JP4616575B2 - Protein membrane immobilization method - Google Patents

Description

  The present invention relates to a method for immobilizing a protein on a membrane, an immobilized membrane, an immunoassay device, and an immunoassay kit.

2. Description of the Related Art Conventionally, an immunoassay method for detecting viruses, bacteria, and other substances in a sample using a membrane on which immune reaction components such as antibodies and antigens are immobilized has been known.
For example, an anti-influenza virus monoclonal antibody can be immobilized on a nitrocellulose filter, and the presence of an influenza virus antigen in a sample such as a body fluid of a subject can be detected using an immune reaction. For example, the following methods are known as methods for immobilizing proteins such as antibodies on the membrane. Prepare purified anti-influenza virus monoclonal antibody by suspending in 50 mM phosphate buffer (pH 7.4) and diluting with 50 mM phosphate buffer (pH 7.4) so that the absorbance at 280 nm is 1.0. The diluted solution is dropped on a nitrocellulose filter so as to be 10 μL / device, then allowed to stand at 45 ° C. for 40 minutes, and then dried (see, for example, Non-Patent Document 1 and Non-Patent Document 2).

A membrane immobilized by such a method exhibits a relatively high detection sensitivity when used immediately after the membrane is formed, but there is a problem that the sensitivity gradually decreases when used after storage for a certain period of time.
Troubleshooting Protein Binding in Nitrocellulose Membranes (Part 1): Principles, 1999-03, 32, Kevin Jones, In-Vitro Diagnostic Technology Magazine Troubleshooting Protein Binding In Nitrocellulose Membranes (Part 2): Common Problems, 1999-05, 26, Kevin Jones, In-Vitro Diagnostic Technology Magazine

  As described above, a film immobilized by a conventional method exhibits a relatively high detection sensitivity when used immediately after film formation, but has a problem that the sensitivity gradually decreases when used after storage for a certain period of time. The cause of the decrease in sensitivity is not clear, but is thought to be due to a decrease in the activity of the immobilized protein.

  Therefore, an object of the present invention is to provide a protein membrane immobilization method in which the activity of a protein immobilized on a membrane is maintained without being lowered even when stored for a long period of time. Another object of the present invention is to provide a protein-immobilized membrane in which the activity of the immobilized protein is maintained without being lowered even after long-term storage. Another object of the present invention is to provide an immunoassay device and an immunoassay kit using the protein-immobilized membrane.

  It has been found that the above problem can be solved by suspending the protein to be immobilized in a saccharide-containing aqueous solution.

  That is, the present invention relates to a method for immobilizing a protein on a membrane, wherein the protein is suspended and immobilized in a saccharide-containing aqueous solution or a saccharide-containing buffer.

  The present invention also relates to a protein-immobilized membrane prepared by the above method. The present invention also relates to an immunoassay device including the membrane. Furthermore, the present invention relates to an immunoassay kit including the above immunoassay device.

  The present invention provides a method for efficiently and stably immobilizing immune reaction components on a diagnostic membrane filter for a long period of time.

  The method of the present invention will be described. The method of the present invention is a method for immobilizing a protein on a membrane, characterized by suspending the protein in a saccharide-containing aqueous solution or a saccharide-containing buffer and immobilizing the protein.

  In the present invention, the “protein” may be any protein as long as it can serve as a ligand for the analyte in a method for measuring the analyte in the sample such as immunoassay. Specific examples include immune reaction components selected from the group consisting of antibodies, antigens, receptors and the like. More specifically, polyclonal antibodies, monoclonal antibodies, receptors, etc. that specifically react with and bind to bacteria, viruses, hormones, other clinical markers, etc., or virus antigens, virus hollow particles, recombinant E. coli expressed proteins, genes Examples include recombinant yeast-expressed proteins.

  In the method of the present invention, the membrane can immobilize proteins and is usually insoluble in water. Moreover, it is preferable to have a certain pore size, thickness, and strength so that the complex of the protein and the analyte in the sample can be separated from substances contained in other samples. Any membrane may be used as long as it is commercially available, but a membrane made of a material such as nitrocellulose, acetate-mixed nitrocellulose, nylon, polyethersulfone, or polyvinylidene difluoride is preferably used.

  The pore size of the membrane depends on the analyte to be measured, the detection reagent (enzyme, gold colloid, colored latex, etc.) and the target sensitivity, and is not particularly defined, but 0.22 μm to 12 μm is most often used.

