JP6411740B2 - Composition, kit and use thereof for determining the effectiveness of a neuraminidase activity inhibitor - Google Patents

Description

  The present invention relates to a technique for diagnosing and treating influenza viruses, and more particularly to a technique for determining the effectiveness of a neuraminidase activity inhibitor.

  Influenza viruses are classified based on the antigenicity of core proteins (nuclear protein (NP protein) and membrane protein (M1 protein)), and it is now clear that there are three types of influenza A, B and C .

  Influenza A virus is further abbreviated as hemagglutinin (HA), which is the two surface glycoproteins of virus particles, and a neuraminidase (NA), hereinafter abbreviated as NA. And subtypes of H1 to H16 in HA and N1 to N9 in NA. HA adsorbs and invades cells such as humans and binds to sialic acid present on the cell surface to play an important role when influenza virus particles are taken into the cell. On the other hand, NA has a function of cleaving sialic acid when virus particles leave the cell surface in the late stage of infection, and is useful for acquiring infectivity.

  Virus separation methods, RT-PCR methods, and the like are used as techniques for the definitive diagnosis of influenza. However, these methods are not suitable for rapid diagnosis because of complicated operations and time required for measurement. In recent years, immunochromatography using a membrane such as nitrocellulose has been used as a rapid and simple test method. In this technique, a complex of a solid phase (for example, a membrane) that specifically binds to a detection target, a detection target (virus particle), and a label (labeled antibody) that specifically binds to the detection target is immobilized. It is a method of forming on a phase. An enzyme (for example, alkaline phosphatase), a metal colloid (for example, gold colloid), a colored latex particle, or the like is bound as a label to the antibody that specifically binds to the detection target. Patent Document 1 discloses a technique based on an immunochromatography method, and a kit using this technique is also commercially available. However, a conventional immunochromatography diagnostic kit has high specificity (98.2%) but low sensitivity (62.3%) (see Non-Patent Document 1).

  Neuraminidase is required for the release of influenza virus from infected cells during the influenza virus growth process. Therefore, by inhibiting neuraminidase, the growth of influenza virus can be suppressed. Neuraminidase activity inhibitors have attracted attention as a new treatment method for influenza viruses, and several compounds have been developed so far. Examples of drugs that have been developed include zanamivir (trade name Relenza), oseltamivir (trade name Tamiflu), peramivir (trade name Rapiacta), and laninamivir (registered trademark Inavir). A neuraminidase activity inhibitor inhibits viral infection in mucus at the respiratory site and is considered effective for early treatment. In addition, a detection method using an N-acetylneuraminic acid derivative as a luminescent reagent for detecting influenza is known (see Patent Documents 2 to 4).

WO2011 / 105537 (September 1, 2011 international publication) Special table 5-505099 (announced on August 5, 1993) US Patent Publication US2008 / 0286758 (published November 20, 2008) US Patent Publication US2011 / 0189655 (published August 4, 2011)

Ann. Intern. Med. 156, 500-511, 2012

  Since neuraminidase activity inhibitors need to be administered early (within 48 hours) after the onset of influenza, it is necessary to quickly determine whether neuraminidase activity inhibitors are effective against infected influenza viruses. It is done. However, when a conventional immunochromatography diagnostic kit is used, there is a possibility that an infected patient at the early stage of onset may be missed due to low sensitivity. Moreover, although the conventional immunochromatography method can determine whether the detected influenza virus is A type or B type, it cannot determine whether it is sensitive to a neuraminidase activity inhibitor.

  In addition, the techniques of Patent Documents 2 to 4 are merely techniques for detecting neuraminidase activity, and not only any technique for determining the effectiveness of a neuraminidase activity inhibitor has been studied. No technique has been studied for knowing whether or not it is sensitive to a neuraminidase activity inhibitor.

  The present invention has been made in view of the above problems. Specifically, the influenza virus is detected with higher sensitivity than the conventional immunochromatography method, and the detected influenza virus is sensitive to a neuraminidase activity inhibitor. The object is to provide a technology that can easily know whether or not there is.

  That is, the present invention provides a method for determining the effectiveness of a neuraminidase activity inhibitor against detected influenza virus, the method comprising N-acetylneuraminic acid-luciferin, luciferase and a first neuraminidase activity inhibitor. A first luminescent reagent containing and a control reagent containing N-acetylneuraminic acid-luciferin and luciferase, respectively, with the subject sample; a first from the first luminescent reagent after incubation; Measuring the amount of luminescence and the amount of control luminescence from the control reagent after incubation; and comparing the first amount of luminescence with the amount of control luminescence; or Contacting with; A first luminescent reagent comprising an acetylneuraminic acid-luciferin, luciferase and neuraminidase activity inhibitor and a control reagent comprising N-acetylneuraminic acid-luciferin and luciferase in contact with a subject sample. Incubating each with a fraction containing an influenza antibody; measuring a first luminescence amount from the first luminescence reagent after incubation and a control luminescence amount from the control reagent after incubation; and The method includes a step of comparing one light emission amount with a control light emission amount. In addition, this method can also be a method of acquiring the data which judge the effectiveness of the neuraminidase activity inhibitor with respect to the influenza virus detected.

