JP2008275511A - Method for measuring immunity of influenza virus antigen and device used for the same - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method capable of measuring an influenza virus antigen with high sensitivity and conveniently measuring it and a device therefor. <P>SOLUTION: The method for measuring immunity of an influenza virus antigen using an anti-influenza virus antibody serves a sample to be tested having a specimen processed by neuraminidase for an antigen antibody reaction with the antibody. For the method for measuring immunity, the sandwich type immunity measurement method that sandwiches the antigen between a first and a second antibody against an influenza virus antigen is preferred, specially an immuno-chromatography measuring method is preferred. Neuraminidase may be included in an extraction liquid to obtain a sample to be tested that serves for the immunity measurement of an influenza virus antigen by diluting a specimen or in a development solvent used for the influenza virus antigen measurement or may be maintained in such a shape as to be impregnated in an impregnation member under a freeze drying state so that it is put in the extraction liquid or the developing solvent when used or can be used by arranging on a film carrier for chromatography. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  The present invention relates to a highly sensitive immunoassay method for influenza virus antigens, and to an object used in the assay method, and in particular, an immunoassay method and apparatus useful for quickly and easily diagnosing influenza virus infection. About.

  Diagnosis of highly pathogenic avian influenza is currently a method of isolating a virus after collecting tracheal swab or cloacaswab (total excretory swab) from a diseased bird suspected of viral infection, inoculating it into a growing chicken egg, and culturing it It is done by. However, this method is disadvantageous in that it takes several days to obtain a result and the result cannot be obtained quickly. In addition, the time taken to obtain results is greatly shortened by using recently developed genetic tests (PCR method, LAMP method). However, because such genetic tests require special equipment and technology, poultry farming It is not possible to inspect on site.

  On the other hand, immunochromatographic measurement methods and diagnostic methods using immunochromatographic test strips have been developed. However, although they are excellent in rapidity, they are inferior in detection sensitivity compared to culture tests and genetic tests. In addition, in such an immunoassay, a nonspecific reaction is likely to occur due to the sample component, and the sensitivity that should originally be obtained cannot often be obtained due to inhibition of the antigen-antibody reaction by the sample component.

  Conventionally, by adding bovine serum albumin (BSA) and other compounds to the developing solvent used in immunochromatographic measurement methods, it has been proposed to improve sensitivity by preventing nonspecific aggregation and nonspecific reactions during measurement. (Patent Document 1). However, it has been confirmed that the sensitivity of specimens collected from birds infected with influenza viruses, particularly influenza virus H5 subtype, is low, and further improvement in sensitivity has been desired.

JP 2003-344406 A

  Then, this invention aims at providing the method and apparatus which can measure an influenza virus antigen with high sensitivity, and also can measure easily.

  As a result of examining the influence on the immunoassay sensitivity of influenza virus antigens by treating the specimen with various enzymes, the present inventors have found that when the specimen is treated with neuraminidase and used as a test sample, the above immunity The present inventors have found that the measurement sensitivity is significantly improved and have completed the present invention.

That is, according to one aspect of the present invention, an immunoassay method for an influenza virus antigen using an anti-influenza virus antibody, comprising subjecting a test sample containing a specimen treated with neuraminidase to an antibody antigen reaction with the antibody. A featured immunoassay for influenza virus antigens is provided.
The immunoassay method in this detection method is not particularly limited, but is a sandwich immunoassay method in which the antigen is sandwiched between the first antibody and the second antibody against influenza virus antigen, particularly ELISA ( High effects can be obtained in the enzyme-linked immunosorbent assay method, the immunochromatography measurement method, and the like.

  Therefore, according to another aspect of the present invention, the influenza virus antigen immunoassay method is a sandwich immunoassay method in which the antigen is sandwiched between a first antibody and a second antibody against the influenza virus antigen. Is provided.

  Thus, according to a preferred embodiment of the present invention, a membrane carrier comprising a capture site formed by preliminarily fixing a first antibody against an influenza virus antigen in a predetermined position is prepared, and a second antibody against the influenza virus antigen is prepared. A mixed solution with a predetermined amount of the test sample is chromatographed on the membrane carrier toward the capture site, and a complex comprising the influenza virus antigen and the second antibody contained in the test sample is obtained. There is provided an immunochromatographic assay method for capturing at a capture site, wherein the test sample is treated with neuraminidase before or simultaneously with chromatographic development, wherein the test sample is treated with neuraminidase.

The treatment with the neuraminidase can be most simply performed by mixing the mixed solution or the test sample with neuraminidase. Specifically, for example, the mixed solution or the test sample is mixed with neuraminidase in a container separate from the membrane carrier before chromatographic development. Neuraminidase may be preliminarily impregnated into an impregnated member in a freeze-dried state, and the test sample may be treated with neuraminidase by introducing the impregnated member into the container.
As another method, neuraminidase may be arranged on a membrane carrier, and the mixture and neuraminidase may be mixed when the mixture is chromatographed.

