sample, and an anti-hCG monoclonal antibody capable of participating in a sandwich reaction with hCG as a first specific binding substance and a second specific binding substance is labeled. An embodiment of the present invention using a gold colloid as a material will be described.
When colored particles such as gold colloid are used here, since the colored particles are fine, the label can be concentrated in a small area or volume, and the first detection unit 5 and the second detection unit 6 are used. Thus, hCG can be qualitatively and / or quantitatively accurately determined using a signal derived from a reaction contributed by colloidal gold, which is a labeling material for the first specific binding substance.
[0031]
First, a sample for quantitative analysis or qualitative analysis is spotted on the sample spotting unit 3. The spotted sample reaches the holding part 4 while penetrating the matrix 2 from the sample spotting part 3 by capillary action. 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 to label. A material-first specific binding substance-analyte conjugate 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 where nitrocellulose is used will be described.
[0032]
Nitrocellulose is superior to other matrix materials such as paper because it has the ability to bind to proteins inherently without prior sensitization. By directly applying a specific binding substance such as an antibody to nitrocellulose, the specific binding substance can be reliably fixed, and no chemical treatment is required to interfere with the specific binding ability of the specific binding substance. For example, when the matrix material is paper, it is necessary to perform chemical bonding using CNBr, carbonyl dimidazole, tresyl chloride or the like to immobilize the antibody.
[0033]
Further, since nitrocellulose having pores of various sizes can be obtained, it becomes easy to select a matrix material in accordance with requirements such as a sample flow rate. When a nitrocellulose sheet is used, it is desirable to increase the strength in handling by attaching (lining) a sheet-like reinforcing material made of a material such as plastic to the back of the sheet. This can be easily manufactured by forming a nitrocellulose thin film on the reinforcement.
[0034]
After the antibody is immobilized on the detection unit, it is preferable to block the matrix and reduce nonspecific 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.
[0035]
The holding unit 4 is provided with an anti-hCG monoclonal antibody which is a first specific binding substance having immunological binding characteristics with hCG which is an analysis object. The anti-hCG monoclonal antibody is labeled with a gold colloid as a labeling material.
The analyte hCG contained in the sample urine flows into the holding unit 4 and specifically binds to the first specific binding substance, whereby a conjugate of labeling material-first specific binding substance-analyte is obtained. . In other words, the colloidal gold is imparted to the hCG that is the analysis object via the anti-hCG monoclonal antibody.
[0036]
Here, the first specific binding substance is preferably provided in the holding unit 4 in a dry state. For example, a reagent containing a first specific binding substance is applied to the matrix after blocking, and a freeze-drying process is performed so that the first specific binding substance is supported on the holding unit 4 in a dry state. Thus, when the holding unit 4 is in a dry state, the first specific binding substance is held in the holding unit 4, and when the matrix 2 is wetted by the liquid sample permeating and developing the matrix 2, This specific binding substance can move freely on the matrix 2.
[0037]
Therefore, the sample including the conjugate having a state in which the analyte and the first specific binding substance are bound to each other is infiltrated and expanded through the matrix 2 while the first detection unit 5 and the second detection in the matrix 2 are detected. Flows into section 6.
An anti-hCG monoclonal antibody that is a second specific binding substance that can bind 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, the anti-hCG monoclonal antibody, does not migrate even when wet.
In the second detection unit 6, the analyte (hCG) or the like that binds to the anti-hCG monoclonal antibody that is the first specific binding substance is substantially immobilized. This also does not move in a wet state.
[0038]
Note that the sample may be spotted on the sample spotting portion after reacting 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 not be bound to the labeling material, and further, the labeling material generates a signal different from the labeled first specific binding substance in the matrix. It does not matter if it is combined with. When the labeled first specific binding substance and the sample are reacted in advance outside the strip, the holding unit 4 provided with the first specific binding substance does not have to be arranged in the matrix 2.
[0039]
The analyte in the sample that reaches the first detection unit 5 specifically binds to the second specific binding substance. As a result, the analysis object is fixed to the first detection unit 5 via the second specific binding substance. That is, the anti-hCG monoclonal antibody that is the first specific binding substance labeled with colloidal gold is the anti-hCG that is the second specific binding substance immobilized on the first detection unit 5 via the analyte hCG. Binds to a monoclonal antibody.