In the method of the present invention, the protein is immobilized after the protein is suspended in a saccharide-containing aqueous solution or a saccharide-containing buffer.
The saccharide in the saccharide-containing aqueous solution or saccharide-containing buffer may be a monosaccharide or a disaccharide, and may be a polysaccharide, but from the viewpoint of ease of handling due to solubility in a solvent and viscosity of the liquid, etc. Disaccharides are preferred. Examples of the saccharide include glucose, sucrose, trehalose, mannitol, or sorbitol, or a mixture of two or more thereof.

  The concentration of saccharides in water or buffer is preferably in the range of 0.1 w / v% to 5 w / v%, more preferably 0.5 w / v% to 5 w / v%, still more preferably Is in the range of 1 w / v% to 5 w / v%. The solvent in the saccharide-containing aqueous solution is usually water, but a trace amount of alcohol (for example, 1 to 5 v / v% methanol) may be added.

It is preferable to use a buffer solution as the solvent to which the saccharide is added because it has a synergistic effect of improving storage stability for a long period of time.
Moreover, from the viewpoint of the effect of the present invention, the saccharide-containing buffer solution is preferably a solution obtained by adding saccharides to an acidic buffer solution having a pH of less than 7.0. Furthermore, the buffer solution is more preferably a buffer solution having a pH of 4.0 to 5.5 from the viewpoint of the effect of the present invention, and further preferably pH 4.0 to 5.0. Specifically, citrate buffer (pH 4.0 to 5.5), oxalate buffer, tartaric acid buffer, acetic acid buffer, phthalate buffer and other organic acid buffers, and phosphoric acid and other inorganic acids A buffer solution may be mentioned. Among these, an organic acid is preferable from the viewpoint of safety in handling and economy, and a citrate buffer is particularly preferable. The concentration of the buffering agent in the acidic buffer is preferably in the range of 1 to 500 mM from the viewpoints of effect and economy.

One embodiment of the method of the present invention will be described taking the immobilization of anti-influenza virus monoclonal antibody (mouse) on a nitrocellulose filter as an example.
The anti-influenza virus monoclonal antibody obtained from the mouse is purified by affinity column chromatography using a protein A column. The obtained antibody is obtained in a state suspended in a buffer solution of pH 4-7. The buffer is replaced with 10 mM citrate buffer (pH 4.0) supplemented with 0.1 w / v% trehalose using a Sephadex G-25 gel filtration column. Dilute with 10 mM citrate buffer (pH 4.0) to which 0.1 w / v% trehalose has been added so that the absorbance at 280 nm is 1.0, and add an appropriate amount (for example, 10 μL / device for flow-through diagnostic equipment). ) Drip onto the nitrocellulose membrane attached to the assay device. Subsequently, it is left to stand at 45 ° C. for 40 minutes and dried to obtain an anti-influenza virus monoclonal antibody-immobilized nitrocellulose membrane.

 Since the immobilization is performed more efficiently by incubating at a constant temperature for a certain time after immobilization on the nitrocellulose membrane as described above, it is preferable. Preferable incubation conditions include, for example, standing at 37 ° C. overnight, standing at 45 ° C. for 60 minutes or more, or standing at 55 ° C. for 30 minutes or more. When drying at a higher temperature than this, the component to be immobilized may be affected by heat and impair its activity (function), so it must be set according to the nature of the immobilized component. On the other hand, incubating at a lower temperature for a shorter time does not sufficiently remove moisture and may adversely affect the storage stability of the component to be immobilized. Therefore, a method of promoting drying under reduced pressure is employed.

An immunoassay device can be created using the protein-immobilized membrane obtained as described above.
Examples of the immunoassay device include devices that can be used in immunoassay methods such as flow-through type or lateral flow type immunoassay.

A flow-through type immunoassay device usually has a housing on which the protein-immobilized membrane is immobilized. In some cases, a liquid absorbing member is installed in the lower part of the protein-immobilized film so as to be in close contact with the protein-immobilized film, so that the liquid dripped from the upper surface is quickly absorbed. Further, an adapter having an opening for introducing a liquid into a predetermined region where the protein on the membrane is immobilized may be installed on the membrane.
In the lateral flow type immunoassay device, the protein-immobilized membrane is usually pressure-bonded to a sample dropping member, a detection reagent holding member, and an absorbent paper, and this is further installed in a housing case, or the contact of each member is not impaired. Each member may be pressure-bonded with an insoluble transparent seal.

