JP2003344408A - Flow-through assay method, kit and device - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a flow-through assay method and a flow-through assay device operable extremely easily and executable more quickly. <P>SOLUTION: This flow-through assay method is characterized by forming a coloring particle/first receptor/analytical object/second receptor composite on the sheet-shaped carrier surface by using the coloring particles with which the first receptor to the analytical object is coupled and the sheet-shaped carrier with which the second receptor to the analytical object is coupled, and detecting the composite. <P>COPYRIGHT: (C)2004,JPO

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a flow-through assay method and a flow-through assay device, which are a type of immunoassay.

[0002]

2. Description of the Related Art In a flow-through assay method using a membrane, which is a type of rapid immunoassay, a method of detecting or measuring a ligand (analyte) by dropping a sample and then a receptor labeled with an enzyme is known. It is known (Japanese Patent Publication No. 7-34016). However, this method requires many steps, such as a step of dropping an enzyme-labeled receptor, a step of removing unreacted enzyme, and a step of reacting a substrate corresponding to the enzyme, which is time-consuming. there were. As a flow-through assay method that does not perform such enzyme-substrate reaction, Alice R. Arr
edondo et al. reported a method of mixing blue latex beads to which an IgG antibody was bound, an IgM antibody and a sample and then filtering the mixture (Biotechnology Letter).
s, 22: 547-550, 2000). However, this method is still inconvenient because it requires an operation of mixing three kinds of substances and then filtering with a filter, which is inconvenient for use in clinical diagnosis.

[0003]

SUMMARY OF THE INVENTION It is an object of the present invention to provide a flow-through assay method, kit and device which are extremely easy to perform a test and can be performed more quickly.

[0004]

The above object is to bring a sample liquid containing an analyte into contact with colored particles to which a first receptor for the analyte is bound, and then to a second receptor for the analyte. A flow-through characterized in that a colored particle / first receptor / analyte / second receptor complex is formed on the surface of the sheet-shaped carrier by passing through the sheet-shaped carrier to which is bound and detected. Solved by the assay method. The above-mentioned problem is also characterized in that a porous pad is arranged above the surface of the sheet-shaped carrier, and the colored particles are contained in the surface or in the porous pad. Be solved by the method.

Further, the above-mentioned problem is a flow-through assay kit comprising a colored particle having a first receptor bound to an analyte bound thereto and a sheet-like carrier having a second receptor bound to the analyte bound thereto, and a porous substrate. The kit further comprising a qualitative pad, wherein the colored particles are contained on or in the surface of the porous pad, and a porous material containing colored particles having a first receptor for an analyte bound thereto. A flow-through assay device comprising a quality pad and a sheet-like carrier having a second receptor for the analyte bound thereto.

The present invention also relates to the above method, kit and device, wherein said colored particles are colloidal gold or latex. The present invention also relates to the above method, kit and device, wherein the sheet-like carrier is a membrane or a filter. The invention also relates to the above method, kit and device, wherein said first and / or second receptor is an antibody. The present invention also relates to the above method, kit and device, wherein the analyte is a virus or bacterium.

A sample liquid containing an analyte to be collected from a patient or the like is brought into contact with, for example, by mixing with colored particles to which the first receptor for the analyte is bound, and then this mixed solution is added to the analyte. By dripping onto the sheet-shaped carrier to which the second receptor is bound, colored particles / first receptor / analyte / second receptor complex are formed on the surface of the sheet-shaped carrier. On the other hand, other components in the sample that have not become a complex and components such as colored particles to which the first receptor is bound pass without being captured by the carrier.
Therefore, only the above composite remains on the surface of the sheet-shaped carrier,
The colored particles allow this complex to be detected visually. As described above, according to the method of the present invention, only the analyte in the sample can be reliably detected by a very simple operation.

Further, when the colored particles bound to the first receptor are arranged on or in the surface of the porous pad arranged on the upper part of the sheet-shaped carrier, the sample liquid containing the analyte is added to the porous pad. The colored particles / first receptor / analyte / analyte / second receptor complex can be similarly formed on the surface of the sheet-shaped carrier to detect the analyte simply by dropping from above, which is very simple.

