JP4976068B2 - Simple immunoassay specimen suspension and assay method - Google Patents

Description

  The present invention relates to a sample suspension used for floating and diluting a sample and causing an antigen-antibody reaction between a detected substance and a capture substance in the sample in a simple test method for a sample using a simple immunoassay method. .

  In recent years, simple tests that detect or quantify various measurement items such as infection with pathogens such as viruses and bacteria, presence of pregnancy, blood glucose level, etc. in a few minutes to tens of minutes using antigen-antibody reactions and enzyme reactions Reagents or kits have been developed. Pathogen constituent proteins, human ciliary gonadotropin (hCG), blood glucose, and the like are targets for detection or quantification. Many of the simple test reagents are characterized by the fact that they do not require special equipment, are easy to operate, and are inexpensive. For example, simple test reagents for pregnancy diagnosis are sold as OTC at general pharmacies. Unlike other test reagents, simple test reagents for measuring infection with pathogens are widely used in general hospitals and clinics in addition to large hospitals and medical test centers. In particular, for influenza viruses, the first time a patient visits a hospital for the presence or absence of the infection, if the specimen collected from the patient is identified on the spot whether it is infected or what type of infection, the symptoms are Therefore, the importance of simple test reagents in medical care is increasing.

  In such a test, in many cases, a specimen such as a body fluid or a solution obtained by diluting the specimen sample is used to infect a pathogen such as a virus or a bacterium, but a component derived from a living body is contained in the specimen sample. It is known that many of these are included, and this affects the examination using the antigen-antibody reaction (see Non-Patent Document 1). For example, in respiratory infections such as influenza virus infection, nasal swabs, nasal aspirates, nasal lavage fluids, etc. may be examined as sample samples. Representative viscous mucopolysaccharides and blood cell components are included (see Non-Patent Document 1). In addition, pathogens and human tissue components are also considered to be included. These components in the specimen sample may affect the test, which is sometimes called a matrix effect (see Non-Patent Document 2). The matrix effect can be roughly divided into two. One is false positive and the other is false negative. For example, as a composition of a specimen suspension for suspending or floating a specimen, which is used in a specimen testing method using a colloidal aggregation method as a measurement principle, the composition includes a surfactant and has a pH of 3 to 8. A specimen suspension composition is known (see Patent Document 1). However, the conventional method including this method describes the effect of reducing false positives, but false negatives are not shown, and a sufficient effect is obtained from the viewpoint of reducing the matrix effect. Absent. In addition, when the specimen is suspended and left in the specimen suspension, false negatives due to the matrix effect may be observed over time. Therefore, it is required to reduce the matrix effect that combines false negatives and false positives and perform more stable and highly accurate diagnosis.

“Nanzan-do Medical University Encyclopedia” Nanzan-do Co., Ltd., (18th edition, 5th edition), April 10, 2001, p1792. HHA White Paper (U.S. Army Press Release, July 23,2002) JP 2006-84351 A

  Body fluids used in simple immunoassay methods that detect or quantify various measurement items such as infection with pathogens such as viruses and bacteria, presence of pregnancy, blood glucose level, etc. in a short time of minutes to hours using antigen-antibody reactions This suppresses the influence of biological components contained in specimens such as those on the assay.

As a result of intensive studies to solve the above problems, the present inventors have found that extremely good test results can be obtained by using a sample suspension having a specific composition. It came to.
That is, the present invention is a simple immunoassay sample suspension containing normal mammalian serum at a concentration of 1 to 10 (v / v)% and having a pH in the range of 4.5 to 6.5. Furthermore, the pH is preferably in the range of 5.0 to 6.0. Moreover, it is preferable to contain 3-5 (v / v)% of normal mammal serum. Furthermore, the specimen suspension of the present invention is preferable as a specimen suspension for simple membrane immunoassay or a specimen suspension for ELISA by immunological specific reaction. Further, the present invention is a simple membrane immunoassay method using the above-mentioned specimen suspension.