  In addition, the present invention further comprises a step of incubating a second luminescent reagent containing N-acetylneuraminic acid-luciferin, luciferase and a second neuraminidase activity inhibitor with the subject sample; A step of measuring a second amount of luminescence from the luminescence reagent; and a step of comparing the second luminescence amount with the first luminescence amount and the control luminescence amount, or N-acetylneuramin Incubating a second luminescent reagent comprising acid-luciferin, luciferase and a second neuraminidase activity inhibitor with a fraction containing an anti-influenza antibody contacted with the subject sample; Measuring a second luminescence amount from the second luminescence reagent; and a second luminescence amount Or a method comprising the step of comparing with the first light emission amount and the control amount of light emission.

  In the present invention, the first neuraminidase activity inhibitor is preferably selected from the group consisting of zanamivir, oseltamivir, peramivir and laninamivir.

  In the present invention, the second neuraminidase activity inhibitor is preferably selected from the group consisting of zanamivir, oseltamivir, peramivir and laninamivir and different from the first neuraminidase activity inhibitor.

In the present invention, the luciferase preferably comprises the following amino acid sequence [I], [II] or [III].
[I] The amino acid sequence shown in SEQ ID NO: 1, or the amino acid sequence in which at least one of the following substitutions (a), (b) and (c) has occurred in the amino acid sequence shown in SEQ ID NO: 1:
(A) substitution of isoleucine at position 423 with leucine, methionine or phenylalanine;
(B) substitution of aspartic acid at position 436 with glycine, alanine or serine; or (c) substitution of leucine at position 530 with arginine, lysine or histidine;
[II] The amino acid sequence shown in SEQ ID NO: 3 or the amino acid sequence in which at least one of the following substitutions (d), (e), and (f) occurs in the amino acid sequence shown in SEQ ID NO: 3:
(D) replacement of isoleucine at position 425 with leucine;
(E) substitution of aspartic acid at position 438 with glycine; or (f) substitution of isoleucine at position 532 with arginine;
[III] The amino acid sequence shown in SEQ ID NO: 5 or the amino acid sequence in which at least one of the following substitutions (g), (h) and (i) has occurred in the amino acid sequence shown in SEQ ID NO: 5:
(G) replacement of isoleucine at position 425 with leucine;
(H) substitution of aspartic acid at position 438 with glycine; or (i) substitution of isoleucine at position 532 with arginine.

  In the present invention, the subject sample is preferably a sample derived from the nasopharynx of the subject, and may be a nasopharyngeal or pharyngeal swab, a nasal or bronchial washing solution, or a nasal aspirate.

  The present invention provides a method for in vitro use of a neuraminidase activity inhibitor to determine the effectiveness of a neuraminidase activity inhibitor against detected influenza virus, wherein the neuraminidase activity inhibitor is N-acetyl. It is characterized by being used in combination with neuraminic acid-luciferin and luciferase, and an anti-influenza antibody may be further combined.

  The present invention further provides a composition for determining the efficacy of a neuraminidase activity inhibitor against detected influenza virus, the composition comprising N-acetylneuraminic acid-luciferin, luciferase and neuraminidase activity inhibitors. It is characterized by including.

  The present invention also provides a kit for determining the effectiveness of a neuraminidase activity inhibitor against detected influenza virus, the kit comprising N-acetylneuraminic acid-luciferin, luciferase and a first neuraminidase activity inhibitor It may be further provided with an anti-influenza antibody. Preferably, the kit comprises a first luminescent reagent comprising N-acetylneuraminic acid-luciferin, luciferase and a first neuraminidase activity inhibitor, and N-acetylneuraminic acid-luciferin and luciferase. And a control reagent. The kit may further comprise a second luminescent reagent comprising N-acetylneuraminic acid-luciferin, luciferase and a second neuraminidase activity inhibitor.

  The present invention relates to a first luminescent reagent comprising N-acetylneuraminic acid-luciferin, luciferase and a first neuraminidase activity inhibitor, N-acetylneuraminic acid-luciferin, luciferase and a second neuraminidase activity inhibitor And a second luminescent reagent containing N, and a control reagent containing N-acetylneuraminic acid-luciferin and luciferase, respectively, with the subject sample; Measuring a first luminescence amount, a second luminescence amount from the second luminescence reagent after incubation and a control luminescence amount from the control reagent after incubation; and a first luminescence amount and a second luminescence amount And a method to compare the amount of control light emission Encompassing, it may be a method of detecting influenza virus.

  By using the present invention, it is possible to detect influenza virus with higher sensitivity than immunochromatography and to know whether or not the detected influenza virus is sensitive to a neuraminidase activity inhibitor easily and quickly.

It is a figure which shows the result of having examined the detection sensitivity of influenza virus using the conventional test | inspection kit based on the immunochromatography method. It is a figure which shows the result of having examined the detection sensitivity of influenza virus using this invention. It is a figure which shows the result of having examined the influence of oseltamivir with respect to different neuraminidase. It is a figure which shows the result of having examined whether it can know whether the detected influenza virus is sensitive to oseltamivir. It is a figure which shows the result of having examined the influence of the zanamivir with respect to a different neuraminidase.

  Hereinafter, embodiments of the present invention will be described in detail. However, the present invention is not limited to this, and can be implemented in a mode in which various modifications are made within the described range. Moreover, all the academic literatures and patent literatures described in this specification are incorporated herein by reference. Unless otherwise specified in this specification, “A to B” indicating a numerical range means “A or more and B or less”.