[0009]

DETAILED DESCRIPTION OF THE INVENTION The method and apparatus of the present invention are described below. (Flow-through assay method) The principle of the flow-through assay method is “Guide to Diagnostic Rapid Test Devic
^{e Components ", 2 nd edition,} published by Schleiche
r & Schuell company, January 2000, Edited by Lisa V
ickers, p6-8, flow through format (Flow Thro
ugh Formats). That is, in an immunoassay method that uses a sheet-shaped carrier such as a membrane, the sample liquid containing the analyte passes through the sheet-shaped carrier, and at that time, the analyte is captured by the receptor immobilized on the carrier. This is a characteristic method. It is preferable that a member that absorbs the sample liquid plays a role of sucking the sample liquid downward under the sheet-shaped carrier, and this member enables more rapid detection. Further, Japanese Examined Patent Publication No. 7-34016 (Hybridtech) is also cited as a document describing one mode of the flow-through assay method.

According to a first method of the present invention, in the above flow-through assay method, colored particles to which the first receptor for the analyte is bound are contacted with a sample liquid containing the analyte, This is a method in which this sample liquid is passed through a sheet-shaped carrier to which a second receptor for the analyte is bound. Colored particles / First receptor /
The analyte / second receptor complex is formed on the surface of the sheet-shaped carrier, and this complex can be detected by the marker. In addition, the second method of the present invention is, in the above flow-through assay method, analyzed on the surface of the porous pad containing colored particles, which is arranged above or above the surface of the sheet-shaped carrier. This is a method of dropping a sample liquid containing an object. A colored particle / first receptor / analyte / second receptor complex is formed on the surface of the sheet-shaped carrier, and this complex can be detected by the marker.

(Colored particles) In the present specification, the colored particles are particulate substances insoluble in a sample suspension such as water and buffer solution, and can be visually detected such as colored labeling substances such as dyes. It means a labeled product. Since the colored particles are used by binding to the first receptor, it is necessary to be able to bind to the receptor by means such as adsorption. Further, since it is necessary that the colored particles to which the receptor is bound pass through the sheet-shaped carrier and do not remain on the surface of the carrier, it is necessary that the particles have a size smaller than the pore size of the carrier. Depending on the type of carrier, the diameter is usually 0.01
It is preferable that the particles have a size of about 1 μm. Examples of such colored particles include colloidal gold and colored latex.

The colored particles bound to the first receptor for the analyte may be added to the analyte liquid containing the analyte as a solid and mixed and then passed through a sheet-shaped carrier, or the colored particles may be passed through a buffer solution. Or the like, and the diluted solution and the sample solution containing the analyte are mixed and then passed through the carrier. As the liquid for diluting the colored particles, those described for the sample suspension described below can be used.
The dilution ratio when diluting the colored particles varies depending on the type of the colored particles and the performance of the antibody, and can be appropriately determined by those skilled in the art so that the concentration becomes appropriate. For example, in the case of a gold colloid bound with an antibody, when a gold colloid solution having an absorbance at 530 nm of 1.0 to 10 is used, for example, a gold colloid solution 1 (volume) is replaced with a sample solution (specimen solution). 0.1 to 20 (volume), that is, about 1
It can be used at about 20 times dilution. When using a colored latex, 0.0001 w / v% to 0.01
It can be used in the range of about w / v%.

The colored particles to which the first receptor is bound may be contained in advance in or on the surface of the porous pad disposed above the surface of the sheet-shaped carrier to which the second receptor is bound. This method is very simple and preferable because the assay can be performed simply by dropping the sample liquid from the porous pad. As a method for incorporating the colored particles into or on the surface of the porous pad, the colored particles may be made into a solution or a suspension and impregnated in the porous pad and then dried, or sprayed on the pad surface. A method of post-drying may be used. Alternatively, the colored particles may be embedded in or on the porous pad in a dry state. When the colored particles are used as a solution or suspension, or as a solvent for diluting when adsorbing to the porous pad, a buffer solution such as phosphate buffered saline or Good's buffer may be mentioned. , More protein (casein,
Bovine serum albumin, gelatin, purified globulin fraction) and sugars (mannitol, trehalose, sorbitol, sucrose, glucose, etc.) are preferably contained.

