JP2007121320A - Flow-through assay method, kit, and system - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a flow-through assay method and a flow-through assay system which are operated very easily and speedily. <P>SOLUTION: Colored particles, bonded to a first receptor to an analyte and a sheet-like carrier bonded to a second receptor to the analyte, are used to form a complex of the colored particles, the first receptor, the analyte, and the second receptor in the surface of the sheet-like carrier. The complex is detected in the flow-through assay method. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to a flow-through assay method and a flow-through assay apparatus which are a kind of immunoassay.

In a flow-through assay method using a membrane which is a kind of rapid immunoassay, a method is known in which a ligand (analyte) is detected or measured by dropping a sample and then dropping a receptor labeled with an enzyme ( Patent Document 1). However, this method requires many steps, such as a step of dropping a receptor labeled with an enzyme, a step of removing an unreacted enzyme, and a step of reacting a substrate corresponding to the enzyme. there were.
As a flow-through assay method in which such enzyme substrate reaction is not performed, Alice R. Arredondo et al. Reported a method of filtering blue latex beads, IgM antibody, and specimen bound with IgG antibody, followed by filtration ( Non-patent document 1). However, since this method requires an operation of mixing three kinds of substances and then performing filtration with a filter, the operation is still complicated and inconvenient to use in a clinical diagnosis.

Japanese Patent Publication No. 7-34016 Biotechnology Letters, 22: 547-550, 2000

  It is an object of the present invention to provide a flow-through assay method, kit and apparatus that are extremely easy to operate for testing and can be performed more quickly.

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

  Furthermore, the above-described problems further include a flow-through assay kit and a porous pad comprising colored particles bound to the first receptor for the analyte and a sheet-like carrier bound to the second receptor for the analyte. The kit containing the colored particles on or in the surface of the porous pad, and the porous pad containing the colored particles bound to the first receptor for the analyte in or on the surface. And a flow-through assay device comprising a sheet carrier to which a second receptor for the analyte is bound.

  The present invention also relates to the above method, kit and apparatus, wherein the colored particles are gold colloid or latex. The present invention also relates to the above method, kit and apparatus, wherein the sheet-like carrier is a membrane or a filter. The present invention also relates to the above methods, kits and devices 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 a bacterium.

  The sample liquid containing the analyte collected from a patient or the like is brought into contact with the colored particles to which the first receptor for the analyte is bound, and this mixed liquid is then contacted with the second analyte for the analyte. By dropping on the sheet-like carrier to which the receptor is bound, a colored particle / first receptor / analyte / second receptor complex is formed on the surface of the sheet-like carrier. On the other hand, other components in the specimen that have not become a complex and components such as colored particles bound to the first receptor pass through without being captured by the carrier. Therefore, only the above complex remains on the surface of the sheet-like carrier, and this complex can be detected visually by the colored particles. 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.

  In addition, when the colored particles bound to the first receptor are arranged on or in the porous pad surface arranged on the upper part of the sheet-like carrier, the specimen liquid containing the analyte is dropped from the porous pad. Similarly, the colored particles / first receptor / analyte / second receptor complex is formed on the surface of the sheet-like carrier and the analyte can be detected, which is very convenient.

  By using the flow-through assay method and apparatus of the present invention, viruses and the like can be detected more easily and quickly than in the past.

The method and apparatus of the present invention are described below.
(Flow-through assay method)
Flow principle of through assay methods, "Guide to Diagnostic Rapid Test Device Components", 2 nd edition, published by Schleicher & Schuell company, January 2000, Edited by Lisa Vickers, p6-8, the flow-through format (Flow Through Formats) As described. That is, in an immunoassay method using a sheet-like carrier such as a membrane, the sample liquid containing the analyte passes through the sheet-like carrier, and at that time, the analyte is captured by the receptor fixed to the carrier. It is a characteristic method. Under the sheet-like carrier, there is preferably a member that absorbs the liquid, which serves to suck the sample liquid downward, and this member enables more rapid detection. Japanese Examined Patent Publication No. 7-34016 (Hybrtech) is also cited as a document describing an embodiment of the flow-through assay method.