[Evaluation of efficacy of neuraminidase activity inhibitor]
The present invention provides a new use for neuraminidase activity inhibitors. Specifically, the present invention provides in vitro use of neuraminidase activity inhibitors to determine the effectiveness of neuraminidase activity inhibitors against detected influenza viruses, and the present invention provides neuraminidase activity inhibitors Is used in combination with N-acetylneuraminic acid-luciferin and luciferase.

  As used herein, the “neuraminidase activity inhibitor” is not particularly limited as long as it inhibits the activity of influenza virus neuraminidase, and does not need to be specific only to influenza virus neuraminidase. Preferably, the neuraminidase activity inhibitor is a compound that specifically inhibits the activity of neuraminidase of influenza virus, such as zanamivir, oseltamivir, peramivir or laninamivir. Such neuraminidase activity inhibitors have been developed as therapeutic agents for influenza viruses, but are used for in vitro determination, specifically for determining whether neuraminidase activity inhibitors are effective. Of course, no such use is envisaged. In particular, it has never been described or suggested to be used for determination in vitro in combination with N-acetylneuraminic acid-luciferin and luciferase.

[Method for determining effectiveness of neuraminidase activity inhibitor]
The present invention may be a method for determining whether a neuraminidase activity inhibitor is effective for a subject suspected of suffering from influenza. The method of the present invention may also be a method for assisting in determining the effectiveness of a neuraminidase activity inhibitor. Specifically, a neuraminidase activity inhibitor is effective for a subject suspected of suffering from influenza. It may also be a method of acquiring data for determining whether or not there is.

  The present invention includes incubating a subject sample with a luminescent reagent comprising N-acetylneuraminic acid-luciferin, luciferase and a neuraminidase activity inhibitor. The incubating step is performed in parallel with the step of incubating the subject sample with a control reagent comprising N-acetylneuraminic acid-luciferin and luciferase.

  When targeting a human subject, the present invention suitably uses a sample (subject sample) obtained from a human subject suspected of suffering from influenza. When used in the context of therapeutic diagnosis, the subject sample is preferably a sample derived from the subject's nasopharynx, such as a nasopharynx or pharyngeal swab, nasal or bronchial lavage fluid, nasal aspirate (e.g. Nasal discharge) and may also be other respiratory tissue or exudate, or a biopsy sample containing lymph nodes. Alternatively, when used from the viewpoint of prognosis, various tissue materials derived from animals (living bodies or cadaver) may be used. Subject samples may be prepared by physically disrupting the tissue or cellular structure of the tissue material, as long as it does not interfere with the activity of influenza virus neuraminidase, including enzymes, buffers, salts, surfactants, etc. It may be prepared as a solution or suspension.

  Incubation of the luminescent reagent or control reagent with the subject sample may be performed at room temperature for a short time, for example, 10 to 40 ° C., preferably 15 to 35 ° C., more preferably 20 to 30 ° C. It may be performed for 60 minutes, preferably 5 to 30 minutes, more preferably 10 to 15 minutes.

  In the present invention, a subject sample is incubated with a luminescent reagent or control reagent containing N-acetylneuraminic acid-luciferin, luciferase and neuraminidase activity inhibitor, and then the amount of luminescence from the luminescent reagent or control reagent is measured. The That is, the present invention includes a step of measuring the first luminescence amount from the luminescent reagent after incubation and the control luminescence amount from the control reagent after incubation.

  The luciferin constituting N-acetylneuraminic acid-luciferin used in the present invention is preferably firefly luciferin. In one embodiment, compound A having the following structure is used as N-acetylneuraminic acid-luciferin.

The neuraminidase of influenza virus hydrolyzes the above compound A to produce the following compound B (N-acetylneuraminic acid) and compound C (firefly luciferin), and this firefly luciferin is present in the presence of Mg ²⁺ and ATP. It emits light when it is catalyzed by luciferase and oxidized.

  In this embodiment, when influenza virus is contained in a subject sample to be incubated with a control reagent containing N-acetylneuraminic acid-luciferin and luciferase, the control reagent after incubation emits light, and N-acetylneuron Luminic acid-luciferin, luciferase and neuraminidase activity inhibitor If the subject sample incubated with influenza virus sensitive to neuraminidase activity inhibitor, the incubated luminescence reagent will not emit light . Furthermore, even if a subject sample not containing influenza virus is incubated with a luminescent reagent or a control reagent, neither the luminescent reagent nor the control reagent after the incubation emits light.

  The amount of luminescence from the luminescence reagent or the control reagent is not particularly limited as long as a conventionally known measurement method used for measuring luminescence of luciferase is used. It should be noted that the luminescence amount of the luminescent reagent or the control reagent may be measured before incubation with the subject sample, and in that case, the luminescence amount before and after the incubation is preferably compared.

  In addition, since influenza A virus infects not only humans but also animals such as birds, pigs, horses, minks, whales, etc., when targeting non-human subjects, the present invention provides samples from subjects. You may include the process to acquire.