This is because the sample that has undergone the step (A) is brought into contact with the first detection unit to which the second specific binding substance is immobilized, and the excess analyte that has not bound to the first specific binding substance is removed from the second detection target. This is a step (B) of binding to the specific binding substance and fixing it to the first detection unit.
[0040]
Here, when the hCG in the sample is excessive, hCG not specifically bound to the anti-hCG monoclonal antibody labeled with the gold colloid and hCG specifically bound to the anti-hCG monoclonal antibody with the gold colloid are the first. In the specific binding reaction with the second specific binding substance in the detection unit 5.
As a result, a single hCG preferentially reacts 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 gold colloid bound to the first detection unit 5 decreases, the signal intensity contributed by the gold colloid decreases, and the signal detected by the first detection unit 5 The intensity no longer reflects the amount of hCG in the sample. As described above, the phenomenon that the signal intensity does not increase according to the amount of the analysis object due to the excessive analysis object is referred to as the prozone phenomenon as described above.
[0041]
Among the anti-hCG monoclonal antibodies that are the first specific binding substances labeled with the colloidal gold that has reached the second detection unit 6, the anti-hCG monoclonal antibodies that are the first specific binding substances that can further bind to the analyte hCG Is specifically bound to the immobilized analyte hCG and immobilized on the second detection unit 6. That is, the anti-hCG monoclonal antibody, which is the first specific binding substance labeled with colloidal gold, is immobilized on the second detection unit 6 via the analyte hCG.
This is caused by bringing the sample that has undergone the step (B) into contact with a second detection unit to which the same or similar substance as the analyte is fixed, and binding the first specific binding substance in the conjugate to the substance. And (c) fixing the combined body to the second detection unit.
[0042]
Here, it is preferable that the sample is developed so that the sample continues to flow even if the sample exceeds the first detection unit 5 and the second detection unit 6. For this purpose, a sufficient 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 present. The first detection unit 5 and the second detection unit 6 are washed away by the sample flow that continues beyond the detection unit. For this purpose, an absorption part may be provided at the end part where the sample of the matrix 2 is developed. As the material of the absorption part, any material having sufficient absorption ability to wash away samples other than the analysis target may be used, and examples thereof include glass fiber filter paper GA200 (manufactured by Toyo Co., Ltd.).
[0043]
When the absorption part is provided in this manner, unreacted substances are washed away together with the flow of the sample. Therefore, after the specific binding reaction, a signal derived from the specific binding reaction is detected without performing the separation operation of the unreacted substances. It can be detected by the second detector 6. 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 where the specific binding reaction is performed. It is possible to detect the signal intensity distribution in the region including the first detection unit 5 and the second detection unit 6 or the region including the first detection unit 5 and the second detection unit 6. It is preferable to qualitatively or quantitatively determine the analyte in the sample from the distribution situation.
[0044]
Then, the step (D) of measuring the intensity 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 the intensity of the signal 1 and the signal 2 Based on this, the step (E) of obtaining the concentration of the analyte in the sample and performing qualitative or quantitative determination is performed.
Quantitative information showing the correspondence between signal intensity distribution and concentration obtained by measurement using a sample containing an analyte with a known concentration was created in advance, and this quantitative information and analysis were obtained. The concentration may be determined from the signal intensity distribution.
[0045]
FIG. 2 is a schematic diagram showing a configuration of an analyzer used together with the strip 1 in the specific binding analysis method according to the present invention. The 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. The first electrode 11 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 a sample is spotted on the sample spotting unit 3 from 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. Further, 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 light to the first detection unit 5 and the second detection unit 6. The photodetector 8 is a device that detects reflected light from the first detection unit 5 and the second detection unit 6.
[0046]
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 sample spotting are measured. Information thus obtained is input to the measurement start detector 10 as measurement start information.
Then, when a sample containing hCG is spotted on the sample spotting part 3 and 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 landing portion changes. The measurement start detector 10 can detect that the sample is spotted on the sample spotting unit 3 by referring to the change in conductivity and the measurement start information.
[0047]
The sample is preferably spotted on the sample spotting unit 3 after the test reagent strip 1 is mounted in the analyzer, but the test reagent strip 1 spotted with the sample may be mounted on the analyzer. .