The first method of the present invention is the above-described flow-through assay method, wherein colored particles to which a first receptor for an analyte is bound are contacted with a sample liquid containing the analyte, and the sample liquid is further contacted. Is passed through a sheet carrier to which a second receptor for the analyte is bound. A colored particle / first receptor / analyte / second receptor complex is formed on the surface of the sheet-like carrier, and this complex can be detected by the marker.
Further, the second method of the present invention is the above-described flow-through assay method, wherein the analysis is performed on the surface or the surface of the porous pad containing colored particles therein, which is disposed above the surface of the sheet-like carrier. This is a method of dropping a specimen liquid containing an object. A colored particle / first receptor / analyte / second receptor complex is formed on the surface of the sheet-like carrier, and this complex can be detected by the marker.

(Colored particles)
In the present specification, the colored particles are particulate substances that are insoluble in a specimen suspension such as water or a buffer, and mean a labeled product that can be detected visually, such as a colored labeled product with a dye or the like. 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 the colored particles to which the receptor is bound need to pass through the sheet-like carrier and do not remain on the surface of the carrier, the particles must be smaller than the pore size of the carrier. Although it depends on the type of the carrier, it is usually preferable to use particles having a diameter of about 0.01 to 1 μm. Examples of such colored particles include gold colloid and colored latex.

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

Further, the colored particles bound to the first receptor may be contained in advance on or in the porous pad surface disposed above the surface of the sheet-like carrier bound to the second receptor. 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 on or in the surface of the porous pad, the colored particles may be made into a solution or suspension, 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 the porous pad surface or in a dry state.
When the colored particles are used as a solution or suspension, or as a solvent for diluting when adsorbed to the porous pad, a buffer solution such as phosphate buffered saline or Good's buffer can be used. Further, it preferably contains a protein (casein, bovine serum albumin, gelatin, purified globulin fraction, etc.), and a saccharide (mannitol, trehalose, sorbitol, sucrose, glucose, etc.).

  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 in which colored particles suspended in a suitable solvent are brought into contact with a receptor for an analyte. After adsorbing the receptor, in order to cover the part of the colored particle surface that was not involved in the adsorption, it was treated with a protein blocking agent such as bovine serum albumin, and then the receptor was bound (adsorbed) by centrifugation or ultrafiltration. The colored particles are separated from the excess receptor that has not been adsorbed. Examples of the chemical bonding method include a method of forming a covalent bond via a functional group such as a carboxyl group or 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 the porous pad, glass fiber filter paper having a pore diameter of 10 to 200 μm and a thickness of 300 to 3000 μm, a polypropylene nonwoven fabric, and other polymer nonwoven fabrics are preferably used. As described above, when a porous pad containing colored particles on the surface or inside thereof is used, it is preferable to dispose the pad above the surface of the sheet-like carrier described later. The porous pad may be stacked so as to be in direct contact with the sheet-like carrier, or the porous pad is disposed above the surface of the sheet-like carrier at regular intervals without contacting the sheet-like carrier. It may be.

(Sheet carrier)
The sheet-like carrier is a water-insoluble support and is used to immobilize the receptor and capture the colored particles / first receptor / analyte / second receptor complex and separate them from other components. Examples include membranes or filters. Such a membrane or filter is not particularly limited as long as it has a certain pore size capable of passing substances other than the analyte contained in the sample liquid and colored particles to which the first receptor binds. . Any commercially available one may be used, but nitrocellulose, acetate mixed nitrocellulose, nylon, polyethersulfone, polyvinylidene difluoride and the like are preferably used.

As described above, the pore size of the sheet-like carrier is not particularly limited because it depends on the size of the colored particles to which the analyte and the first receptor bind, but 0.22 μm to 12 μm is most often used.
The method for immobilizing (binding) the receptor on the sheet-like carrier may be physical adsorption or chemical bonding. Preparation of the sheet-like carrier to which the receptor is bound is carried out, for example, by adsorbing a solution obtained by diluting the receptor in a buffer solution or the like to the sheet-like carrier and then drying it.