  In addition, according to the present invention, as an apparatus suitable for carrying out the immunochromatographic measurement method of the present invention, the apparatus comprises at least a first antibody against an influenza virus antigen, a second antibody, and a membrane carrier, The antibody is preliminarily fixed at a predetermined position on the membrane carrier to form a capture site, and the second antibody is labeled with an appropriate labeling substance and chromatographed on the membrane carrier at a location separated from the capture site. An influenza virus antigen detection kit comprising at least an immunochromatographic test strip arranged as possible and a developing solvent for preparing a test sample so that the specimen can be diluted and chromatographed on the membrane carrier. And further comprising a neuraminidase to be added to the developing solvent. Detection kit is provided.

In this kit, the sample is mixed with a neuraminidase-containing developing solvent before chromatographic development, and treatment with neuraminidase is performed reliably, and the immunochromatographic test strip can be used as it is. Convenient.
In this kit, neuraminidase is impregnated in advance in a freeze-dried state on an impregnated member, and the test sample treated with neuraminidase is obtained by placing the impregnated member in a container containing a specimen. Good.

Further, according to the present invention, at least a first antibody against influenza virus antigen, a second antibody, and a membrane carrier are provided, and the first antibody is fixed in advance on a predetermined position of the membrane carrier to form a capture site. The second antibody is an immunochromatographic test strip labeled with an appropriate labeling substance and arranged so as to be chromatographically developed on the membrane carrier at a position separated from the capture site, the neuraminidase An immunochromatographic test strip for detecting influenza virus antigens is provided, wherein the second antibody is disposed at or near the position where the second antibody is disposed on the membrane carrier. Neuraminidase is preferably placed in a lyophilized state. The neuraminidase may be disposed on the membrane carrier in the state of being impregnated in the impregnation member. For example, the impregnation member connected to the membrane carrier may be impregnated with the second antibody.
In this immunochromatographic test strip, the sample is treated with neuraminidase placed on the membrane carrier at the same time as the chromatographic development, so that the measurement method of the present invention can be carried out in the same manner as in the conventional immunochromatographic test strip. It is.

Furthermore, according to the present invention, as an object suitable for carrying out the above-described immunoassay, an extract for obtaining a test sample for use in immunoassay of influenza antigen by diluting a specimen, which contains neuraminidase An extract for immunoassay characterized by the above is provided.
Furthermore, according to the present invention, there is provided an extract preparation kit for obtaining a test sample to be used for immunoassay of an influenza antigen by diluting a specimen, as another suitable for carrying out the above immunoassay method, There is provided an extract preparation kit for immunoassay comprising a solvent and an impregnated member impregnated with neuraminidase in a lyophilized state, and capable of preparing an extract by adding the impregnated member to the solvent during use.
When the extract has a composition as a developing solvent for immunochromatography measurement, it can be used as it is as a test sample for immunochromatography measurement by diluting the specimen with the extract.

According to the present invention, in the immunoassay method of influenza virus antigen using anti-influenza virus antibody, the sample was treated with neuraminidase and subjected to antibody antigen reaction with the antibody. Nonspecific reaction can be prevented and immunoassay sensitivity can be improved.
As a cause of the low immunoassay sensitivity of specimens collected from birds infected with influenza virus, particularly influenza virus H5 subtype, viruses in biological components (cell components, mucosal components, etc.) contained in specimens such as tracheal swab and cloacaswab It was speculated that they were confined while being coupled.
According to the present invention, since the specimen is pre-enzymatically treated with neuraminidase having an activity of cleaving the sugar chain on the surface of the biological component contained in the specimen, more hemagglutinin molecules and virus particles are obtained from the biological component. Is released and becomes involved in the antibody-antigen reaction, and is considered to have improved sensitivity. This effect is considered to be effective in a sandwich immunoassay, particularly an immunochromatographic assay, in which the antigen is sandwiched between a first antibody and a second antibody against an influenza virus antigen, and in particular, an influenza virus hemagglutinin antigen. It is considered effective in a detection system using an antibody that recognizes.

  In the immunoassay method of the present invention, in order to treat a sample with neuraminidase, the sample and neuraminidase may be mixed in an appropriate aqueous solvent, for example, water, physiological saline, or a buffer solution. The neuraminidase enzyme activity is highest at 37 ° C., but the treatment can usually be performed at room temperature. It is preferable to adjust the pH of the solvent to the optimum pH (4.5 to 7.0) of neuraminidase using an appropriate pH adjuster as necessary. However, in the immunochromatography measurement method, the pH of the developing solvent is preferably in the range of 6.7 to 7.7. Examples of the buffer include Tris buffer and phosphate buffer, and HEPES (2-hydroxypiperazine-N′-2-ethanesulfonic acid) buffer can also be used. As a pH adjuster, hydrochloric acid, sodium hydroxide, etc. can be used, for example.