  As described above, by using the sample suspension of the present invention, it is possible to suppress the influence on the test of a biological component contained in a sample such as a body fluid, and to significantly reduce both false positives and false negatives that can occur in the test. This makes it possible to perform more accurate diagnosis. In particular, it occurs due to uncertain factors in the test, such as false negatives that occur over time after mixing the specimen and specimen suspension, and false negatives that occur at dilution rates close to the sensitivity limit value (dilution limit value). A reliable membrane assay method and kit that can reliably suppress false negatives are provided.

  In simple immunoassay, the mechanism of false positives and false negatives due to biologically derived components is not simple, but the false positive hypothesis is that biologically-derived components are attached to a trapped substance immobilized by a nonspecific reaction. However, it is considered that the aggregation of the labeling reagent occurs at that position. In addition, the false negative hypothesis is that the biological component adheres to the detection target by a non-specific reaction and inhibits the antigen-antibody reaction between the labeled reagent or the immobilized capture substance and the detection target. It is thought that. As a method of reducing these false positives and false negatives, the occurrence of these decreases when the pH of the sample suspension is set within the above range, but the suppression of false positives is not necessarily sufficient, and The matrix effect cannot be comprehensively suppressed. The specimen suspension of the present invention has a pH in the above range and contains normal mammalian serum, and its presence significantly suppresses false positives and synergistically suppresses the matrix effect of biological substances. can do.

<Sample suspension>
The specimen suspension for simple immunoassay of the present invention contains normal mammalian serum at a concentration of m1 to 10 (v / v)%, and has a pH in the range of 4.5 to 6.5.
In the present invention, “normal mammal serum” refers to serum collected from mammals not infected with viruses or bacteria. For example, “infected serum” is not included in “normal mammalian serum” of the present invention. The normal serum of the mammal is not particularly limited, and examples thereof include normal serum of mammals such as sheep, goat, horse, cow, mouse, rat, and rabbit. Among these, normal rabbit serum is preferable because it can be easily obtained. As the normal serum of mammals, commercially available ones may be used as appropriate, or they may be collected from mammals by a conventionally known serum preparation method. As a commercial item, it can obtain from Funakoshi Co., Ltd., for example.
The mammalian serum can be used as it is or after being degreased. The concentration needs to be a concentration that does not inhibit the measurement system, and the concentration range is 1 to 10 (v / v)%, and preferably 3 to 5 (v / v)%. If the amount is less than 1 (v / v)%, the above effect cannot be obtained. If the amount exceeds 10 (v / v)%, the antigen-antibody reaction between the immobilized capture substance and the detected substance and the labeled substance and the detected substance antigen May inhibit antibody response.

  The pH range of the sample suspension of the present invention varies depending on the measurement principle used for the test, but it must be in the range of 4.5 to 6.5, and is included in the sample within this range. The effect which suppresses the influence which the component derived from a biological body has on the test | inspection of this invention can be acquired. More preferably, the pH is in the range of 5-6. If the pH is less than 4.5, the activity of a capture substance such as an antibody used in the present invention is reduced, and the capture power of the detection object is reduced, so that the effect of the present invention cannot be obtained. Moreover, when pH exceeds 6.5, the influence which the component derived from a living body has on a test | inspection will become large, and a false positive and a false negative will generate | occur | produce.

  The type of the buffer that determines the pH of the specimen suspension of the present invention is not particularly limited as long as it is a reagent having a buffer capacity in the range of pH 4.5 to 6.5. Of these, ADA (N- (2-acetamido) iminodiacetic acid) or MES (2-morpholinoethanesulfonic acid) is preferred in view of its performance and economy.

  In addition to the normal serum of mammals described above, the sample suspension of the present invention includes protein components such as BSA and casein used as a blocking agent, and KCl that may affect the stability of the measurement target after suspension. Inorganic salts such as NaCl and organic salts such as arginine salts can also be added. A surfactant can also be added depending on the measurement object. For example, in the case of influenza virus, it can be added for the purpose of inactivating and cleaving the virus.