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 intensity of the signal accompanying the scanning, and obtain the chromatograms shown in FIGS.
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 the coloration of the sample and the labeling material in the first detection unit 5 and the second detection unit 6 may be selected.
[0048]
Here, the signal may be any signal that is generated by a reaction contributed by the labeling material and can be detected. For example, fluorescence that can be measured with a fluorometer, luminescence that can be measured with a luminescence photometer, or first detection. The visual determination in the unit 5 and the second detection unit 6 and coloration that can be measured with a color difference meter may be used. In this case, the first detection unit 5 and the second detection unit 6 detect the reflected light, the intensity of fluorescence or light emission generated in the first detection unit 5 and the second detection unit 6, and the like. .
This signal detection is continuously performed while the positional relationship between the test reagent strip 1 and the light source 7 and the photodetector 8 is changed in parallel with the penetration direction of the sample, that is, relative to 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.
[0049]
The photodetector 8 sends this signal to the analyzer 9. The analyzer 9 measures the intensity of the signal by analyzing the signal from the photodetector 8. The analyzer 9 creates a corresponding curve consisting of the measured signal intensity and the scanning distance, that is, a chromatogram, and analyzes it.
FIGS. 3 and 4 are schematic diagrams of corresponding curves (chromatograms) drawn by analyzing the signal intensity distribution in the vicinity of the first detector 5 and the second detector 6, respectively. This chromatogram is drawn by irradiating light from the light source 7 onto the matrix 2 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). .
[0050]
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 the chromatogram may be the signal intensity based on the specific binding reaction. The heights of the chromatograms of the first detector 5 and the second detector 6 are Hs and Hr, respectively, and the areas of the chromatograms of the first detector 5 and the second detector 6 are Ss and Sr, respectively. Show.
Here, the test reagent strip 1 or the light source 7 is scanned, and the reflected light in the region including the first detection unit 5 and the second detection unit 6 is continuously measured to obtain a chromatogram. For example, the first detection unit 5 and the second detection unit 6 are connected to each other with a straight line connecting the upstream signal strength near the spotting unit 3 and the downstream signal intensity on the opposite side. The distance obtained by subtracting the height of the straight line from the height of the maximum value of the chromatogram in section 6 is set as the height H of the chromatogram, and the area of the area surrounded by the chromatogram and the straight line is chromatographed. By setting the area S in grams, the signal intensity based on the specific binding reaction obtained by subtracting the signal intensity derived from the background from the signal intensity in the first detection unit 5 and the second detection unit 6 can be obtained. 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 or the contaminants.
[0051]
At this time, the signal intensity at an arbitrary location on the matrix 2 other than the first detection unit 5 and the second detection unit 6 may be the intensity derived from the background. For example, the first detection unit 5 and You may choose the minimum value among the signal strengths in the 2nd detection part 6 vicinity.
Background is the behavior of a sample that does not contain the analyte. For example, the non-specific adsorption of specific binding substances or the like in the first detection unit 5 and the second detection unit 6 or the coloration of the sample itself when measuring a signal due to coloration affects the background, and the measurement accuracy is reduced. It is decreasing. 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 the same as the analyte in the binding reaction with the specific binding substance. A substance that exhibits behavior (for example, a structurally related substance of the analysis target) or a substance that interferes with the specific binding reaction between the analysis target and the specific binding substance by binding to the analysis target.
The analyzer 9 has quantitative information in a memory (not shown), and specifies the concentration of the analyte by referring to the analytical values Hs and Hr or Ss and Sr and the quantitative information.
Hereinafter, the specific binding analysis method according to the present invention will be described on the basis of an example carried out using the strip shown in FIG. 1 and the analyzer shown in FIG. 2, but the present invention is not limited to these. .
[0052]
【Example】
"Example"
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 specifies the concentration of the analysis object from Hs and Hr.
A graph showing the relationship between the signal intensity Hs and the hCG concentration when the first detection unit 5 is irradiated with light having a wavelength of 650 nm is shown in FIG. A sample obtained by adding hCG to a control urine for accuracy control whose hCG concentration is substantially zero was used. The 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), 1500 (IU / L), 2000 (IU / L), 3000 (IU / L), 5000 (IU / L), 7500 (IU / L) or 10000 (IU) / L) and measured 2 or 3 times at each concentration.