In the present invention, a solvent containing a predetermined amount of neuraminidase can be provided in the form of a specimen extract. In this case, treatment with neuraminidase can be carried out by adding the sample to the extract and diluting it. This treatment solution can be used for immunoassay as a test sample as it is, which is convenient.
In order to prevent inactivation of neuraminidase during storage, the extract is impregnated with neuraminidase in a freeze-dried state, and the extract is added by adding the impregnation member to a solvent during use. The extract may be prepared in the form of an immunoassay extract preparation kit. As the impregnated member, for example, glass fiber nonwoven fabric, cellulose cloth (filter paper, nitrocellulose membrane, etc.), porous plastic cloth such as polyethylene and polypropylene, and the like can be used.

When the immunoassay method of the present invention is an immunochromatography assay method, a solvent containing a predetermined amount of neuraminidase can be provided in the form of a developing solvent for immunochromatography assay. In this case, treatment with neuraminidase can be performed by adding the sample to a developing solvent and diluting it. This treatment solution can be conveniently used as it is for measurement using a conventional immunochromatographic test strip as a test sample.
In this developing solvent for immunochromatography, in order to prevent inactivation of neuraminidase during storage, neuraminidase is impregnated into the impregnated member in a freeze-dried state, and the impregnated member is added to the developing solvent during use. It is good also as a form of the developing solvent preparation kit for immunoassay which can prepare the said developing solvent by this. As the impregnated member, for example, glass fiber nonwoven fabric, cellulose cloth (filter paper, nitrocellulose membrane, etc.), porous plastic cloth such as polyethylene and polypropylene, and the like can be used.
The extract and the developing solvent may contain various additives such as surfactants, preservatives, inorganic salts, and polymers described in JP-A-2003-344406 as necessary.

The anti-influenza virus antibody used in the present invention is not particularly limited, and polyclonal antibodies and monoclonal antibodies usually used in various immunoassays can be used. Of these, from the viewpoint of reaction specificity, it is usually preferable to use a monoclonal antibody. When the immunoassay method of the present invention is a sandwich-type immunoassay method in which the antigen is sandwiched between a first antibody and a second antibody, particularly an immunochromatographic assay method, the first antibody and the second antibody It is preferable that at least one of the antibodies is a monoclonal antibody.
Since the immunoassay method of the present invention is effective for improving the detection sensitivity of influenza virus H5 subtype, it is preferable to use an antibody that recognizes influenza virus H5 subtype as an anti-influenza virus antibody.

A polyclonal antibody can be obtained, for example, from an antiserum obtained by immunizing an animal according to a conventional method using influenza virus as an antigen.
For example, after immunizing an animal such as a mouse with an influenza virus as an antigen, the monoclonal antibody was selected from fused cells obtained by cell fusion of spleen cells and myeloma cells of the immunized animal in a HAT-containing medium. It can be obtained by growing the strain later and selecting the grown strain by, for example, enzyme-labeled immunization using the purified protein obtained as described above.

The immunochromatographic measurement method of the present invention can be easily carried out in accordance with the configuration of a known immunochromatographic test strip.
In general, such an immunochromatographic test strip is labeled with a first antibody capable of reacting with an antibody at the first antigenic determinant of the antigen and an antibody antigen reactive with the second antigenic determinant of the antigen. At least a second antibody and a membrane carrier, wherein the first antibody is preliminarily fixed at a predetermined position of the membrane carrier to form a capture site, and the second antibody is separated from the capture site. The membrane carrier is arranged and configured to be chromatographed. As described above, each of the first antibody and the second antibody may be a polyclonal antibody or a monoclonal antibody, but at least one of them is preferably a monoclonal antibody. Usually, the first antibody and the second antibody are used in a “hetero” combination, that is, the first antibody and the second antibody, each recognizing each antigenic determinant differing in position and structure on the antigen. These antibodies are used in combination. However, the first antigenic determinant and the second antigenic determinant may be structurally identical if they have different positions on the antigen, in which case the first antibody and the second antibody are homologous. In combination, i.e., the same monoclonal antibody can be used for both the first antibody and the second antibody.