<Simple immunoassay method>
In the present invention, the specimen suspension refers to a liquid medium used in a simple immunoassay method, and is a solution for diluting and / or extracting a specimen that can contain a specimen to be suspended, and at the same time, a specimen to be measured in the specimen. It is a solution that serves as a field for causing an antigen-antibody reaction between the substance and the capture substance. The simple immunoassay method shown here refers to a simple membrane immunoassay method, an ELISA (Enzyme Linked Immuno-Sorbent Assay) method, and a method similar to ELISA. The specimen suspension of the present invention is preferably used for simple membrane immunoassay.

  The simple membrane immunoassay method is a method that uses a simple assay device equipped with a membrane, does not require special equipment, is easy to operate, and is used in general hospitals and clinics other than large hospitals and medical examination centers. This inspection method can be easily performed in a short time. The simple membrane immunoassay method can be roughly divided into two, one is an immunochromatography method (or a lateral flow method), and the other is a flow-through method. Both methods capture and detect a target substance contained in a specimen by an antigen-antibody reaction using a solid-phase capture substance. In the former method, a sample containing a target substance is removed from a strip-shaped membrane. While the latter is developed in the longitudinal direction, the latter is a method of developing in a direction perpendicular to the surface of the membrane. As these methods, known general methods can be used, and a detection target can be detected by a method such as a sandwich immunoassay method using a so-called labeling reagent.

  The ELISA method is a method for analyzing an object to be detected with high sensitivity by labeling an antibody or an antigen with an enzyme based on an antigen-antibody reaction. There are various types of ELISA methods, all of which prepare a plastic plate or the like on which a capture substance is solid-phased, capture the detected object, and capture the detected object with the enzyme-labeled antibody or antigen. It is to detect. In particular, as an antigen protein detection method, it is widely used as an analysis method for detecting an antigen protein in a specimen or, conversely, an antibody that binds to a specific antigen protein. This is a test method that can assay multiple specimens simultaneously and obtain results in a few hours. As a method similar to ELISA, FIA method (Fluorescence Immunoassay) using a fluorescent substance for labeling, CLIA method (Chemiluminescence Immunoassay, chemiluminescence immunoassay) using a luminescent substance for labeling, and radioisotope labeling RIA method (Radioimmunoassay, radioimmunoassay) used in

The specimen suspension of the present invention is used as a medium for performing an immunologically specific antigen-antibody reaction with an antibody and an antigen in any of the above-described methods, and in particular, a specimen sample that may contain an object to be detected is suspended. It can be used as a specimen suspension for dilution and / or extraction.
As a result of intensive investigations to solve the problem of suppressing the occurrence of false positives and false negatives due to biologically-derived components contained in the specimen sample in the simple immunoassay, the present inventors have conducted a specimen suspension. It is possible to significantly suppress both false positives and false negatives that occur due to the influence of substances derived from living organisms in the test by setting the pH of the product to a specific range and containing normal serum of a specific concentration of mammals. Heading, reaching the present invention.

<Detection object>
Examples of the detection object to be detected by the simple immunoassay method of the present invention include various antigens and antibodies. Preferably, pathogenic microorganisms, biomarkers, hormones, environmental hormones and antibodies thereto can be mentioned, more preferably viruses and bacteria causing respiratory infections, digestive infections, cardiovascular infections, and against them Examples thereof include antibodies, tumor markers, circulatory system markers, diabetes markers, bone metabolism markers, and antibodies to these. More preferably, virus antigens such as influenza virus antigens, adenovirus antigens, RS virus antigens, HA antigens, HBc antigens, HCV antigens, HIV antigens, EBV antigens, NLV antigens and the like, antibodies against these, pathogenic E. coli antigens, Chlamydia traco Bacterial antigens such as Mathis antigen, Streptococcus antigen, Pneumococcal antigen, Bordetella pertussis antigen, Helicobacter pylori antigen, Leptospira antigen, Treponema pallidum antigen, Toxoplasma gondii antigen, Borrelia antigen, Bacillus anthracis antigen, MRSA antigen, and antibodies thereto Examples include mycoplasma lipid antigens, toxin antigens produced by bacteria, and antibodies to these, and more preferably influenza virus antigen, adenovirus antigen, RS virus antigen, rotavirus antigen, NV antigen, pneumonia Plasma antigen, A group streptococcus antigens, pneumococcal antigens, chlamydia antigens, pathogenic E. coli antigens, there may be mentioned a Campylobacter antigens, and the like.