The immunoassay kit of the present invention includes the above-described immunoassay device. The kit of the present invention can be used for a flow-through type immunoassay method or a lateral flow type immunoassay method, and contains reagents necessary for these methods.
Specifically, it is preferable to include the following (1) to (3).
(1) Immunoassay device including protein-immobilized membrane (2) Labeled detection reagent (3) Sample suspension composition and / or washing composition

  Here, the labeled detection reagent is capable of specifically binding to the analyte captured on the membrane and forming a complex with the analyte, and after forming the complex with the analyte. It means a detection reagent labeled so that it can be detected by any means. For example, when the analyte is an antigenic substance such as a virus, it means an antibody against the virus and labeled with an enzyme or the like. When labeled with an enzyme in this way, the complex can be detected by adding a substrate of the enzyme that produces a substance detectable by a colorimetric method or a fluorescence method by a reaction catalyzed by the enzyme. It can be carried out. Examples of the detection reagent before labeling are the same as those described for the capture reagent. Examples of the label include an enzyme, a fluorescent label, a magnetic label, a radioisotope, a gold colloid, and a colored latex. Usually, an enzyme label is used from the viewpoint of convenience and economy. When an enzyme label is used, examples of the enzyme used include alkaline phosphatase, peroxidase, and glucose-6-phosphate dehydrogenase.

When an enzyme label is used, the kit may contain an enzyme substrate that develops color by enzymatic degradation, a reaction stop solution, and the like, if necessary.
The enzyme substrate is usually a substrate for the enzyme, and generates a substance detectable by a colorimetric method or a fluorescence method by a reaction catalyzed by the enzyme. Specific examples include 5-bromo-4-chloro-3-indolyl phosphate, nitrotetrazolium blue, tetramethylbenzidine, and glucose-6-phosphate NAD +.
The reaction stop solution is a solution for stopping the reaction between the enzyme and the substrate. Specific examples include citric acid and sulfuric acid.

  Further, if necessary, it may contain a filter tube for specimen sample, a negative control solution consisting only of 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. . Further, a filter for filtering the sample and a sterilized cotton swab may be included.

Below, an example of a more specific procedure is demonstrated and demonstrated about the flow through assay method which detects an analyte using the said kit.
(1) A specimen sample collected from the pharynx or nasal cavity of a patient infected with a virus or bacteria is suspended in the specimen suspension composition.
(2) The suspended liquid is put into a specimen sample filtration tube and filtered.
(3) The filtrate is dropped onto a membrane (protein-immobilized membrane) on which an antigen such as a virus or a bacterium is immobilized in an immunoassay device, and the analyte (antigen such as a virus or bacterium) is placed on the membrane. To capture.
(4) A labeled detection reagent that specifically binds to the analyte is dropped on the membrane to form a protein (antibody) / analyte / labeled detection reagent complex.
(5) The presence / absence of the analyte in the sample is measured by detecting the presence of the complex with the labeled detection reagent in the complex. Specifically, when an enzyme-labeled detection reagent is used, a substrate that produces a substance that can be detected by a colorimetric method or a fluorescence method is added by a reaction catalyzed by the enzyme. In some cases, a reaction stop solution is added to stop the reaction, and then the presence or absence of the analyte is detected by a colorimetric method or the like.

Hereinafter, the present invention will be described more specifically based on examples. However, the present invention is not limited to the following examples.
Example 1 Detection of influenza A virus using a flow-through type assay device (1) Preparation of colloidal gold antibody
Take 10 mL of gold colloid and adjust the pH to 7.0 with 100 mM potassium carbonate. Prepare anti-influenza A virus monoclonal antibody purified by dialysis, centrifugation and purification with 2 mM boric acid solution to a concentration of 100 μg / mL with 2 mM boric acid solution. An amount of 10 μg / mL final concentration of the prepared anti-influenza A virus monoclonal antibody is added to the colloidal gold with sufficient stirring. After 5 minutes, add 1 mL of 10% BSA and gently agitate with a rotator for 10 minutes. Transfer the entire volume to a centrifuge tube and centrifuge at 14000 rpm for 30 minutes at 4 ° C. After centrifugation, the supernatant is aspirated and discarded, and a solution containing 10 mM Tris-HCl buffer, 1% BSA, 150 mM sodium chloride in a sensitized version of colloidal gold colloid and anti-influenza A virus monoclonal antibody. Float with 1 mL.