A specific example of the immunochromatographic test strip is, for example, the test strip shown in FIG. In FIG. 1, numeral 1 is an adhesive sheet, 2 is an impregnation member, 3 is a membrane carrier, 31 is a capture site, 4 is an absorption member, and 5 is a sample addition member.
In the example shown in the figure, the membrane carrier 3 is made of a strip-like nitrocellulose membrane filter having a width of 5 mm and a length of 36 mm.
The first antibody is fixed to the membrane carrier 3 at a position 7.5 mm from the end on the side of the chromatographic expansion start point, and a specimen capture site 31 is formed.
In the illustrated example, the membrane carrier 3 uses a nitrocellulose membrane filter. However, the membrane carrier 3 should be capable of chromatographic development of the specimen contained in the test sample and immobilize the antibody forming the capture site 31. Any cellulose membrane, nylon membrane, glass fiber membrane, etc. can be used.

The impregnated member 2 is a member impregnated with a second antibody that reacts with the antigen with the second antigen determinant located at a different site from the first antigen determinant to which the first antibody binds. Become. The second antibody is previously labeled with an appropriate labeling substance.
In the illustrated example, a 5 mm × 15 mm strip-shaped glass fiber nonwoven fabric is used as the impregnating member 2, but is not limited thereto, and examples thereof include cellulose cloth (filter paper, nitrocellulose membrane, etc.), polyethylene, Porous plastic cloths such as polypropylene can also be used.

The labeling substance for the second antibody may be any substance as long as it can be used, and examples thereof include a coloring labeling substance, an enzyme labeling substance, and a radiation labeling substance.
Among these, it is preferable to use a color labeling substance from the viewpoint that the color change at the capturing site 31 can be determined quickly and easily by observing with the naked eye.
Examples of the color labeling substance include metal colloids such as gold colloid and platinum colloid, synthetic latex such as polystyrene latex colored with respective pigments such as red and blue, and latex such as natural rubber latex. Of these, metal colloids such as gold colloid are particularly preferred.
The impregnated member 2 can be produced by impregnating the labeled second antibody suspension into a member such as the glass fiber non-woven fabric and drying it.

As shown in FIG. 1, the membrane carrier 3 is stuck in the middle of the pressure-sensitive adhesive sheet 1 and the chromatographic development start side of the membrane carrier 3 (that is, the left side of FIG. 1, hereinafter referred to as “upstream side”, The downstream side of the impregnation member 2 is overlapped and connected to the opposite end, that is, the right side of FIG. Can be attached to the pressure-sensitive adhesive sheet 1 to produce the immunochromatographic test strip of the present invention.
Further, if necessary, the downstream portion of the sample addition member 5 may be placed on the upper surface of the impregnation member 2, and the upstream portion of the sample addition member 5 may be attached to the adhesive sheet 1. Further, the upstream portion of the absorbing member 4 can be placed on the upper surface of the downstream portion of the membrane carrier 3, and the downstream portion of the absorbing member 4 can be attached to the adhesive sheet 1.

As the sample addition member 5, for example, a porous synthetic resin sheet or film such as porous polyethylene or porous polypropylene, and a paper or woven or non-woven fabric made of cellulose such as filter paper and cotton cloth are used. be able to.
The absorbing member 4 may be of any material that can absorb and hold liquid quickly, and examples thereof include cotton cloth, filter paper, and porous plastic nonwoven fabric made of polyethylene, polypropylene, etc., and filter paper is particularly suitable. is there.

  Further, in the case of a commercially available product, the immunochromatographic test strip of FIG. Provided.

Thus, after a specimen composed of a biological sample or the like is mixed with a developing solvent containing neuraminidase to obtain a mixed solution that can be chromatographed, the sample is added to the sample addition member 5 of the immunochromatographic test strip shown in FIG. When injected above, the mixed solution passes through the sample addition member 5 and mixes with the labeled second antibody in the impregnation member 2.
At that time, if the target antigen is present in the mixed solution, a complex of the target antigen and the second antibody is formed by the antigen-antibody reaction.
This complex is chromatographed in the membrane carrier 3 to reach the capture site 31, where it is captured by an antigen-antibody reaction with the first antibody immobilized thereon.
At this time, if a colored labeling substance such as colloidal gold is used as the labeling substance, the capture site 31 develops color due to the accumulation of the colored labeling substance, so that the antigen to be detected is immediately measured qualitatively or quantitatively. can do.

In the present invention, neuraminidase can be placed, for example, in a lyophilized state at an appropriate location on the immunochromatographic test strip so that the test sample is treated with neuraminidase simultaneously with chromatographic development. In this case, neuraminidase does not have to be contained in the developing solvent as described above, and the highly sensitive measurement of the present invention can be performed by using the same developing solvent as in the conventional method, which is convenient. is there.
The location where the neuraminidase is arranged is not particularly limited as long as it is upstream of the capture site 31 of the immunochromatographic test strip. However, in order to ensure a long processing time, neuraminidase such as the sample addition member 5 or the impregnation member 2 may be used. It is preferable to arrange it on a member that can be impregnated.