Further, in order to bind the receptor for the analyte to the colored particles, a physical adsorption method, a chemical binding method or the like can be used. Examples of the physical adsorption method include a method of bringing colored particles suspended in a suitable solvent into contact with a receptor for an analyte. After the receptor is adsorbed, it is treated with a protein blocking agent such as bovine serum albumin to cover the part of the colored particle surface that was not involved in adsorption, and then the receptor is bound (adsorbed) by centrifugation or ultrafiltration. The colored particles formed are separated from the excess receptor that has not been adsorbed. In addition, examples of the chemical bonding method include a method of forming a covalent bond through a functional group such as a carboxyl group and an amino group. The receptor concentration capable of binding to each colored particle varies depending on the material, size (particle size, surface area), etc. of the colored particle, and can be appropriately determined by those skilled in the art.

(Porous Pad) As a porous pad, the pore diameter is 10 to 200 μm, and the thickness is 300 to 3
000μm glass fiber filter paper, polypropylene non-woven fabric,
Other polymer-based nonwoven fabrics are preferably used. When a porous pad having colored particles contained on or in the surface thereof is used as described above, it is preferable to dispose the pad above the surface of the sheet-shaped carrier described later. The porous pad may be loaded in direct contact with the sheet-shaped carrier, or the porous pad may be placed above the surface of the sheet-shaped carrier at regular intervals without contacting the sheet-shaped carrier. May be.

(Sheet-shaped carrier) The sheet-shaped carrier is a support which is insoluble in water, immobilizes the receptor, and captures the colored particles / first receptor / analyte / second receptor complex to form another component. Used to separate from. Examples include membranes or filters. There is no particular limitation on such a membrane or filter as long as it has a certain pore size that allows substances other than the analyte contained in the sample liquid and colored particles to which the first receptor binds to pass through. . Any commercially available one may be used, preferably nitrocellulose,
Acetate-mixed nitrocellulose, nylon, polyether sulfone, polyvinylidene difluoride and the like are used.

Further, the pore size of the sheet-like carrier depends on the size of the colored particles to which the analyte and the first receptor bind, as described above, and is not particularly limited, but is 0.
22 μm to 12 μm is most often used. The method of immobilizing (bonding) the receptor to the sheet-shaped carrier may be physical adsorption or may be by chemical bonding. The sheet-shaped carrier to which the receptor is bound is prepared, for example, by adsorbing a solution prepared by diluting the receptor in a buffer solution or the like on the sheet-shaped carrier and then drying.

(Flow-through Assay Device) The device used in the first flow-through assay method of the present invention comprises a sheet-like carrier having at least a receptor for an analyte bound thereto. The method using this apparatus is as follows. A sample liquid containing an analyte is brought into contact with colored particles to which the first receptor for the analyte is bound, and then passed through a sheet-shaped carrier in the apparatus, whereby the surface of the sheet-shaped carrier is colored particles. /
A first receptor / analyte / second receptor complex is formed and detected. A schematic view of this device is shown in FIGS. 1 is a plan view of the apparatus, and FIG. 2 is a plan view of FIG.
FIG. 3 is a sectional view taken along line II ′ of FIG. 1 and 2, a
Has an opening for dropping the prepared sample (sample solution),
It is an adapter provided with holes (A hole and B hole) for a sample to pass through on the bottom surface. b is a sheet-like carrier to which a receptor for an analyte is bound, and c is a member that absorbs liquid.

The apparatus used in the second flow-through assay method of the present invention is a porous pad containing colored particles having the first receptor for an analyte bound on or in the surface thereof, and the analyte. A flow-through assay device comprising a sheet-shaped carrier having a second receptor for an object bound thereto. The method using this apparatus is as follows. A sample liquid containing an analyte is dropped on the surface of the porous pad and then passed through the sheet-shaped carrier to give colored particles /
A first receptor / analyte / second receptor complex is formed and detected. FIG. 3 shows the cutting of the flow-through assay device of the present invention in which colored particles having a receptor bound thereto are contained on or in the surface of the porous pad, and the porous pad is arranged above the surface of the sheet-shaped carrier. Shows an end view. In FIG. 3, d is a porous pad containing colored particles having a receptor for an analyte bound thereto on or in the surface thereof.