[0053]
As can be seen from FIG. 5, when the hCG concentration in the sample is low, most of the analyte binds to the labeled first specific binding substance and is immobilized on the first detection unit 5. Bound to two specific binding substances. 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. When the hCG concentration in the sample is high, an analyte that is not specifically bound to the labeled first specific binding substance is excessively present in the reaction system, and therefore the labeled first specific binding substance And the second specific binding substance immobilized on the first detection unit 5 by competing with the labeled first specific binding substance and the non-specific binding analyte. As a result of the specific binding reaction, the signal intensity Hs derived from the reaction contributed by the labeling material detected by the first detector 5 decreased as the concentration increased.
By using the dotted line in FIG. 5 as a calibration curve, the concentration of the analysis object could be specified. That is, when the signal intensity Hs on the vertical axis indicates a certain value, the horizontal axis of the dotted line indicating this value corresponds to the density. For example, when the signal intensity Hs is 5.0, it can be specified as about 1200 (IU / L).
[0054]
However, there are cases where there are a plurality of concentrations specified when the analyzer 9 refers to the dotted line in FIG. 5 as a calibration curve, and the concentration of the analysis object relative to the signal intensity may not be uniquely determined. When the hCG concentration may be 6000 (IU / L) or higher, when the signal strength Hs is 10, the hCG concentration is about 3500 (IU / L) or about 7600 (IU / L). There wasn't. When the concentration of the analysis object with respect to the signal intensity Hs is not uniquely determined, the signal intensity Hr is obtained simultaneously from the chromatogram of the second detector 6 as follows, and the Hr and Hs are analyzed simultaneously. The concentration could be specified.
[0055]
FIG. 6 shows 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.
A sample obtained by adding hCG to a control urine for accuracy control whose hCG concentration is substantially zero was used. The 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), 1500 (IU / L), 2000 (IU / L), 3000 (IU / L), 5000 (IU / L), 7500 (IU / L) or 10000 (IU) / L), and was measured 2 or 3 times at each concentration.
[0056]
As can be seen from FIG. 6, since the labeled first specific binding substance that is not bound to the analyte binds to the analyte or similar substance immobilized on the second detector 6, The signal intensity Hr derived from the contributing reaction decreased with increasing concentration. That is, when there is a labeled first specific binding substance that is reduced in concentration and not bound to the analyte, these are the analyte or similar substance immobilized on the second detector 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 large excess of the analyte having a high concentration, substantially all of the labeled first specific binding substances are bound to the analyte, so that the second detection unit 6 is reached. However, the signal intensity Hr derived from the reaction contributed by the labeling material was not substantially generated because it could not be bound to the immobilized analyte or the like and was washed away.
[0057]
Here, more strictly, when the first specific binding substance has a plurality of binding sites as in the case of an antibody (two in the case of an IgG antibody) as in this example, the binding state of each of these binding sites Reflected in signal strength. That is, since the first specific binding substance in which all the binding sites are buried with the analysis object has a very low probability of being able to newly bind to the analysis object, the analysis object immobilized on the second detection unit 6 The probability of combining with was also very low. However, if at least one binding site that can bind to the analyte remains, the probability of binding to the analyte immobilized on the second detection unit 6 depends on the ratio of the residual binding site. Increased. In any case, the signal increase Hr indicated by the dotted line in FIG. 6 decreased as the analyte concentration increased.
[0058]
By using the dotted line in FIG. 6 and the dotted line in FIG. 5 as quantitative information, the concentration of the analysis object could 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 referenced, and the concentration of the analyte with respect to the signal intensity may not be uniquely determined. For example, when there is a possibility that the hCG concentration may be 6000 (IU / L) or more, when the signal intensity Hs is 10, the hCG concentration is about 3500 (IU / L) or about 7600 (IU / L). Could not be distinguished. However, the concentration could be specified by further referring to the dotted line in FIG. That is, if the signal intensity Hr in FIG. 6 is 1.2, the density is about 3500 (IU / L) from the dotted line, and if the signal intensity Hr is 0.45, the density is about 7600 (IU / L) from the dotted line. ).