(2) Immobilization of antibody on diagnostic membrane filter and production of assay device An example of immobilization of anti-influenza virus monoclonal antibody (mouse) on a nitrocellulose filter will be described.
Prepare an anti-influenza virus monoclonal antibody (mouse) affinity-purified with a protein A column. Use a Sephadex G-25 gel filtration column to buffer the antibody in a 0.1 w / v%, 0.3 w / v% or 1 w / v% trehalose aqueous solution or 1 w / v% trehalose-added 10 mM citrate buffer. Replaced.
As controls, purified water (Control 1) and 25 mM sodium phosphate buffer (pH 7.0) (Control 2) were used.
At this time, at pH 4.0, the antibody was slightly insolubilized during long-term liquid storage, so the protein concentration was measured, diluted, and filtered immediately before the solid phase.

Dilute with each solution so that the absorbance at 280 nm is 1.0, and after 0.22 μm filtration, apply the appropriate amount (for example, 10 μL / device for a flow-through diagnostic device) onto the nitrocellulose membrane attached to the assay device. The solution was added dropwise, then left at 45 ° C. for 40 minutes and dried.
The device after fabrication was sealed in an aluminum pouch with a desiccant and stored under each condition until used in the assay.
Storage is usually performed at about 15 to 30 ° C., but a test for storage at 45 ° C. was also conducted in order to examine the stability under severe conditions.

(3) Detection method
Sample 1 (strong positive) containing influenza A virus at 10 ⁷ pfu / ml or more, sample 2 (medium positive) containing around 10 ⁶ pfu / ml, and sample 3 (weak positive) containing 10 ⁴ to 10 ⁵ pfu / ml are appropriate Suspended in various buffers. A buffer solution containing nothing was used as a negative sample. 500 μL of the solution and 100 μL of colloidal gold antibody were mixed and reacted for a certain time (for example, 10 minutes). After reacting for a certain period of time, the solution was filtered through a filter (for example, 0.22 μm), and then dropped entirely into the assay device (device). After the liquid is completely absorbed by the membrane member, if the membrane member where the anti-influenza virus monoclonal antibody is adsorbed is colored in the color of colloidal gold (eg, red to reddish brown), the influenza A virus in the sample Is confirmed to be present (+). If there is no change in color tone and the color of the membrane member remains, influenza A virus is not present in the sample (-).

[Table 1]

[Table 2]

[Table 3]

[Table 4]

As is apparent from Table 1, when immobilized using purified water or sodium phosphate buffer (25 mM, pH 7.0), medium positive specimens could not be detected after storage at 45 ° C. for 10 days. . After 5 days and 10 days storage at 45 ° C., weak positive specimens could not be detected.
On the other hand, when suspended in purified water (0.1-1 w / v%) with trehalose and immobilized, the medium positive sample should be detected even after storage at 45 ° C. for 10 days. I was able to. In addition, when suspended in purified water (1 w / v%) added with trehalose and immobilized, weak positive specimens could be detected even after storage at 45 ° C. for 5 and 10 days (Table). 2).
In addition, when suspended in 10 mM sodium citrate buffer (pH 4.5) supplemented with 1 w / v% trehalose, it was possible to detect weakly positive specimens even after storage at 45 ° C. for 5 and 10 days. Could be done (Table 3).

As is clear from Table 4, when immobilized using 25 mM sodium phosphate buffer (pH 7.0), weak positive specimens cannot be detected after storage at 8 ° C. for 3 and 7 months. It was.
On the other hand, when suspended in 10 mM sodium citrate buffer (pH 4.5) supplemented with 1 w / v% trehalose, weak positive specimens can be detected after storage at 8 ° C. for 7 months. did it.

Example 2 Detection of influenza A virus using a lateral flow apparatus (1) Preparation of colloidal gold antibody
10 mL of gold colloid was taken and adjusted to pH 7.0 with 100 mM potassium carbonate. Anti-influenza A virus monoclonal antibody purified by dialysis, centrifugation and purification with 2 mM boric acid solution was prepared with 2 mM boric acid solution to a concentration of 100 μg / mL. An amount of the prepared anti-influenza A virus monoclonal antibody at a final concentration of 10 μg / mL was added to the gold colloid with sufficient stirring. After 5 minutes, 1 mL of 10% BSA was added and gently stirred for 10 minutes with a rotator. Transfer the entire volume to a centrifuge tube and centrifuge at 14000 rpm for 30 minutes at 4 ° C. After centrifugation, the supernatant is aspirated and discarded, and a solution containing 10 mM Tris-HCl buffer, 1% BSA, and 150 mM sodium chloride is sensitized to the precipitated gold colloid and anti-influenza A virus monoclonal antibody. It floated at 1 mL.