  In order to rapidly test for A / B influenza viruses, simple test reagents using immunochromatography are widely used. The above-mentioned simple test reagent allows the solid phase (for example, a cotton swab) wiped from the subject's nasal cavity or pharynx to be put into the extract, and the immune complex produced by the immune reaction using the extracted virus-derived protein as an antigen is visible. Viruses are detected by emitting light automatically. Alternatively, the simple test reagent may be an embodiment in which the immune reaction is performed on a test strip, and in this case, the test strip may be a solid phase for wiping the nasal cavity or pharynx of the subject. Since the neuraminidase activity inhibitor needs to be administered early after the onset of influenza, it is preferable to determine the effectiveness of the neuraminidase activity inhibitor at the early stage of influenza infection. The present invention may be carried out in combination with such an immunochromatography method.

  That is, the present invention may include a step of bringing an anti-influenza antibody into contact with a subject sample. The anti-influenza antibody that can be used in the present invention is not particularly limited as long as it can form an immune complex with influenza particles of type A or B or neuraminidase. In the contact between the anti-influenza antibody and the subject sample, even if both are brought into contact with each other in the liquid form, the subject sample in the liquid form is absorbed to spread on the test strip on which the anti-influenza antibody is immobilized. Both may be brought into contact with each other.

  The present invention includes a step of incubating the formed immune complex with a luminescent reagent containing N-acetylneuraminic acid-luciferin, luciferase and neuraminidase activity inhibitor. When the anti-influenza antibody and the subject sample are contacted in a liquid form, the immune complex can be obtained as a fraction containing the antibody by collecting the antibody using a conventionally known technique. . When the anti-influenza antibody and the subject sample are brought into contact on the test strip, the immune complex can be obtained as a fraction containing the antibody by cutting out the region where the antibody is immobilized. That is, the step of incubating the step of incubating the fraction containing the anti-influenza antibody brought into contact with the subject sample with a luminescent reagent containing N-acetylneuraminic acid-luciferin, luciferase and neuraminidase activity inhibitor. It is. The incubating step is performed in parallel with the step of incubating the formed immune complex with a control reagent containing N-acetylneuraminic acid-luciferin and luciferase.

  Incubation of the fraction with the luminescent reagent or the control reagent may also be performed at room temperature for a short time, for example, 10 to 40 ° C., preferably 15 to 35 ° C., more preferably 20 to 30 ° C. It can be performed for -60 minutes, preferably 5-30 minutes, more preferably 10-15 minutes.

  In the present invention, the amount of luminescence from the luminescent reagent or the control reagent is measured after the above fraction is incubated with the luminescent reagent or the control reagent containing N-acetylneuraminic acid-luciferin, luciferase and neuraminidase activity inhibitor. The That is, the present invention includes a step of measuring the first luminescence amount from the luminescent reagent after incubation and the control luminescence amount from the control reagent after incubation.

  The measurement of the amount of luminescence from the luminescence reagent is not particularly limited as long as a conventionally known measurement method used for measuring luminescence of luciferase is used. In addition, before the incubation with the said fraction, you may measure the light-emission quantity of a luminescent reagent, and it is preferable to compare the light-emission quantity before and behind incubation in that case.

  Type A or type B influenza has neuraminidase. That is, according to the present invention, it is possible to determine whether or not a subject suffers from influenza by providing a method for determining whether the detected influenza virus is type A or type B influenza. It becomes like this. As shown in Examples described later, the present invention can detect influenza viruses with higher sensitivity than immunochromatography. If the present invention is used in combination with a simple test reagent for immunochromatography, it can be easily and quickly known whether or not the detected influenza virus is sensitive to a neuraminidase activity inhibitor.

  As used herein, “luciferase” is intended to be firefly luciferase, and more specifically, firefly luciferase selected from the group consisting of North American firefly luciferase, Heike firefly luciferase and Genji firefly luciferase. The luciferase may be a wild type or a mutant type. Examples of the mutant luciferase include: (a) substitution of isoleucine at position 423 with leucine, methionine or phenylalanine of wild type North American firefly luciferase; (b) aspartic acid at position 436 with glycine, alanine or serine Or (c) a protein comprising a mutated amino acid sequence in which at least one substitution of leucine at position 530 with arginine, lysine or histidine has occurred. Further mutant luciferases include, for example, wild-type Heike firefly luciferase (d) substitution of isoleucine at position 425 with leucine; (e) substitution of aspartic acid at position 438 with glycine; or (f) position 532 A protein comprising a mutant amino acid sequence in which at least one substitution of isoleucine with arginine has occurred is preferred. Still further mutant luciferases include, for example, (g) substitution of isoleucine at position 425 with leucine, (h) substitution of aspartic acid at position 438 with glycine, or (i) 532 of wild type geniferal luciferase. And a protein comprising a mutated amino acid sequence in which at least one substitution of isoleucine at the position to arginine has occurred. These mutant luciferases have increased luminescence intensity compared to the wild type. The amino acid sequence and base sequence of wild-type North American firefly luciferase are shown in SEQ ID NOs: 1 and 2, respectively. The amino acid sequence and base sequence of wild-type Heike firefly luciferase are shown in SEQ ID NOs: 3 and 4, respectively. The amino acid sequence and base sequence are shown in SEQ ID NOs: 5 and 6, respectively. In addition, the mutant luciferase that can be used in the present invention is not limited to the above-described one, and Japanese Patent Application Laid-Open No. 2007-97577 (published on April 19, 2007) has improved thermal stability. Proteins listed as those having improved substrate affinity, those having changed emission wavelength, those having improved luminescence persistence, and surfactant resistance may be used.