The flow-through assay device preferably has a liquid absorbing member, which absorbs a liquid such as a sample liquid dropped on the sheet-shaped carrier, below the sheet-shaped carrier. Examples of such liquid absorbing member include materials such as glass fiber, cellulose, or a mixture thereof. Further, it is preferable that the liquid absorbing member is in contact with or in close contact with the sheet-shaped carrier to which the receptor is bound so that the liquid can be absorbed quickly. Due to the presence of the liquid absorbing member in contact with or in close contact with such a sheet-like carrier,
The passage of the sample liquid, such as a liquid, into a sheet-shaped carrier is promoted, which enables rapid detection.

(Receptor) The first and second receptors for the analyte are receptors that react specifically with the analyte to form a complex. Therefore, it is natural that the receptor to be used differs depending on the analyte, but generally, the analyte is bacteria, viruses, hormones,
In the case of other clinical markers and the like, polyclonal antibodies, monoclonal antibodies and the like which specifically react with and bind to these are mentioned. In addition, viral antigens, viral hollow particles, recombinant E. coli expressed proteins, recombinant yeast expressed proteins and the like can be mentioned. The first and second receptors used may be the same or different.

(Analyte) By the flow-through assay method of the present invention, various antigens such as bacteria, viruses, hormones and other clinical markers, antibodies, etc. can be analyzed. In particular, it is effective for analysis of influenza virus, small microsphere virus and the like.

(Sample suspension) The sample can be collected from the patient's pharynx, nasal cavity, etc., using a cotton swab or the like, or nasal aspirate, etc. can be used. Ordinarily, suspend the sample in suspension and perform the assay. As the sample suspension, buffers or the like usually used in sample detection or quantification methods by immunological methods such as immunodiffusion method, enzyme immunoassay method and agglutination method can be used. More specifically, physiological saline, phosphate buffered saline (PBS), PBS with gelatin, PBS with bovine serum albumin (BSA), Good's buffer,
Calf Infusion Broth (VIB), Heart Infusion Broth, Eagle's Minimum Essential Medium (EME
M), BSA-added EMEM, and the like, but not limited thereto. Moreover, you may use the said buffers in combination of 2 or more types. Further, in addition to the above composition, a basic amino acid, an inorganic salt and / or a surfactant can be added. By using a sample suspension containing at least two kinds of basic amino acids, inorganic salts and surfactants, non-specificity to the carrier of the components other than the analyte contained in the sample and the receptor bound to the carrier It is preferable because it can reduce binding. In addition, in the method of the present invention, filtration may be performed after suspending the sample in the sample suspension and before mixing with the colored particles.

(Kit for flow-through assay) The kit for flow-through assay of the present invention is a kit used in the above-mentioned flow-through assay method. The flow-through assay kit of the present invention comprises at least the following and. A pad containing colored particles to which the first receptor for the analyte is bound or colored particles to which the first receptor for the analyte is bound Sheet-like carrier to which the second receptor for the analyte is bound It may contain a liquid or a colored particle diluent. And / or may be included in the kit in the form of the flow-through assay device described above.

If necessary, a negative control solution consisting of only a buffer for testing the activity of the kit, and a positive control consisting of a buffer containing an analyte such as an antigenic substance may be included. Further, a filter for filtering the sample or a sterile cotton swab may be included.

[0026]