  In the present invention, the specimen is not particularly limited. For example, cloacaswab, tracheal swab, stool, nasal aspirate, nasal swab and throat swab, blood (whether whole blood, serum, or plasma), saliva Urine, organ emulsions and the like. In the case of immunochromatography measurement, the specimen is usually diluted with a developing solvent and injected as a test sample onto a membrane carrier.

  When whole blood is used as a test sample, and particularly when a colored labeling substance such as gold colloid is used as the labeling substance of the labeled antibody, it is preferable to dispose a blood cell capturing membrane member on the sample addition member. . The blood cell trapping membrane member is preferably laminated between the impregnation member and the sample addition member. This prevents red blood cells from being developed on the membrane carrier, so that it is easy to confirm the accumulation of the colored label at the capture site of the membrane carrier. As the blood cell capturing membrane member, a carboxymethylcellulose membrane is used. Specifically, ion exchange filter paper CM (trade name) sold by Advantech Toyo Co., Ltd. or ion exchange cellulose sold by Whatman Japan Co., Ltd. Paper or the like can be used.

  The present invention will be described more specifically with reference to the following examples, but the present invention is not limited to these examples.

Reference Example 1 (Preparation of immunochromatographic test strip)
(1) Preparation of gold colloid solution Add 1 ml of 1% (v / w) chloroauric acid aqueous solution to 99 ml of ultrapure water boiled by heating, and 1 minute later, 1% (v / w) sodium citrate After 1.5 ml of an aqueous solution was added and heated to boil for 5 minutes, it was allowed to cool to room temperature. Subsequently, 200 mM potassium carbonate aqueous solution was added to this solution to adjust to pH 9.0, and ultrapure water was added thereto to make a total volume of 100 ml to obtain a gold colloid solution.

(2) Preparation of colloidal gold-labeled anti-influenza virus H5 subtype antibody solution Colloidal gold labeling was carried out on antibodies having binding ability to hemagglutinin protein of influenza virus H5 subtype (hereinafter, anti-H5 antibody) by the following procedure.
The protein equivalent weight of the anti-H5 antibody is 1 μg (hereinafter, when the protein equivalent weight of the antibody is indicated, it is simply indicated by the weight value by weight analysis of the purified protein) and 1 ml of the colloidal gold solution of (1) above, Let the antibody stand for 2 minutes to bind all of these antibodies to the gold colloid particle surface, and then add 10% bovine serum albumin (hereinafter referred to as “BSA”) aqueous solution so that the final concentration in the gold colloid solution is 1%. Then, all the remaining surfaces of the gold colloid particles were blocked with the BSA to prepare a gold colloid-labeled anti-H5 antibody (hereinafter referred to as “gold colloid-labeled antibody”) solution. This solution was centrifuged (5600 × G, 30 minutes) to precipitate colloidal gold labeled antibody, and the supernatant was removed to obtain colloidal gold labeled antibody. The colloidal gold labeled antibody was suspended in a 50 mM Tris-HCl buffer (pH 7.4) containing 10% sucrose, 1% BSA, 0.5% Triton-X100 to obtain a colloidal gold labeled antibody solution.

(3) Preparation of immunochromatographic test strip The immunochromatographic test strip shown in FIG. 1 was prepared by the following procedure.

(3-1) Formation of capture site An elongated strip-shaped nitrocellulose membrane having a width of 5 mm and a length of 36 mm was prepared as a membrane carrier 3 for chromatographic development of a chromatographic medium.
0.5 μl of a solution containing 1.0 mg / ml of anti-H5 antibody was applied in a line at a position 7.5 mm from the end of the chromatographic development starting point side in the membrane carrier 3 for chromatographic development and dried at room temperature. The capture site 31 of the complex of influenza virus H5 subtype and colloidal gold labeled antibody was used.

(3-2) Colloidal gold labeled antibody impregnated member
A 5 mm × 15 mm strip-shaped glass fiber nonwoven fabric was impregnated with 37.5 μl of a colloidal gold labeled antibody solution, which was dried at room temperature to obtain a colloidal gold labeled antibody impregnated member 2.
(3-3) Preparation of immunochromatographic test strip In addition to the chromatographic development membrane carrier 3 and the labeled antibody-impregnated member 2, a cotton cloth as a sample addition member 5 and a filter paper as an absorbing member 4 were prepared. Then, an immunochromatographic test strip similar to that shown in FIG. 1 was prepared using these members.