  The method for determining the effectiveness of the neuraminidase activity inhibitor may be performed using a plurality (at least two or more types) of neuraminidase activity inhibitors. In the present specification, the first neuraminidase activity inhibitor, the second neuraminidase activity inhibitor, the first neuraminidase activity inhibitor, the first neuraminidase activity inhibitor, the first neuraminidase activity inhibitor, the first neuraminidase activity inhibitor, In some cases, the luminescent reagent is referred to as a second luminescent reagent. That is, for example, in the present invention, (i) a step of incubating a subject sample with a first luminescent reagent containing N-acetylneuraminic acid-luciferin, luciferase and a first neuraminidase activity inhibitor; ii) incubating the subject sample with a second luminescent reagent comprising N-acetylneuraminic acid-luciferin, luciferase and a second neuraminidase activity inhibitor; (iii) subject sample to N- Incubating with a control reagent comprising acetylneuraminic acid-luciferin and luciferase may be performed in parallel. Then, the present invention provides a first luminescence amount from the first luminescence reagent after incubation, a second luminescence amount from the second luminescence reagent after incubation, and a control reagent after the incubation. A step of measuring the amount of control light emission may be included. Furthermore, the present invention may include a step of comparing the first light emission amount, the second light emission amount, and the control light emission amount.

  Here, the second neuraminidase activity inhibitor is preferably different from the first neuraminidase activity inhibitor. For example, when the first neuraminidase activity inhibitor is oseltamivir, the second neuraminidase activity inhibitor is preferably a neuraminidase activity inhibitor other than oseltamivir (eg, zanamivir, peramivir or laninamivir).

  Thereby, it can be accurately determined that the subject sample contains an influenza virus having resistance to any neuraminidase activity inhibitor. Specifically, when light is emitted by the control reagent, light is emitted by the first light-emitting reagent to which the first neuraminidase activity inhibitor is added, and light is not emitted by the second light-emitting reagent to which the second neuraminidase activity inhibitor is added. It can be determined that the influenza has resistance to the first neuraminidase activity inhibitor (sensitive to the second neuraminidase activity inhibitor). In addition, if the first luminescence reagent added with the first neuraminidase activity inhibitor does not emit light and the second luminescence reagent added with the second neuraminidase activity inhibitor does not emit light, It can be determined that the influenza is sensitive to one neuraminidase activity inhibitor (also sensitive to a second neuraminidase activity inhibitor).

  The neuraminidase activity inhibitor is preferably a compound (for example, oseltamivir, zanamivir, peramivir, or laninamivir) that specifically inhibits the neuraminidase activity of influenza virus. In this case, the specificity of the inhibitor makes it possible to reliably distinguish bacteria from influenza that is resistant to the inhibitor.

  For example, in the case of a subject sample of oseltamivir-resistant virus, light is emitted when a control reagent is used, and light is emitted even when a luminescent reagent containing oseltamivir is used. Therefore, if it can be identified as influenza by immunochromatography etc., it can be determined that it is oseltamivir resistant influenza, but if it cannot be identified as influenza, is it a microorganism with neuraminidase that is oseltamivir resistant influenza or is not derived from influenza? It may not be possible to distinguish. In recent years, oseltamivir resistant influenza virus has been reported, and oseltamivir resistant virus became a problem in Japan during the 2008/09 season. In the future, there is a high possibility that oseltamivir-resistant virus will be prevalent, and in that case, it is very significant if it can be surely identified as oseltamivir-resistant virus.

  Therefore, the present inventors considered the following as a method for determining whether a virus has resistance to a specific neuraminidase activity inhibitor. For example, although oseltamivir resistant virus has been reported all over the world, reports of zanamivir resistant virus are extremely limited (Clin. Infect. Dis. 52, 432-437, 2011). Therefore, it is considered extremely unlikely that influenza viruses that are resistant to both oseltamivir and zanamivir will appear. Therefore, the present inventors considered that it is possible to determine whether a virus is resistant to oseltamivir, for example, by conducting tests using two types of oseltamivir and zanamivir. Specifically, when it emits light with the control reagent, emits light with the luminescent reagent added with oseltamivir, and does not emit light with the luminescent reagent added with zanamivir, it can be determined that it is oseltamivir-resistant influenza (zanamivir sensitive). Moreover, when it emits light with a control reagent, it does not light with the light-emitting reagent which added oseltamivir, and it does not light even with the light-emitting reagent which added zanamivir, it can determine with oseltamivir sensitivity influenza (it is also zanamivir sensitivity). In addition, when light is emitted in any of the control reagent, the luminescent reagent to which oseltamivir is added, and the luminescent reagent to which zanamivir is added, it can be determined that the bacterium has neuraminidase.

  Of course, two or more neuraminidase activity inhibitors may be used in the same manner even when the above-described anti-influenza antibody includes a step of contacting the subject sample.

[Composition for determining efficacy of neuraminidase activity inhibitor]
The present invention further provides a composition for determining the efficacy of a neuraminidase activity inhibitor against detected influenza virus, the composition comprising N-acetylneuraminic acid-luciferin, luciferase and neuraminidase activity inhibitors. It is characterized by including. The composition of the present invention is preferably used in a method for determining whether or not a neuraminidase activity inhibitor is effective. N-acetylneuraminic acid-luciferin, luciferase and neuraminidase activity constituting the composition of the present invention The inhibitor only needs to have the characteristics described above.