<Sample>
As a matter of course, a sample obtained by experimentally cultivating the above-mentioned detection object and diluted with physiological saline or the like does not cause false negatives and false positives, which are problems in the present invention. Samples used in the simple immunoassay of the present invention include, for example, whole blood, urine, feces, nasal swab, nasal aspirate, throat swab, nasal wash, alveolar wash, sputum, tympanic incision / reservoir, and swab Secreted secretions and the like. The components contained in these specimens are, for example, blood cell components such as red blood cells and white blood cells in the case of whole blood, and in the case of various wiping liquids, cellular components in which biological components mixed in the specimen are peeled or decomposed and aggregated and so on. These samples may contain various biological components such as viscous substances such as proteoglycans and glycolipids, and may contain microorganisms such as viruses and bacteria. The specimen suspension of the present invention is used as the specimen suspension of the specimen as described above. These components may induce non-specific reactions such as false positives and false negatives depending on the principle used for the test. Non-specific reactions due to the above components are also targets to be suppressed with the composition of the specimen suspension of the present invention.

<Captured substance>
In the simple immunoassay method of the present invention, the capture substance that captures the detection target is, if the detection target is an antigen, an antibody corresponding thereto or a substance that undergoes substantially the same antigen-antibody reaction. In the case where the detection target is an antibody, it may be an antigen corresponding thereto or a substance that undergoes substantially the same antigen-antibody reaction.

The present invention will be specifically described with reference to examples.
(Sample preparation)
1. Preparation of monoclonal antibody (1) Preparation of anti-influenza A virus NP monoclonal antibody (mouse) The spleen was excised from BALB / c mice that had been immunized with purified influenza A virus antigen and maintained for a certain period of time (Kohler et al. et al., Nature, vol, 256, p495-497 (1975)) and fused with mouse myeloma cells (P3 × 63).
The obtained fused cells (hybridomas) are maintained in a 37 ° C. incubator, and purification of the cells (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 two cell lines were each 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 affinity purification using Protein A column chromatography (Amersham) to obtain two types of purified anti-influenza A virus NP monoclonal antibodies.

(2) Anti-influenza B virus NP monoclonal antibody (mouse)
The 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 obtained by the method of Keller et al., Nature, vol. 256, p495-497 (1975). Fused with cells (P3x63). The obtained fused cells (hybridomas) are maintained in an incubator at 37 ° C., and purification of the cells (monocloning) while confirming the antibody activity of the supernatant by ELISA using an influenza B virus NP antigen solid phase plate Went. The obtained two cell lines were each 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 Protein A column chromatography (Amersham) to obtain two types of purified anti-influenza B virus NP monoclonal antibodies.

2. Preparation of labeled anti-influenza antibody (1) Preparation of latex-labeled anti-influenza A antibody One type of anti-influenza A virus NP monoclonal antibody was mixed with 50 mM MES (2-Morpholinoethanesulfonic acid, monohydrate) buffer (pH 6. 0) After dialysis with the solution, it was mixed with red polystyrene latex particles (particle size: 0.394 μm, surface modifying group was carboxyl group; Merck) and reacted. Next, EDAC (N- (3-Dimethlaminopropyl) -N′-ethylcarbodiimide hydrochloride; Sigma) was added to a final concentration of 0.1%, followed by reaction for 2 hours. After washing, it floats in the final suspension (5mM Tris, 0.04 (W / V)% BSA (bovine serum albumin)), and is applied to an ultrasonic dispersion device (Olympus) to disperse latex particles and latex-labeled anti-influenza A influenza Antibodies were prepared.