Example 1 (Selection of enzyme)
For the purpose of improving the addition recovery rate, the enzyme used as an additive was evaluated. Protease, TypeXIV (150mU / mL), Proteinase, TypeXXIV (120mU / mL), Proteinase K (420mU / mL), Thermolysin (160mU / mL), Subtilisin A, TypeVIII (180mU / mL), Trypsin (75mU / mL) mL), Trypsin, TypeI (28mU / mL), α-Chymotrypsin (850mU / mL), Protease, TypeXX (442mU / mL), Protease (130mU / mL), Neuraminidase (Arthrobactor ureafaciens) (50mU / mL) .

  A tracheal swab collected from a negative chicken without influenza infection was suspended in 400 mM Tris buffer (pH 7.5), and A / Dk / Mong / 54 / 01-Dk / Mong / 47/01 (influenza virus) H5N1) strain was added to 400 ng / ml to prepare a false positive sample. After the aforementioned enzyme was added to a false positive sample and allowed to react at room temperature for 15 minutes, 100 μL of the enzyme was micropided on the sample addition member 5 of the immunochromatographic test strip for detection of influenza virus H5 subtype antigen prepared in Reference Example 1. It was dropped with a pipette and chromatographed. After standing at room temperature for 15 minutes, the amount captured at the capture site 31 was observed with the naked eye and an immunochromatographic reader (manufactured by Otsuka Electronics). The same experiment was performed without adding virus (Blank). Further, as a control, a buffer alone (Control 1) and a buffer solution (Control 2) in which only a tracheal swab was added without adding an enzyme were used.

  The amount of capture is in four stages:-(no coloration), ± (weak coloration), + (clear coloration), and ++ (significant coloration). (However, the weak coloring at each stage was marked with w), and the color intensity was digitized by an immunochromatographic reader. The results are shown in Table 1.

  From the results in Table 1, it was shown that the enzyme that contributed to the improvement of the addition recovery rate was neuraminidase. Nonspecific coloration in Blank by addition of neuraminidase was not confirmed. In addition, the tendency of agglutination of colloidal gold-labeled antibody was observed even when other enzymes contributed to the sensitivity increase.

Example 2 (Investigation of relationship between neuraminidase and antibody-antigen reaction and non-specific reaction)
A specimen extract was prepared by adding neuraminidase (Arthrobactor ureafaciens) or neuraminidase (III) as an enzyme to a final concentration of 1.0 mU, 2.5 mU, 5.0 mU and 10 mU in 400 mM Tris buffer (pH 7.5). Then, the influenza virus A / Dk / Mong / 54 / 01-Dk / Mong / 47/01 (H5N1) strain was diluted with the sample extract to prepare 100 ng / mL and 200 ng / mL, did. Then, 100 μl of the test sample was dropped with a micropipette on the sample addition member 5 of the immunochromatographic test strip prepared in Reference Example 1, and chromatographed. After standing at room temperature for 15 minutes, the amount captured at the capture site 31 was observed with the naked eye and an immunochromatographic reader (manufactured by Otsuka Electronics).

  The capture amount is determined by classifying the degree of reddish purple color that increases or decreases in proportion to the amount into three stages:-(no coloration), ± (weak coloration), + (clear coloration), In each case, the color intensity was quantified by an immunochromatographic reader. As a control, a test sample without addition of neuraminidase (concentration 0 mU / T) was used. The results are shown in Tables 2 and 3.

  From the results of Tables 2 and 3, it was confirmed that when the above two types of neuraminidase were used, the coloration did not significantly decrease, and neither caused reaction inhibition or nonspecific reaction. In addition, the aggregation of colloidal gold-labeled antibody that occurs when protease is used as an enzyme was not confirmed.

Example 3 (Examination of addition amount of neuraminidase)
A tracheal swab collected from a negative chicken without influenza infection was suspended in 400 mM Tris buffer (pH 7.5), and A / Dk / Mong / 54 / 01-Dk / Mong / 47/01 (influenza virus) A predetermined amount of H5N1) strain was added to prepare a false positive specimen. Neuraminidase (Arthrobactor ureafaciens) or neuraminidase (III) was added to the false positive specimens at final concentrations of 0, 1.0, 2.5, 5.0, and 10 mU / T to prepare test samples. A similar test sample (Example 2) except that no tracheal swab was added was used as a control. And by the method similar to Example 2, the test sample was dripped at the immunochromatography test strip, and the capture amount was observed.

The capture amount is determined by classifying the degree of reddish purple color that increases or decreases in proportion to the amount into three stages:-(no coloration), ± (weak coloration), + (clear coloration), In each case, the color intensity was quantified by an immunochromatographic reader.
The addition recovery rate is the percentage of the immunochromatographic reader value in each test sample with respect to the numerical value of the control immunochromatographic reader at the same concentration (same as those in Example 2 and Table 3). The greater the reaction inhibition by the analyte component, the lower the recovery rate. The results are shown in Tables 4 and 5.