  As used herein, a “composition” is intended to be a form in which various components are contained in one substance.

[Kit for determination of effectiveness of neuraminidase activity inhibitor]
The present invention also provides a kit for determining the effectiveness of a neuraminidase activity inhibitor against detected influenza virus, the kit comprising N-acetylneuraminic acid-luciferin, luciferase and a neuraminidase activity inhibitor. And a control reagent containing N-acetylneuraminic acid-luciferin and luciferase, and may further comprise an anti-influenza antibody. Here, the “kit” includes various components to be contained in the composition in a container (for example, a bottle, a plate, a tube, a dish, etc.), and all of the containers are packed together as a whole. Are intended. Moreover, this kit may be equipped with two or more types of luminescent reagents. That is, this kit may include, for example, a first luminescent reagent and a second luminescent reagent as luminescent reagents, and the first luminescent reagent and the second luminescent reagent are different types of neuraminidase activity inhibitors. May be included.

  As used herein in the context of a kit, “comprising” is intended to mean that the component is contained within any of the individual containers that make up the kit. . In addition, the kit may be a package in which a plurality of different compositions are packaged in one, or may be a package in a solution form that is contained in a container, and two or more different components are the same. It is also possible to prepare a mixture of these containers. The kit preferably comprises instructions describing the procedure using the above components (ie, the procedure for determining the effectiveness of a neuraminidase activity inhibitor against the detected influenza virus). The “instructions” may be written or printed on paper or other media, or may be affixed to electronic media such as magnetic tape, computer readable disk or tape, CD-ROM, etc. . The kit may be used to constitute the above-described composition, and may comprise separately the substances contained in the above-described composition, or may comprise separately the above-described composition and additional components. .

  In addition, this kit may be equipped with a N-acetylneuraminic acid-luciferin, luciferase, and neuraminidase activity inhibitor for preparing a luminescent reagent and / or a control reagent in separate containers.

  The present invention is not limited to the above-described embodiments, and various modifications are possible within the scope shown in the claims, and embodiments obtained by appropriately combining technical means disclosed in different embodiments. Is also included in the technical scope of the present invention.

  EXAMPLES Hereinafter, although an Example demonstrates this invention concretely, this invention is not limited by an Example.

  In the following examples, the detection sensitivity of influenza virus was compared between the method of the present invention and the conventional immunochromatography method. Specifically, in Example 1, the detection sensitivity of influenza virus was examined using a conventional test kit based on a commercially available immunochromatography method. In Example 2, a luminescence detection method using a luminescent reagent was used. The detection sensitivity of influenza virus was examined. Moreover, in Example 3, the influence of oseltamivir on different neuraminidases was examined, and in Example 4, it can be known whether influenza viruses detected by conventional immunochromatography are sensitive to neuraminidase activity inhibitors. I examined whether or not. Specifically, influenza virus was detected by immunochromatography, the detection part was cut out, and then the effect of oseltamivir was verified by incubating the detection part cut out in a luminescent reagent with or without oseltamivir added. In Example 5, the influence of zanamivir on different neuraminidases was examined.

[Example 1] Detection sensitivity of influenza virus by immunochromatography Rapid tester color FLU stick (manufactured by Sekisui Medical) was used as an influenza virus test kit by immunochromatography. As influenza virus, Human influenza A / PR / 8/34 (H1N1) UV-inactivated (manufactured by Advanced Biotechnology) was used. After adding 10 μL of water or 6.1 × 10 ^{4 to} 6.1 × 10 ⁶ vp (virus particle) / 10 μL of influenza virus sample solution to 300 μL of dilution solution, immerse the test stick in the sample dilution solution did. Ten minutes after the immersion, the test stick was taken out of the specimen diluent, and the determination area of the test stick was taken in with a scanner ES-2200 (manufactured by EPSON) and imaged. The results are shown in FIG. Since the influenza virus used this time is type A, a blue line (lower arrow in the figure) was detected in the judgment area of the test stick. In the figure, the upper arrow indicates the control.

When the number of viruses was 6.1 × 10 ⁶ or more 10 minutes after immersion of the test stick, a clear blue line was detected in the judgment area. When the number of viruses was 6.1 × 10 ^5, a weak but blue line was detected, and when the number of viruses was 6.1 × 10 ⁴ , no blue line was detected. From the above results, it was found that 6.1 × 10 ⁵ influenza viruses can be detected by the conventional method based on immunochromatography.

[Example 2] Detection sensitivity of influenza virus with luminescent reagent As influenza virus, Human influenza A / PR / 8/34 (H1N1) UV-inactivated (manufactured by Advanced Biotechnologies) was used. As a luminescent reagent, luminescent reagent A (50 mM Tricine buffer (pH 8.0), 5 μg / mL firefly luciferase (source: Promega or Bio-Enex)), 10 μg / mL N-acetylneuraminic acid-luciferin (patent) (Synthesized according to the method described in Document 3) 1 mg / mL BSA, 12 mM ATP, 10 mM DTT, 1 mM coenzyme A, 1 mM Mg (CH ₃ COO) ₂ , 4 mM CaCl ₂ , 5% trehalose, 4% mannitol, 1% sucrose 0.5% Triton X-100). After adding 10 μL of water or 6.1 × 10 ^{2 to} 6.1 × 10 ⁷ vp / 10 μL of influenza virus specimen solution to 40 μL of luminescent reagent A, mixing using Lumitester C110 (manufactured by Kikkoman) The amount of luminescence in the liquid was measured. The results are shown in FIG.