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

In addition, the apparatus used in the second flow-through assay method of the present invention includes a porous pad having a colored particle bound with a first receptor bound to an analyte on the surface, and a first pad for the analyte. A flow-through assay device comprising a sheet-like carrier to which two receptors are bound. The method using this apparatus is as follows. A sample liquid containing an analysis object is dropped on the surface of the porous pad, and then passed through the sheet-like carrier, whereby the colored particles / first receptor / analyte / second A bireceptor complex is formed and detected.
FIG. 3 shows the cutting of the flow-through assay device of the present invention in which colored particles to which the receptor is bound are contained in or on the surface of the porous pad, and the porous pad is arranged above the surface of the sheet-like carrier. An end view is shown. In FIG. 3, d is a porous pad that contains colored particles on the surface or in which receptors for the analyte are bound.

  The flow-through assay device preferably has a liquid absorbing member that absorbs a liquid such as a sample solution dropped on the sheet-like carrier at the lower part of the sheet-like carrier. Examples of such a 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-like carrier to which the receptor is bound so that liquid absorption proceeds rapidly. The presence of such a liquid absorbing member that is in contact with or in close contact with the sheet-like carrier facilitates the passage of the sample liquid, such as a liquid, through the sheet-like carrier and enables rapid detection.

(Receptor)
The first and second receptors for the analyte are receptors that specifically react with the analyte to form a complex. Therefore, it is natural that the receptor to be used differs depending on the analyte, but in general, when the analyte is a bacterium, virus, hormone, other clinical marker, etc., it specifically reacts and binds to these. A polyclonal antibody, a monoclonal antibody, etc. are mentioned. In addition, virus antigens, virus hollow particles, recombinant E. coli expressed protein, recombinant yeast expressed protein and the like can be mentioned.
The first and second receptors used may be the same or different.

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

(Sample suspension)
The specimen can be collected from the patient's pharynx, nasal cavity, etc. using a cotton swab, etc., or a nasal aspirate can be used. The specimen thus obtained is usually suspended in the specimen suspension. Perform the assay. As the specimen suspension, buffers or the like usually used in sample detection or quantification methods by immunological techniques such as immunodiffusion method, enzyme immunoassay method, and aggregation method can be used.
More specifically, physiological saline, phosphate buffered saline (PBS), gelatin-added PBS, bovine serum albumin (BSA) -added PBS, Good's buffer, calf infusion broth (VIB), heart-in Examples include, but are not limited to, fusion broth, Eagle's minimum essential medium (EMEM), and BMEM-added EMEM. Moreover, you may use the said buffers in combination of 2 or more types.
Further, in addition to the above composition, basic amino acids, inorganic salts and / or surfactants may be added. By using a sample suspension containing at least two types of basic amino acids, inorganic salts, and surfactants, non-specificity to the carrier other than the analyte contained in the sample and the receptor bound to the carrier Bonding can be reduced, which is preferable.
In the method of the present invention, after the sample is suspended in the sample suspension, filtration may be performed before mixing with the colored particles.

(Flow-through assay kit)
The flow-through assay kit of the present invention is a kit used in the above-described flow-through assay method. The kit for flow-through assay of the present invention comprises at least the following (i) and (ii).
(i) a pad containing colored particles bound to the first receptor for the analyte or colored particles bound to the first receptor for the analyte
(ii) A sheet-like carrier to which a second receptor for the analyte is bound. If necessary, a specimen suspension and a colored particle diluent may be included. (i) and / or (ii) may be included in the kit in the form of the flow-through assay device described above.

  Further, if necessary, a negative control solution consisting only of a buffer for examining 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 and a sterilized cotton swab may be included.