(2) Preparation of latex-labeled anti-influenza B antibody One type of anti-influenza A virus NP monoclonal antibody was dialyzed with 50 mM MES buffer (pH 6.0) solution, and then blue polystyrene latex particles (particle size: 0.394 μm, surface) The modifying group was mixed with a carboxyl group (Merck) and reacted. Next, EDAC was added to a final concentration of 0.1%, and then reacted for 2 hours. After washing, it floats in the final suspension (5mM Tris, 0.04 (W / V)% BSA (bovine serum albumin)), and is applied to an ultrasonic dispersion device (Olympus) to disperse latex particles and latex-labeled anti-type B influenza Antibodies were prepared.

3. 1. Drying of latex labeled antibody (1) Mixing of latex labeled antibody The latex-labeled anti-influenza A antibody and latex-labeled anti-type B influenza antibody prepared in (1) and (2) were diluted so that the latex concentrations were equal to each other, and then mixed at the same temperature at room temperature.
(2) Production of latex labeled antibody pad The latex labeled antibody prepared in (1) was sprayed on the whole surface of a cellulose nonwoven fabric having a width of 15 mm wound in a reel shape using a positive pressure spraying device (BioJet; BioDot). After spraying, warm air of 50 ° C. was blown for 1 minute to dry, thereby preparing a latex labeled antibody pad.

4). Preparation of antibody for membrane solid phase (1) Preparation of anti-influenza A antibody for solid phase The purified anti-influenza A virus NP monoclonal antibody prepared in (1), which was not used for labeling, was 0.22 μm filtered and diluted with MQ water to prepare a solid phase anti-influenza A influenza antibody.
(2) Preparation of anti-influenza B antibody for solid phase The purified anti-influenza B virus NP monoclonal antibody prepared in (2), which was not used for labeling, was 0.22 μm filtered and diluted with MQ water to prepare a solid phase anti-influenza B influenza antibody.

5. Production of Lateral Flow Membrane Assay Device for Detection of Influenza Virus A lateral flow membrane assay device for detection of influenza virus was used having the same configuration as that shown in FIGS.
As the membrane, a nitrocellulose membrane (Millipore) sheet having a width of 3 cm and a length of 10 cm was used. Using a positive pressure spray device (BioJet; BioDot) for solid phase anti-influenza A antibody at a position 8 mm away from one end of the long axis side (this end is the upstream end and the opposite side is the downstream end) The solid-phase anti-influenza B influenza antibody was applied in a line using a positive pressure spray apparatus (BioJet; BioDot) at a position e 12 mm away. After coating, warm air of 45 ° C. was blown for 10 minutes to dry.

Next, in order to fix the member and increase the strength, a plastic backing sheet (manufactured by BioDot) is adhered to the opposite side (this surface is the lower surface) of the antibody-coated surface (this surface is the upper surface). did.
Next, 3. The latex-labeled antibody pad prepared in (2) was cut to a width of 15 mm x a length of 10 cm, and placed on the upper surface of the membrane so that the upstream end of the membrane overlapped by 2 mm, and was used as a sample dropping pad.
Next, a cellulose filter paper (Whatman) having a width of 30 mm x a length of 10 cm was placed on the upper surface of the membrane so as to overlap the downstream end of the membrane by 5 mm, and used as a sample absorption pad.
Next, the entire upper surface was covered with a transparent plastic laminate (Adhesive Research) except for a width of 7 mm at the upstream end of the sample dropping pad.
Finally, the membrane assay apparatus shown in FIGS. 1 and 2 was produced by cutting along the long axis direction by 5 mm.