The amount of luminescence 10 minutes after the addition of 6.1 × 10 ⁴ influenza viruses was 703, which was distinguishable from the background luminescence amount 274 before the addition. This indicates that 6.1 × 10 ⁴ influenza viruses can be detected. Further, this result shows that the sensitivity is about 10 times higher than the immunochromatography method shown in Example 1. Furthermore, the amount of luminescence 60 minutes after the addition of 6.1 × 10 ³ influenza viruses was 596, which could be distinguished from the background luminescence amount 268 before the addition. From this, it was found that the influenza virus detection sensitivity was 6.1 × 10 ³ .

[Example 3] Inhibition effect specific to influenza virus by neuraminidase activity inhibitor Human influenza A / PR / 8/34 (H1N1) UV-inactivated (manufactured by Advanced Biotechnology) was used as an influenza virus. Luminescent reagent B (50 mM imidazole buffer (pH 7.0), 5 μg / mL firefly luciferase, 10 μg / mL N-acetylneuraminic acid-luciferin, 1 mg / mL BSA, 12 mM ATP, 10 mM DTT, 1 mM coenzyme A, 15 mM MgSO ₄ , 4 mM CaCl ₂ , 4% mannitol, 1% sucrose, 0.5% Triton X-100). After adding 0-10 mM / 5 μL (final concentration 0-1000 μM) of oseltamivir phosphate (commonly known as Tamiflu, manufactured by LKT Laboratories) to 40 μL of luminescent reagent B, 0.1 U / 5 μL of Clostridium Perfingens-derived neuraminidase (New) (England Biolabs) or 6.1 × 10 ⁵ vp / 5 μL of influenza virus specimen solution was added, and the amount of luminescence in the mixture was measured using Lumitester C110 (Kikkoman). The results are shown in FIG.

  C. In the case of using Perfingens-derived neuraminidase, even when 1000 μM oseltamivir was added, no decrease in the amount of luminescence in the mixture was observed (FIG. 3 (a)). However, when neuraminidase derived from influenza virus was used, a decrease in the amount of luminescence in the mixture was observed depending on the concentration of oseltamivir, and luminescence was completely suppressed in the presence of 1000 μM oseltamivir (FIG. 3 (b)). . From this, oseltamivir is C.I. It was found that it does not inhibit nephrminidase from Perfingens but specifically inhibits neuraminidase of influenza virus.

[Example 4] Susceptibility test for oseltamivir against influenza virus detected by immunochromatography Rapid tester color FLU stick (manufactured by Sekisui Medical Co., Ltd.) was used as an influenza virus test kit by immunochromatography. As influenza virus, Human influenza A / PR / 8/34 (H1N1) UV-inactivated (manufactured by Advanced Biotechnology) was used. After adding 3.05 × 10 ⁸ vp / 5 μL of influenza virus to 300 μL of the diluting solution, the test stick was immersed in the specimen diluent. Ten minutes after the immersion, the test stick was taken out from the specimen diluent, and the determination area of the test stick was cut out and washed with water. Next, for 40.5 μL of Luminescent Reagent B, 4.5 μL of water, or 10 mM / 4.5 μL (final concentration 1 mM) of oseltamivir phosphate (trade name Tamiflu, LKT)
(Laboratories) was added, and the determination area after washing was incubated in the mixed solution, and the amount of luminescence in the mixed solution was measured with a Lumitester C110 (Kikkoman) (see FIGS. 3 and 4).

  Luminescence was confirmed in the absence of oseltamivir, but luminescence was almost completely suppressed in the presence of oseltamivir (see FIG. 4). This result correlates with the influenza virus used being an oseltamivir sensitive strain. That is, it was found that the sensitivity of the detected influenza virus to oseltamivir can be known. In addition, this method can be similarly tested not only on oseltamivir but also on other neuraminidase activity inhibitors (eg Relenza), and the sensitivity of the detected influenza virus to neuraminidase activity inhibitors can be known and detected. It is thought that a therapeutic agent suitable for influenza virus can be selected.

Example 5 Influenza virus-specific luminescence suppression effect by neuraminidase activity inhibitor-2
As influenza virus, Human influenza A / PR / 8/34 (H1N1) UV-inactivated (manufactured by Advanced Biotechnology) was used. Luminescent reagent B (50 mM imidazole buffer (pH 7.0), 5 μg / mL firefly luciferase, 10 μg / mL N-acetylneuraminic acid-luciferin, 1 mg / mL BSA, 12 mM ATP, 10 mM DTT, 1 mM coenzyme A, 15 mM MgSO ₄ , 4 mM CaCl ₂ , 4% mannitol, 1% sucrose, 0.5% Triton X-100). After addition of 0-10 mM / 5 μL (final concentration 0-1000 μM) of zanamivir (commonly known as Relenza, manufactured by Tokyo Chemical Industry Co., Ltd.) to 40 μL of luminescent reagent B, 0.2 U / 5 μL of Clostridium Perfingens-derived neuraminidase (New England) Biolabos) or 1.22 × 10 ⁶ vp / 5 μL of influenza virus specimen solution was added, and the amount of luminescence in the mixture was measured using Lumitester C110 (Kikkoman). The results are shown in FIG.