[Example 1]
The following reagents and equipment were used.
1. Production of monoclonal antibody (production of anti-influenza A virus NP monoclonal antibody (mouse))
Spleens were excised from BALB / c mice immunized with purified influenza A virus antigen and maintained for a certain period of time, and mouse myeloma was prepared by the method of Keller et al. (Kohler et al., Nature, vol, 256, p495-497 (1975)). Fused with cells (P3x63).
The obtained fusion cell (hybridoma) is maintained in a 37 ° C. incubator, and purification of the cell (monocloning) is performed while confirming the antibody activity of the supernatant by ELISA using an influenza A virus NP antigen solid phase plate. went.
The obtained cell line was intraperitoneally administered to pristane-treated BALB / c mice, and about 2 weeks later, antibody-containing ascites was collected. IgG was purified from the obtained ascites by an ammonium sulfate fractionation method.
(Preparation of anti-type B influenza virus NP monoclonal antibody (mouse))
Spleen was excised from BALB / c mice immunized with purified influenza B virus antigen and maintained for a certain period of time, and mouse myeloma was prepared by the method of Keller et al. (Kohler et al., Nature, vol. 256, p495-5497 (1975)). Fused with cells (P3x63). 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 about 2 weeks later, antibody-containing ascites was collected. IgG was purified from the obtained ascites by an ammonium sulfate fractionation method.

2. Binding of gold colloid and monoclonal antibody Take 10 mL of gold colloid solution (BBI, particle size 40 nm, 0.01% chloroauric acid concentration) and adjust the pH to 5.5 with 100 mM potassium carbonate. The purified anti-type A or type B influenza virus NP monoclonal antibody was dialyzed against a 2 mM sodium borate solution (pH 9.2) to prepare a concentration of 100 μg / mL. To the colloidal gold solution adjusted to pH 5.5, an amount such that the final concentration of the antibody was 10 μg / mL was added, and gently stirred by hand so that the colloidal gold and the antibody were sufficiently bound. After 5 minutes, 1 mL of 10% BSA was added and gently stirred by hand for 10 minutes. The whole amount was transferred to a centrifuge tube and centrifuged at 14,000 rpm for 30 minutes at 4 ° C. After centrifugation, the supernatant was aspirated and discarded, and 1 mL of 10 mM Tris-HCl buffer pH 8.5 containing 1% BSA and 150 mM sodium chloride was added to the precipitated antibody-bound gold colloid to float.
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 carrier Nitrocellulose was used as the sheet carrier. Immobilization was performed by spotting two antibody solutions onto a nitrocellulose membrane. 1 μL of the solution containing 1 mg of the anti-influenza A virus NP monoclonal antibody was spotted in the A hole of the apparatus, and 12 μL of the solution containing 1 mg of the anti-B influenza virus NP monoclonal antibody was spotted in the B hole. As a buffer for diluting the antibody, a 10 mM phosphate buffer (pH 7.0) was used. After spotting, drying was performed for 40 minutes in a 45 ° C. drying room.

5. 1. Antigen detection method The antibody-bound gold colloid prepared in step 1 was diluted with 5 mM phosphate buffer (pH 7.4) so that the absorbance was 1.0 at OD 530 nm, and 5% BSA, 4.5% Triton X-100, 2% fish. A sample diluted with 5 mM phosphate buffer (pH 7.4) containing gelatin was diluted and mixed 1: 4, and 500 μL was dropped onto the assay device. Detection was carried out using the influenza A virus and influenza B virus diluted as positive specimens and the above buffer as a negative specimen. The specimen was dropped into the A hole and B hole in such a way that 500 μL of bubbles were not added, and allowed to stand at room temperature until there was no liquid on the membrane. Thereafter, the adapter was removed and judged visually. As a result of the determination, regarding A hole, influenza A virus could be detected up to 10 4 pfu / mL, and negative for influenza B virus. On the other hand, regarding the B hole, the influenza B virus could be detected up to 10 4 pfu / mL, and it was judged negative for the influenza A virus.