6). Preparation of labeled antibody-loaded sample filtration filter Prepare a nozzle for filtration as shown in Fig. 3, punch the glass filter paper for filtration (Advantech Toyo Co., Ltd.) into a circle (diameter 0.7 cm), and load it on the bottom of the nozzle. An antibody loaded sample filtration filter was made.

(Reference Example 1) Detection of influenza virus (i) (Influence on false negative of pH)
(1) Preparation and dilution of cultured virus Type A and type B influenza viruses cultured in embryonated chicken eggs were diluted 100-fold with physiological saline, and further used in a 2-fold serial dilution series with physiological saline ( 100 times to 1600 times) were prepared. In Table 2, for example, “A × 100” means a solution obtained by diluting the solution added with influenza A virus 100 times (2) Detection by membrane assay From patients with no clinically recognized influenza virus infection Samples were collected from the collected nasal aspirate using a sterile cotton swab and suspended in 0.4 mL of each sample suspension (composition shown in Table 1 below) dispensed into a tube as shown in FIG. A test sample was prepared.
5. To this test sample, add 30 μL of the diluted A or B influenza virus virus diluted and cultured in (1), and attach it to the tip of the tube (preparation of sample). The labeled antibody-loaded sample filtration filter prepared in (1) was attached.

  4. Collect the filtrate that has been filtered by passing the entire amount of the test sample through the filter nozzle, and then (sample preparation) The sample dropping pad side of the lateral flow membrane assay device for detecting influenza virus prepared in 1 was immersed in the solution and developed. Ten minutes later, the assay device was observed. If red coloration was observed at position d in FIG. 1 or 2, positive for influenza A virus, and if blue coloration was observed at position e, B was detected. If the influenza virus was positive and color development was not observed in both positions, it was determined as negative.

The abbreviations in Table 1 mean the following compositions.
Tx-100: TritonX-100 (polyethylene glycol mono-p-isooctylphenyl ether)
PB-Na: Sodium phosphate buffer
MES: 2-morpholinoethanesulfonic acid
ADA: N- (2-acetamido) iminodiacetic acid
PIPES: Piperazine-1,4-bis (2-ethanesulfonic acid)
MOPS: 3-morpholinopropanesulfonic acid
BSA: Bovine Serum Albumin
(3) Results The detection results using each suspension liquid listed in Table 1 are summarized in Table 2 below.

  In Table 2, “+” means clear color development (positive), and “−” means no color development at all (negative). Further, “±” means that some color is seen in the determination unit, but the result is unknown because it is not clear color development. Further, in Table 2, a portion where the cell is colored means “−”, “false negative” in which color development is not observed although it should be determined as positive (+) originally.

  In a control experiment using suspension No. 1 (pH 8.0) to which no nasal aspirate was added, samples diluted 100, 200, 400 and 800 times the influenza A virus culture were clearly determined to be positive and 1600 times. Diluted samples were judged negative. This is probably because the virus detection limit was reached at a dilution concentration exceeding 800 times. In the case of influenza B virus culture, the detection limit was reached when the dilution ratio exceeded 400 times. By reducing the pH of the sample suspension to 6.5 or less at such a dilution rate close to the detection limit, the sensitivity decrease due to the matrix effect derived from the nasal aspirate was significantly reduced (References 3 to 5-11). However, the matrix effect (false negative) could not be completely eliminated.