EXAMPLES [Example 1] The following reagents and devices were used. 1. Preparation of Monoclonal Antibody (Preparation of Anti-Influenza A Virus NP Monoclonal Antibody (Mouse)) Purified influenza A virus antigen was immunized and the spleen was excised from BALB / c mice maintained for a certain period of time and the method of Keller et al. (Kohler et al.
al. , Nature, vol, 256, p495-
497 (1975)) by mouse myeloma cells (P
3 x 63). The obtained fused cells (hybridomas) were maintained in a 37 ° C. incubator, and E using influenza A virus NP antigen solid phase plate.
The cells were purified (monocloned) while confirming the antibody activity of the supernatant by LISA. The obtained cell line was intraperitoneally administered to BALB / c mice treated with pristane,
After a week, antibody-containing ascites was collected. IgG was purified from the obtained ascites by the ammonium sulfate fractionation method. (Preparation of anti-influenza B virus NP monoclonal antibody (mouse)) Purified influenza B virus antigen was immunized and the spleen was excised from a BALB / c mouse maintained for a certain period of time, and the method of Keller et al. (Kohler et al.
al. , Nature, vol. 256, p49
5-4397 (1975)) and fused with mouse myeloma cells (P3 × 63). The obtained fused cells (hybridomas) were maintained in an incubator at 37 ° C., and influenza B virus NP antigen solid phase plates were used. The cells were purified (monocloned) while confirming the antibody activity of the supernatant by ELISA. The obtained cell line was intraperitoneally administered to pristane-treated BALB / c mice, and after about 2 weeks, antibody-containing ascites was collected. IgG was purified from the obtained ascites by the ammonium sulfate fractionation method.

2. Binding of gold colloid and monoclonal antibody 10 mL of gold colloid solution (BBI, particle size 40 nm,
0.01%) as a chloroauric acid concentration, and the pH is adjusted to 5.5 with 100 mM potassium carbonate. The purified anti-A or B influenza virus NP monoclonal antibody was dialyzed against a 2 mM sodium borate solution (pH 9.2) to prepare a solution having a concentration of 100 μg / mL. p
To the gold colloid solution in which H was adjusted to 5.5, an amount such that the final concentration of the antibody was 10 μg / mL was added, and the mixture was gently stirred by hand so that the gold colloid and the antibody were sufficiently bound. 5
After 10 minutes, 1 mL of 10% BSA was added and gently stirred by hand for 10 minutes. Transfer the whole amount to a centrifuge tube, 14000 rpm,
Centrifuged for 30 minutes at 4 ° C. After centrifugation, discard the supernatant by suction,
Add 1% BSA, 15 to the precipitated antibody-bonded gold colloid.
1 mL of 10 mM Tris-HCl buffer solution pH 8.5 containing 0 mM sodium chloride was added and suspended. 3. Assay Device An assay device having the same structure as that shown in FIGS. 1 and 2 was used.

4. Immobilization of antibody on sheet-shaped carrier Nitrocellulose was used as a sheet-shaped carrier. Immobilization
The two antibody solutions were spotted on a nitrocellulose membrane. 1 μL of a solution containing 1 mg of anti-influenza virus NP monoclonal antibody in the A-hole of the device, and 12 solutions of 1 mg containing anti-influenza B virus NP monoclonal antibody in the B-hole
Each μL was spotted. 1 buffer for antibody dilution
0 mM phosphate buffer (pH 7.0) was used. After the spot, it was dried in a drying room at 45 ° C. for 40 minutes.

5. 1. Method for detecting antigen The antibody-bonded gold colloid prepared in Step 5 was adjusted so that the absorbance was 1.0 at OD 530 nm in 5 mM phosphate buffer (pH
7.4% diluted with 5% BSA, 4.5% Trit
onX-100, a sample diluted with 5 mM phosphate buffer (pH 7.4) containing 2% fish gelatin was diluted 1: 4 and mixed,
500 μL was dropped onto the assay device. The detection was carried out using the diluted influenza A virus and influenza B virus as a positive sample and the above buffer solution as a negative sample. 500 μL of sample in A hole and B hole
Each of them was dropped so that no air bubbles were introduced, and the mixture was allowed to stand at room temperature until there was no liquid on the membrane. After that, the adapter was removed and visually judged. As a result of the judgment, regarding the A hole, the influenza A virus was 10 4 pfu /
It was possible to detect up to mL, and it was determined to be negative for influenza B virus. On the other hand, regarding B hole, influenza B virus could be detected up to 10 4 pfu / mL, and it was determined to be negative for influenza A virus.

[Second Embodiment] Similar to the first embodiment, 1. Preparation of monoclonal antibody, 2. Binding of gold colloid and monoclonal antibody, 4. The antibody was immobilized on the carrier.
3. The assay device used was a device in which a porous pad containing colored particles was loaded on the surface of a sheet-shaped carrier as shown in FIG. That is, an assay device manufactured by drying a gold colloid bound with a monoclonal antibody as described below was used to detect the antibody as described below.