(2) Production of lateral flow type equipment
A very small amount of anti-influenza virus monoclonal antibody (about 2 μL / cm at a concentration of 1 mg / mL) is dropped on a membrane member for detecting influenza A virus (High Flow Membrane Plus 135 manufactured by Millipore) and at a constant temperature for a certain period of time ( (45 ° C., 10 minutes to 60 minutes) and allowed to adsorb to the membrane member.

(3) Detection method
Sample 1 (strong positive) containing influenza A virus at 10 ⁷ pfu / ml or more, sample 2 (medium positive) containing around 10 ⁶ pfu / ml, and sample 3 (weak positive) containing 10 ⁴ to 10 ⁵ pfu / ml are appropriate Suspended in various buffers. A buffer solution containing nothing was used as a negative sample. 200 μL of the solution and 50 μL of colloidal gold antibody were mixed and reacted for a predetermined time (10 minutes). After reacting for a certain period of time, the mixture was filtered with a filter (0.22 μm), and then the entire amount was dropped onto the pad. If the liquid expands the membrane member and the membrane member where the anti-influenza virus monoclonal antibody is adsorbed is colored in the color of gold colloid (eg, red to reddish brown), influenza A virus is present in the sample. (+) Was confirmed. If there is no change in color tone and the color of the membrane member remains, influenza A virus is not present in the sample (-).

[Table 5]

[Table 6]

[Table 7]

As is apparent from Table 5, when immobilized using purified water or sodium phosphate buffer (25 mM, pH 7.0), medium positive specimens could not be detected after storage at 45 ° C. for 10 days. . After 5 days and 10 days storage at 45 ° C., weak positive specimens could not be detected.
On the other hand, when suspended in purified water (0.1-1 w / v%) to which trehalose has been added and immobilized, the medium positive sample should be detected even after storage at 45 ° C. for 10 days. I was able to. In addition, when suspended in purified water (1 w / v%) added with trehalose and immobilized, weak positive specimens could be detected even after storage at 45 ° C. for 5 days and 10 days (Table). 6).
In addition, when suspended in 10 mM sodium citrate buffer (pH 4.5) supplemented with 1 w / v% trehalose, it was possible to detect weakly positive specimens even after storage at 45 ° C. for 5 and 10 days. Could be done (Table 7).

Claims (12)

A method for immobilizing a protein on a membrane,
step of floating the protein to the sugar analog-containing buffer below pH 7.0,
Dropping the protein suspension on the membrane, and drying the membrane on which the protein suspension is dropped;
Including the above method. The method of claim 1, wherein the saccharide is selected from the group consisting of glucose, sucrose, trehalose, mannitol, and sorbitol. The method according to claim 1 or 2, wherein the saccharide is contained in the saccharide-containing aqueous solution or saccharide-containing buffer in the range of 0.1 w / v% to 5 w / v%. The method according to any one of claims 1 to 3 , wherein the saccharide-containing buffer is an acidic buffer having a pH of 4.0 to 5.5. The method according to any one of claims 1 to 4 , wherein the concentration of the buffer in the saccharide-containing buffer is in the range of 1 to 500 mM. The method according to any one of claims 1 to 5 , wherein the saccharide-containing buffer is an organic acid buffer. The method according to claim 6 , wherein the organic acid buffer is a citrate buffer. The method according to any one of claims 1 to 7 , wherein the protein is an immune reaction component selected from the group consisting of an antibody, an antigen, and a receptor. Film to immobilize the protein, nitrocellulose, acetate mixed nitrocellulose, nylon, polyethersulfone, and a film of a material selected from the group consisting of polyvinylidene difluoride, claim 1-8 one The method according to item. A protein-immobilized membrane prepared by the method according to any one of claims 1 to 9 . An immunoassay device comprising the membrane according to claim 10 . An immunoassay kit comprising the immunoassay device according to claim 11 .