  From the results of Tables 4 and 5, in any case where neuraminidase (Arthrobactor ureafaciens) and neuraminidase (III) were used, a significant sensitivity improvement effect was confirmed at a final concentration of 1.0 mU / T or more. The optimum concentration was 2.5 mU / T. The same effect was observed when 10 mU / T was added, and no non-specific reaction was confirmed.

Example 4 (Example in which neuraminidase was impregnated in an impregnated member)
An enzyme-impregnated pad was prepared by dissolving neuraminidase (III) in an enzyme-dissolved solution and freeze-drying an impregnated member made of a 15 mm × 5 mm glass fiber pad (glass fiber nonwoven fabric). Neuraminidase concentrations were 0, 1.5, 3.0, 4.0 and 5.0 mU / T per pad.

  A tracheal swab collected from a negative chicken without influenza infection was suspended in 400 mM Tris buffer (pH 7.5), and A / Dk / Mong / 54 / 01-Dk / Mong / 47/01 (influenza virus) A predetermined amount of H5N1) strain was added to prepare a false positive specimen. 1 mL of a false positive specimen was placed in a flexible plastic tube, the above-described enzyme-impregnated pad was added to the tube, and the extraction operation and reaction were carried out by pinching the tube to prepare a test sample. The test sample was also used as a control except that no tracheal swab was added. And by the method similar to Example 2, the test sample was dripped at the immunochromatography test strip, and the capture amount was observed.

The amount of capture is in four levels:-(no coloration), ± (weak coloration), + (clear coloration), and ++ (significant coloration). The color intensity was quantified using an immunochromatographic reader.
The addition recovery is the percentage of the immunochromatography reader in each test sample relative to the value of the control immunochromatography reader at each concentration. The greater the reaction inhibition by the analyte component, the lower the recovery rate. The results are shown in Table 6.

  From the results of Table 6, by adding an enzyme-impregnated pad containing neuraminidase to a false positive specimen, a significant sensitivity increase effect (improvement of addition recovery rate) was confirmed, and the freeze-dried enzyme was added as an enzyme addition form. It was found that the same effect as in Example 3 was obtained even when the impregnated pad contained was used.

Example 5 (Example in which neuraminidase was impregnated in an impregnated member of an immunochromatographic test strip)
Neuraminidase (Arthrobactor ureafaciens) or neuraminidase (III) was dissolved in an enzyme solution, impregnated on the gold colloid-labeled antibody-impregnated member of the immunochromatographic test strip of Reference Example 1, and lyophilized. The neuraminidase concentration was 0, 1.5, 3.0, 4.0 and 5.0 mU / T per impregnated member.

  A tracheal swab collected from a negative chicken without influenza infection was suspended in 400 mM Tris buffer (pH 7.5), and A / Dk / Mong / 54 / 01-Dk / Mong / 47/01 (influenza virus) A predetermined amount of H5N1) strain was added to prepare a false positive sample, which was used as a test sample. A similar test sample was used as a control except that no tracheal swab was added. And by the method similar to Example 2, the test sample was dripped at the immunochromatography test strip, and the amount of capture was observed and evaluated.

The capture amount is determined by classifying the degree of reddish purple color that increases or decreases in proportion to the amount into three stages:-(no coloration), ± (weak coloration), + (clear coloration), In each case, the color intensity was quantified by an immunochromatographic reader.
The addition recovery is the percentage of the immunochromatography reader in each test sample relative to the value of the control immunochromatography reader at each concentration. The greater the reaction inhibition by the analyte component, the lower the recovery rate. The results are shown in Table 7 and Table 8.

  From the results of Tables 7 and 8, it can be seen that even when neuraminidase is placed on the impregnated member of the immunochromatographic test strip, a significant sensitivity increase effect (improvement of addition recovery) can be obtained.

  The present invention enables highly sensitive immunoassay, in particular immunochromatographic measurement, of influenza virus antigens, and can rapidly and rapidly prevent diseases such as birds and humans caused by influenza virus H5 subtype. Useful for simple diagnosis.

a is a plan view of an immunochromatographic test strip, and b is a longitudinal sectional view of the immunochromatographic test strip indicated by a.