5. Drying gold colloid 2. 10% sucrose was added to the antibody-bonded gold colloid prepared in Step 10 so that the absorbance was 1.0 at OD 530 nm.
Dilute with Tris-HCl buffer (pH 8.0) and pad (W
Made by hatman: Conjugate Release
Pad F075-14 (pore size: diameter 23μ
m, thickness 355 μm) and dried with warm air. The anti-influenza A virus NP antibody adsorption pad after drying with warm air was placed in the A hole of the assay device, while the anti-influenza B virus NP antibody adsorption pad after drying with warm air was placed in the B hole of the assay device.

6. Antigen detection method Specimen dilution is 5% in 5 mM phosphate buffer (pH 7.4)
BSA, 4.5% Triton X-100, and 2% fish gelatin were used. For detection, influenza A virus and B diluted as positive samples
Influenza virus was performed using the above buffer as a negative sample. 500 samples in A hole and B hole
Each μL was dropped so that air bubbles would not be introduced, and the mixture was allowed to stand at room temperature until the liquid remained on the membrane. After that, the adapter was removed and visually judged. As a result of the judgment, regarding the A hole, the influenza A virus was 10 4 pf
Up to u / mL was detected, and it was determined to be negative for influenza B virus. On the other hand, regarding hole B,
Influenza B virus is 10 4 pfu / mL
Was detected, and it was determined to be negative for influenza A virus.

[0033]

EFFECTS OF THE INVENTION By using the flow-through assay method and device of the present invention, it is possible to detect viruses and the like more simply and quickly than ever before.

[Brief description of drawings]

FIG. 1 is a plan view of an example of an assay device of the present invention.

FIG. 2 is a sectional view taken along the line I-I ′ of FIG.

FIG. 3 is a cut end view of an example of the assay device of the present invention.

[Explanation of symbols]

A: hole B: hole a: Adapter b: sheet carrier c: Liquid absorbing member d: Porous pad containing colored particles

Claims (18)

[Claims] 1. A sample liquid containing an analyte is brought into contact with colored particles bound to a first receptor for the analyte, and then passed through a sheet-shaped carrier bound to a second receptor for the analyte. By letting
On the surface of the sheet-shaped carrier, colored particles / first receptor /
A flow-through assay method, which comprises forming an analyte / second receptor complex and detecting it. 2. The method according to claim 1, wherein a porous pad is arranged above the surface of the sheet-shaped carrier, and the colored particles are contained on or in the surface of the porous pad. 3. The method according to claim 1, wherein the colored particles are colloidal gold or colored latex. 4. The method according to claim 1, wherein the sheet-shaped carrier is a membrane or a filter. 5. The method according to any one of claims 1 to 4, wherein the first and / or second receptor is an antibody. 6. The method according to any one of claims 1 to 5, wherein the analyte is a virus or a bacterium. 7. A kit for a flow-through assay, which comprises colored particles to which a first receptor for an analyte is bound and a sheet-shaped carrier to which a second receptor for the analyte is bound. 8. The kit according to claim 7, further comprising a porous pad, wherein the colored particles are contained in or on the surface of the porous pad. 9. The kit according to claim 7, wherein the colored particles are colloidal gold or colored latex. 10. The kit according to claim 7, wherein the sheet-shaped carrier is a membrane or a filter. 11. The kit according to any one of claims 7 to 10, wherein the first and / or second receptor is an antibody. 12. The kit according to any one of claims 7 to 11, wherein the analyte is a virus or a bacterium. 13. A flow comprising a porous pad containing colored particles bound to a first receptor for an analyte on or in the surface thereof, and a sheet-shaped carrier bound for a second receptor for the analyte. Through assay device. 14. The device according to claim 13, wherein the porous pad is disposed above the surface of the sheet-shaped carrier. 15. A device according to claim 13 or 14, wherein the colored particles are colloidal gold or colored latex. 16. The device according to claim 13, wherein the sheet-shaped carrier is a membrane or a filter. 17. A device according to any one of claims 13 to 16, wherein the first and / or second receptor is an antibody. 18. The apparatus according to claim 13, wherein the analyte is a virus or a bacterium.