(Example 1) Detection of influenza virus (ii) (Influence on false negative of normal serum of mammals)
(1) Preparation and dilution of cultured virus Type A and type B influenza viruses cultured in embryonated chicken eggs were diluted 100-fold with physiological saline, and further used to prepare samples containing type A virus in physiological saline. The B-type virus-added sample was diluted 400 times, respectively.
(2) Detection by membrane assay A sample was collected from a nasal aspirate collected from a patient with no clinical influenza virus infection using a sterile cotton swab and dispensed into a tube as shown in FIG. A test sample was prepared by floating in 0.4 mL of the sample suspension (Table 3). 5. To this test sample, add 30 μL of the diluted chicken egg culture virus diluted solution prepared in (1), and add (sample preparation) to the tip of the tube. The labeled antibody-loaded sample filtration filter prepared in (1) was attached. In this state, it was allowed to stand for 5 minutes, 10 minutes, 15 minutes, and 30 minutes.
4. Filter after standing and collect the filtrate in tubes, respectively (Preparation of sample) The sample dropping pad side of the lateral flow membrane assay device for detecting influenza virus prepared in 1 was immersed in the sample solution and developed. Ten minutes later, the assay device was observed. If red coloration was observed at position d in FIG. 1 or 2, positive for influenza A virus, and if blue coloration was observed at position e, B was detected. If the influenza virus was positive and color development was not observed in both positions, it was determined as negative.

(3) Results Table 4 shows the determination results of the samples that were allowed to stand for 5 minutes, 10 minutes, 15 minutes, and 30 minutes as described above.

  In Table 4, “+” means clear color development (positive), and “−” means no color development at all (negative). Further, in Table 4, a portion where the cell is colored means “−”, “false negative” in which color development is not observed although it should be determined as positive (+) originally.

  In the case of using the suspension of suspension No. 6 to which normal rabbit serum (NRS) is not added, it is considered that the sample that is allowed to stand for 10 minutes or more is caused by biological components in both cases of type A and type B. A false negative was observed (Experiment No. 1 (comparison)). On the other hand, if a specimen suspension containing 1 to 5 (v / v)% normal rabbit serum (NRS) is used as normal serum for mammals, false negatives generated by detection of samples left for 10 minutes or more can be suppressed. Became clear (experiment numbers 2 to 4). In particular, the inhibitory effect when 3 to 5 (v / v)% of NRS was added was strong (experiment numbers 3 to 4).

(Example 2) Detection of influenza virus (iii) (Influence on false positives of normal mammals)
Specimen suspensions 6 and 14 (Table 3) were collected from nasal aspirates collected from patients with no clinical influenza virus infection using sterile cotton swabs and dispensed into tubes as shown in FIG. Reference) Floating in 0.4 mL to prepare a test sample. 6. At the tip of this tube. The labeled antibody-loaded sample filtration filter prepared in (1) was attached.

  4. Collect the filtrate obtained by passing the entire amount of the test sample through a filtration nozzle and collect in a tube; The sample dropping pad side of the lateral flow membrane assay device for detecting influenza virus prepared in 1 was immersed in the solution and developed. Ten minutes later, the assay device was observed. If red coloration was observed at position d in FIG. 1 or 2, positive for influenza A virus, and if blue coloration was observed at position e, B was detected. If the influenza virus was positive and color development was not observed in both positions, it was determined as negative. The determination results are shown in Table 5.

  In Table 5, “+” means clear color development (positive), and “−” means no color development at all (negative). Further, in Table 5, a portion where the cell is colored means (+) “false positive” in which color development was observed although it should be determined as negative (−).

  When the suspension 6 containing no normal rabbit serum (NRS) was used, 2 samples out of 5 samples showed false positives. On the other hand, when the suspension 14 in which normal rabbit serum (NRS) is added to the specimen suspension as normal mammal serum, the matrix effect (false positive) caused by the biological component can be suppressed. Became clear (experiment number 6).

(Example 3) Detection of influenza virus (iv)
(1) Preparation and dilution of cultured virus Type A and type B influenza viruses cultured in embryonated chicken eggs were diluted 100-fold with physiological saline, and further used in a 2-fold serial dilution series with physiological saline ( 100 times to 1600 times) were prepared.
(2) Detection by membrane assay A sample was collected from a nasal aspirate collected from a patient with no clinical influenza virus infection using a sterile cotton swab and dispensed into a tube as shown in FIG. A test sample was prepared by floating in 0.4 mL of a sample suspension (shown in Table 6 below). 5. To this test sample, add 30 μL of the diluted chicken egg culture virus diluted solution prepared in (1), and add it to the tip of the tube (preparation of sample). The labeled antibody-loaded sample filtration filter prepared in (1) was attached. In this state, they were allowed to stand for 0 minutes, 5 minutes, 10 minutes, 15 minutes, and 30 minutes, respectively (Table 8).