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 Adhesive sheet 2 Impregnation member 3 Membrane carrier 31 Capture part 4 Absorption member 5 Sample addition member

Claims (26)

An immunoassay method for influenza virus antigens using an anti-influenza virus antibody, comprising subjecting a test sample containing a specimen treated with neuraminidase to an antibody antigen reaction with the antibody, . The immunoassay method for influenza virus antigens according to claim 1, wherein the immunoassay method is a sandwich immunoassay method in which the antigen is sandwiched between a first antibody and a second antibody against an influenza virus antigen. The method for immunoassay of an influenza virus antigen according to claim 1 or 2, for detecting an influenza virus hemagglutinin antigen. A membrane carrier provided with a capture site formed by preliminarily fixing a first antibody against an influenza virus antigen in a predetermined position, and a mixture of a second antibody against the influenza virus antigen and a predetermined amount of a test sample, An immunochromatographic measurement method in which a chromatographic development is performed on the membrane carrier toward a capture site, and a complex comprising the influenza virus antigen and the second antibody contained in the test sample is captured at the capture site. An immunochromatographic assay method for influenza virus antigens, characterized in that the test sample is treated with neuraminidase before or simultaneously with chromatographic development. The immunochromatography measurement method according to claim 4, wherein the test sample is treated with neuraminidase by mixing neuraminidase into the mixed solution or the test sample. 6. The immunochromatographic measurement method according to claim 5, wherein neuraminidase is mixed with the mixed solution or the test sample in a container separate from the membrane carrier, and then this is chromatographed. The immunochromatographic assay method according to claim 6, wherein neuraminidase is impregnated in a freeze-dried state in advance in an impregnated member, and the mixed solution or the test sample is treated with neuraminidase by introducing the impregnated member into the container. . 6. The immunochromatographic measurement method according to claim 5, wherein neuraminidase is placed on a membrane carrier, and the mixture and neuraminidase are mixed when the mixture is chromatographed. The immunochromatographic assay method according to claim 4, wherein the second antibody is labeled with a metal colloid or latex. The immunochromatographic assay method according to claim 9, wherein the membrane carrier is a nitrocellulose membrane. The immunochromatographic assay method according to any one of claims 5 to 10, for detecting an influenza virus hemagglutinin antigen. A first antibody against an influenza virus antigen, a second antibody, and a membrane carrier, wherein the first antibody is preliminarily fixed at a predetermined position of the membrane carrier to form a capture site, and the second antibody is An immunochromatographic test strip that is labeled with an appropriate labeling substance and arranged so that it can be chromatographed on the membrane carrier at a position separated from the capture site, and a sample is diluted and chromatographed on the membrane carrier An influenza virus antigen detection kit comprising at least a developing solvent for preparing a test sample so as to be capable of further comprising a neuraminidase to be added to the developing solvent. Detection kit. 13. The influenza virus antigen detection kit according to claim 12, wherein neuraminidase is impregnated in a freeze-dried state in advance on the impregnated member, and the impregnated member is used in a container containing a developing solvent. The influenza virus antigen detection kit according to claim 12, wherein the second antibody is labeled with a metal colloid or latex. The influenza virus antigen detection kit according to claim 14, wherein the membrane carrier is a nitrocellulose membrane. The kit for detecting an influenza virus antigen according to any one of claims 12 to 15, for detecting an influenza virus hemagglutinin antigen. A first antibody against an influenza virus antigen, a second antibody, and a membrane carrier, wherein the first antibody is preliminarily fixed at a predetermined position of the membrane carrier to form a capture site, and the second antibody is An immunochromatographic test strip labeled with an appropriate labeling substance and arranged so as to be chromatographed on the membrane carrier at a position separated from the capture site, wherein neuraminidase is the second of the membrane carrier. An immunochromatographic test strip for detecting influenza virus antigens, characterized in that the antibody is placed at or near the place where the antibody is placed. The immunochromatographic test strip according to claim 17, wherein the neuraminidase is arranged in a lyophilized state. The immunochromatographic test strip according to claim 17, wherein the neuraminidase is disposed on the membrane carrier in a state of being impregnated in the impregnation member. The immunochromatographic test strip according to claim 17, wherein the second antibody is labeled with a metal colloid or latex. The immunochromatographic test strip according to claim 20, wherein the membrane carrier is a nitrocellulose membrane. The immunochromatographic test strip according to any one of claims 17 to 21, for detecting an influenza virus hemagglutinin antigen. An extract for immunoassay, comprising a neuraminidase, which is an extract for diluting a specimen to obtain a test sample to be used for immunoassay of influenza antigen. The extract for immunoassay according to claim 23, which is used as an immunochromatographic developing solvent. An extract preparation kit for diluting a specimen to obtain a test sample to be used for immunoassay of influenza antigen, comprising a solvent and an impregnation member impregnated with neuraminidase in a lyophilized state, and the impregnation into the solvent at the time of use An extract preparation kit for immunoassay, in which an extract can be prepared by adding members. The extract preparation kit for immunoassay according to claim 25, which is used as an immunochromatographic developing solvent.

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