[Example 2]
Similar to Example 1, 1. Production of monoclonal antibodies 3. Binding of gold colloid and monoclonal antibody The antibody was immobilized on a carrier.
3. As the assay device, a device in which a porous pad containing colored particles was loaded on the surface of a sheet-like carrier as shown in FIG. 3 was used. That is, an antibody was detected as described below using an assay device prepared by drying gold colloid bound with a monoclonal antibody as described below.

5. 1. Drying of gold colloid The antibody-bound gold colloid prepared in step 1 was diluted with 10 mM sucrose-added 10 mM Tris-HCl buffer (pH 8.0) so that the absorbance was 1.0 at an OD of 530 nm, and a pad (Whatman: Conjugate Release Pad F075-14 ( The pore size was 23 μm in diameter and 355 μm in thickness) and dried with warm air.The anti-influenza A virus NP antibody adsorption pad after warm air blowing was placed in the A hole of the assay device, while anti-B type after drying with warm air The influenza virus NP antibody adsorption pad was placed in the B hole of the assay device.

6). Antigen detection method The specimen was diluted to 5% BSA, 4.5% Triton X-100, and 2% fish gelatin in 5 mM phosphate buffer (pH 7.4). Detection was carried out using the influenza A virus and influenza B virus diluted as positive specimens and the above buffer as a negative specimen. The sample was dropped into the A hole and B hole in 500 μL portions without bubbles, and allowed to stand at room temperature until no liquid was left on the membrane. Thereafter, the adapter was removed and judged visually. As a result of the determination, regarding A hole, influenza A virus could be detected up to 10 4 pfu / mL, and negative for influenza B virus. On the other hand, regarding the B hole, the influenza B virus could be detected up to 10 4 pfu / mL, and it was judged negative for the influenza A virus.

It is a top view of an example of the assay device of the present invention. FIG. 2 is an end view taken along the line I-I ′ of FIG. 1. It 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-like carrier c: Liquid absorbent member d: Porous pad containing colored particles

Claims (18)

  The sample liquid containing the analyte is brought into contact with the colored particles bound to the first receptor for the analyte, and then passed through a sheet-like carrier bound to the second receptor for the analyte. A flow-through assay method characterized by forming a colored particle / first receptor / analyte / second receptor complex on the surface of a sheet-like carrier and detecting the complex.   The method according to claim 1, wherein a porous pad is disposed above the surface of the sheet-like carrier, and the colored particles are contained in or on the surface of the porous pad.   The method according to claim 1 or 2, wherein the colored particles are gold colloid or colored latex.   The method according to any one of claims 1 to 3, wherein the sheet-like carrier is a membrane or a filter.   The method according to any one of claims 1 to 4, wherein the first and / or second receptor is an antibody.   The method according to any one of claims 1 to 5, wherein the analyte is a virus or a bacterium.   A kit for flow-through assay, comprising colored particles bound to a first receptor for an analyte and a sheet-like carrier bound to a second receptor for the analyte.   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.   The kit according to claim 7 or 8, wherein the colored particles are gold colloid or colored latex.   The kit according to any one of claims 7 to 9, wherein the sheet-like carrier is a membrane or a filter.   The kit according to any one of claims 7 to 10, wherein the first and / or second receptor is an antibody.   The kit according to any one of claims 7 to 11, wherein the analyte is a virus or a bacterium.   A flow-through assay device comprising a porous pad containing colored particles bound or bound to a first receptor for an analyte, and a sheet-like carrier bound to a second receptor for the analyte.   The device according to claim 13, wherein the porous pad is disposed above the surface of the sheet-like carrier.   15. An apparatus according to claim 13 or 14, wherein the colored particles are gold colloid or colored latex.   The device according to any one of claims 13 to 15, wherein the sheet-like carrier is a membrane or a filter.   The device according to any one of claims 13 to 16, wherein the first and / or second receptor is an antibody.   The apparatus according to any one of claims 13 to 17, wherein the analyte is a virus or a bacterium.

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