As described above, the signal intensity Hr is obtained from the chromatogram of the second detection unit 6 even when the concentration of the analyte is not uniquely determined only from the signal intensity Hs in FIG. 5, and both the Hr and Hs are obtained. It was possible to specify the concentration by analyzing in combination.
[0059]
Here, it was possible to specify the concentration only from Hr in FIG. 6, but since the inclination with respect to the density of the dotted line in the low concentration region is small, the accuracy when specifying the concentration from the measured Hr is shown in FIG. It was lower than when Hs of 5 was specified. Therefore, it is preferable to use Hr only for determining which of the two concentrations derived from Hs is true, and to use only Hs for specifying the concentration.
For simplicity, it is possible to specify the concentration from Hs in FIG. 5 only when Hr in FIG. 6 is a predetermined value or more. For example, in the case of this embodiment, the concentration is specified from Hs in FIG. 5 only when Hr is 0.6 or more, and when Hr is 0.6 or less, it is determined that the prozone phenomenon has occurred, and the concentration Was only limited to 6000 (IU / L) or more, and it was simple if the concentration was not specified. That is, Hr was effective for simplification if it was used only for pro-zone determination.
[0060]
Further, in the present embodiment, the second detection unit 6 is arranged downstream of the first detection unit 5 in the direction in which the liquid sample is developed (Z direction). Improved accuracy. This is because when a liquid passes through a matrix on which a protein such as an antibody is immobilized, the flow is disturbed and a liquid sample tends to flow only in a specific part of the matrix, and the degree of binding at the downstream detection unit is increased. Unevenness occurred. As a result, reproducibility decreased. The obstruction of the flow of the liquid sample by the protein immobilized on the matrix occurred due to spatial obstacles or uneven hydrophilicity due to uneven immobilization density. Since the quantification accuracy can be improved by ensuring the reproducibility of the signal generated by the first detection unit 5 that specifies the concentration, that is, quantifies, it is preferable to arrange the quantification detection unit upstream. won.
[0061]
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 this embodiment, an example in which Hs and Hr are used has been described. However, the same effect can be obtained by using Ss and Sr shown in FIGS. 3 and 4 in the same manner. That is, it was quantified with Ss, and prozone determination could be performed with Sr.
As described above, according to the specific binding analysis method according to the present embodiment, 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]
【The invention's effect】
As described above, according to the present invention, the analysis value of the signal intensity based on the specific binding reaction of the specific binding substance generated in the two detection units and the quantitative information indicating the correspondence between the concentration of the analysis target are obtained. Since the amount of the analyte in the sample is specified with reference, accurate and qualitative or quantitative analysis of the analyte in the sample can be performed easily and quickly without being affected by the prozone phenomenon.
[Brief description of the drawings]
FIG. 1 is a schematic perspective view of an embodiment of a strip for a test reagent used in a 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 signal intensity distribution state of the first detection unit 5 of the strip 1 detected by the analysis apparatus shown in FIG. 2;
FIG. 4 is a schematic diagram showing a signal intensity distribution state of the second detection unit 6 of the strip 1 detected by the analyzer shown in FIG. 2;
FIG. 5 is a graph showing the relationship between hCG concentration and 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 hCG concentration and signal intensity Hr when the second detection unit 6 emits light having a wavelength of 650 nm.