4. Each test sample is passed through a filtration nozzle and the filtered filtrate is collected in a tube, and then (sample preparation). The sample dropping pad side of the lateral flow membrane assay device for detecting influenza virus prepared in 1 was immersed in the solution and developed. Ten minutes later, the assay device was observed. If red coloration was observed at position d in FIG. 1 or 2, positive for influenza A virus, and if blue coloration was observed at position e, B was detected. It was determined to be negative when the influenza A virus was positive and no color was observed at both positions, and was false positive when a purple color was observed at positions d and / or e in FIG.

(3) Results Table 7 shows the dilution detection limits for each specimen suspension obtained using the samples containing the viruses with the respective dilution ratios (still standing 0 minutes). Table 8 shows the results of determination after each sample containing virus diluted at a dilution rate close to the detection limit was allowed to stand for 5 minutes, 10 minutes, 15 minutes, and 30 minutes.
In addition, using the nasal aspirate collected from a patient with no clinical influenza virus infection used in Example 2, the occurrence of false positives at 0 minutes of stationary was verified in the same manner as in Example 2. The results are shown in Table 9.

By reducing the pH of the specimen suspension to 6.5 or less, the sensitivity decrease due to the matrix effect derived from the nasal aspirate, that is, false negatives, could be reduced. In addition, normal rabbit serum was reduced to 5 (v / v)% under this condition. By adding, the matrix effect (false negative) could be suppressed completely (Table 7, experiment number 10).
The stability of the body fluid sample after suspension was measured at a dilution rate close to the detection limit. By using a suspension containing pH 6.0 and normal rabbit serum 5 (v / v)%, the matrix effect ( (False negative) was suppressed, and it became clear that high-accuracy testing can be performed in high sensitivity and in a short time (Table 8, experiment number 14).
Moreover, when it tested without adding the hatching hen egg culture influenza virus, the matrix effect (false positive) was able to be suppressed by adding NRS (Table 9, experiment number 16 and 18).
The effects of pH and normal mammalian serum were insufficient for either, and it became clear that the combination of both could completely suppress matrix effects (false negatives and false positives).

It is a top view of a lateral flow type membrane assay device. FIG. 2 is an end view taken along the line I-I ′ of FIG. 1. It is the structure of a sample filtration filter. It is a structural diagram of a sample tube.

Explanation of symbols

a: sample dropping pad b: sample absorption pad c: nitrocellulose membrane d: anti-influenza A virus NP monoclonal antibody application line e: anti-B influenza virus NP monoclonal antibody application line f: backing sheet g: laminate h: for filtration Nozzle i: Glass filter paper for filtration j: Latex label pad k: Tube

Claims (5)

A specimen suspension for use in a method for detecting an influenza virus in a specimen, which is a nasal swab, nasal aspirate, or nasal wash, in a membrane assay, and having a concentration of 1 to 10 (v / v)% normal serum contains, simple immuno membrane assay for the analyte suspension is in the range of pH is 4.5 to 6.5 of. The specimen suspension for simple membrane immunoassay according to claim 1, wherein the pH is in the range of 5.0 to 6.0. The specimen suspension for simple membrane immunoassay according to claim 1 or 2, which contains 3 to 5 (v / v)% normal mammalian serum. The specimen suspension for simple membrane immunoassay according to any one of claims 1 to 3, which is a specimen suspension for ELISA, FIA, CLIA or RIA. The simple membrane immunoassay method which detects the influenza virus in the sample which is a nasal swab liquid, a nasal cavity aspirate, or a nasal cavity washing liquid using the sample suspension liquid of any one of Claims 1-4 by a membrane assay .

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