[Explanation of symbols]
1 strip
2 Matrix
3 Sample spotting part
4 Holding part
5 1st detection part
6 Second detector
7 Light source
8 Photodetector
9 Analyzer
10 Measurement start detector
11 First electrode
12 Second electrode

Claims (17)

(A) A sample containing an analyte is brought into contact with a first specific binding substance labeled with a detectable labeling substance, and the analyte is bound to the first specific binding substance to thereby label the first Specific binding substance--obtaining a conjugate of the analyte;
(B) The sample is brought into contact with the first detection unit to which the second specific binding substance is immobilized, and the excess analyte that has not been bound to the first specific binding substance and the conjugate are second specific. A step of binding to a binding substance and fixing to a first detection unit;
(C) The sample is brought into contact with a second detection unit on which a substance the same as or similar to the analysis target is fixed, and the first specific binding substance in the conjugate is bound to the substance, thereby the conjugate. Fixing to the second detection unit,
(D) measuring the intensity of signal 1 and signal 2 derived from the labeling material obtained in the first detection unit and the second detection unit, respectively, and (E) based on the intensity of signal 1 and signal 2 A specific binding analysis method comprising the step of qualitatively or quantitatively determining the concentration of the analyte in the sample. In the step (E), when specifying the concentration of the analyte based on the intensity of the signal 1 obtained in the step (D), when two different concentrations 1 and 2 are derived, The specific binding analysis method according to claim 1, wherein 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). The first detection unit to which the second specific binding substance is immobilized is brought into contact with the sample that has undergone the step (A), and the excess analyte that has not bound to the first specific binding substance and the conjugate are converted into the first detection part. The step (B) of binding to the first specific binding substance by binding to the two specific binding substances is performed, and then the step (B) is applied to the second detection part to which the same or similar substance as the analyte is fixed. The step (C) is performed, wherein the passed sample is brought into contact, the first specific binding substance in the conjugate is bound to the substance, and the conjugate is fixed to the second detection unit. 2. The specific binding analysis method according to 1. The second detection unit to which the same or similar substance as the analyte is fixed is brought into contact with the sample that has undergone the step (A), and the first specific binding substance in the conjugate is bound to the substance. The step (C) of fixing the conjugate to the second detection unit is performed, and then the sample that has undergone the step (C) is brought into contact with the first detection unit to which the second specific binding substance has been immobilized. The excessive analysis object not bound to the specific binding substance and the step (B) of binding the conjugate to the second specific binding substance and immobilizing it on the first detection unit are performed. 2. The specific binding analysis method according to 1. The step (A) is performed in advance, the first detection unit and the second detection unit are provided on the sheet-like chromatographic matrix, and the sample after the step (A) is used as the first detection unit and the second detection unit. And the steps (B) to (E) are performed by moving the sample to the first detection unit and the second detection unit by osmotic force due to capillary action in the matrix. The specific binding analysis method according to claim 1. A holding unit that holds 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-like chromatographic matrix, and the sample is placed in the holding unit or Dropping on the upstream side, and moving the sample to the holding unit, the first detection unit and the second detection unit by osmotic force due to capillary action in the matrix, and performing steps (A) to (E) The specific binding analysis method according to claim 1. The specific binding analysis method according to claim 1, wherein the signal derived from the labeling material is a signal indicating coloration, fluorescence, or luminescence. 2. 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. 2. The specific binding analysis method according to claim 1, wherein the labeling material is a particle containing a metal sol, a dye sol or a fluorescent substance, or a colored latex particle. 2. The specific binding analysis method according to claim 1, wherein in the step (D), the signal 1 and the signal 2 are continuously measured in this order along the moving direction of the analysis object. In the step (D), a corresponding curve indicating the relationship between the signal 1 and the signal 2 continuously measured and the position in the moving direction of the analysis object is created, and in the step (E), based on the corresponding curve. The specific binding analysis method according to claim 11, wherein the method is qualitative or quantitative. In the step (E), the analyte is quantified based on the quantitative information obtained from the sample whose concentration of the analyte is known and the signals 1 and 2 obtained in the step (D). The specific binding analysis method according to claim 1. A sheet-like chromatographic matrix, a first detection unit provided on the matrix and fixed with a specific binding substance, and a second detection unit provided on the matrix and fixed with the same or similar substance as the analyte And
A sample containing an analyte and a specific binding substance is moved to the first detection unit and the second detection unit by osmotic force due to capillary action to cause a specific binding reaction, and a signal derived from the specific binding reaction is detected. A specific binding analysis device characterized in that the analyte is qualitatively or quantitatively determined. A holding unit provided on the upstream side of the first detection unit on the matrix, the holding unit holding a specific binding substance labeled with a detectable labeling material;
The specific binding analysis device according to claim 14, wherein the sample is moved from the holding unit to the first detection unit and the second detection unit by osmotic force due to capillary action. A sample spotting part provided on the upstream side of the holding part on the matrix;
The specific binding analysis device according to claim 15, wherein the sample is moved from the sample spotting unit to the holding unit, the first detection unit, and the second detection unit by an osmotic force due to capillary action. A sample spotting unit provided on the upstream side of the first detection unit on the matrix;
The specific binding analysis device according to claim 14, wherein the sample is moved from the sample spotting unit to the first detection unit and the second detection unit by osmotic force due to capillary action.

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