  C. In the case of using Perfingens-derived neuraminidase, even when 1000 μM zanamivir was added, there was almost no reduction in the amount of luminescence in the mixed solution (FIG. 5A). However, when neuraminidase derived from influenza virus was used, the amount of luminescence in the mixture decreased according to the concentration of zanamivir, and luminescence was completely suppressed in the presence of 10 μM zanamivir (FIG. 5 (b)). . From this, Zanamivir is C.I. It was found that it does not inhibit nephrminidase from Perfingens but specifically inhibits neuraminidase of influenza virus.

  As can be seen from the results of Examples 3 and 5, various types of neuraminidase activity inhibitors can be used in the present invention. Therefore, as described above, if a test is performed using a plurality of types of neuraminidase activity inhibitors, even an influenza virus having resistance to any neuraminidase activity inhibitor can be identified.

  According to the present invention, it is possible to detect influenza virus with high sensitivity and to detect whether or not the detected influenza virus is sensitive to a neuraminidase activity inhibitor. Can contribute.

Claims (6)

A first luminescent reagent comprising N-acetylneuraminic acid-luciferin, luciferase and a first neuraminidase activity inhibitor and a control reagent comprising N-acetylneuraminic acid-luciferin and luciferase, and a subject sample Incubating each;
Measuring a first luminescence amount from the first luminescence reagent after incubation and a control luminescence amount from the control reagent after incubation;
Comparing the first light emission amount with the control light emission amount;
Incubating with a subject sample a second luminescent reagent comprising N-acetylneuraminic acid-luciferin, luciferase and a second neuraminidase activity inhibitor;
Measuring a second luminescence amount from the second luminescence reagent after incubation; and comparing the second luminescence amount with the first luminescence amount and the control luminescence amount,
The first neuraminidase activity inhibitor is selected from the group consisting of zanamivir, oseltamivir, peramivir and laninamivir, and the second neuraminidase activity inhibitor is selected from the group consisting of zanamivir, oseltamivir, peramivir and laninamivir; The neuraminidase activity inhibitor is different from the first neuraminidase activity inhibitor, and it is determined whether or not the subject sample contains influenza virus, and when it is determined that the influenza virus is contained, neuraminidase for the influenza virus is determined. Methods for determining the effectiveness of an activity inhibitor :
Here, the method of determining the effectiveness of a neuraminidase activity inhibitor against the influenza virus is
(1) Whether there is an influenza virus resistant to either the first neuraminidase activity inhibitor or the second neuraminidase activity inhibitor in the subject sample, or
(2) This is a method for determining whether or not an influenza virus having sensitivity to both the first inhibitor and the second inhibitor exists in the subject sample. Contacting an anti-influenza antibody with a subject sample;
A first luminescent reagent comprising N-acetylneuraminic acid-luciferin, luciferase and a first neuraminidase activity inhibitor and a control reagent comprising N-acetylneuraminic acid-luciferin and luciferase, and a subject sample Incubating each with a fraction containing the contacted anti-influenza antibody;
Measuring a first luminescence amount from the first luminescence reagent after incubation and a control luminescence amount from the control reagent after incubation;
Comparing the first light emission amount with the control light emission amount;
Incubating a second luminescent reagent comprising N-acetylneuraminic acid-luciferin, luciferase and a second neuraminidase activity inhibitor with a fraction containing an anti-influenza antibody contacted with the subject sample ;
Measuring a second luminescence amount from the second luminescence reagent after incubation; and comparing the second luminescence amount with the first luminescence amount and the control luminescence amount,
The first neuraminidase activity inhibitor is selected from the group consisting of zanamivir, oseltamivir, peramivir and laninamivir, and the second neuraminidase activity inhibitor is selected from the group consisting of zanamivir, oseltamivir, peramivir and laninamivir; The neuraminidase activity inhibitor is different from the first neuraminidase activity inhibitor, and it is determined whether or not the subject sample contains influenza virus, and when it is determined that the influenza virus is contained, neuraminidase for the influenza virus is determined. Methods for determining the effectiveness of an activity inhibitor :
Here, the method of determining the effectiveness of a neuraminidase activity inhibitor against the influenza virus is
(1) Whether there is an influenza virus resistant to either the first neuraminidase activity inhibitor or the second neuraminidase activity inhibitor in the subject sample, or
(2) This is a method for determining whether or not an influenza virus having sensitivity to both the first inhibitor and the second inhibitor exists in the subject sample. The method according to claim 1 or 2, wherein the luciferase comprises the following amino acid sequence [I], [II] or [III]:
[I] An amino acid sequence represented by SEQ ID NO: 1, or an amino acid sequence in which at least one substitution of the following (a), (b) and (c) has occurred in the amino acid sequence represented by SEQ ID NO: 1:
(A) substitution of isoleucine at position 423 with leucine, methionine or phenylalanine;
(B) substitution of aspartic acid at position 436 with glycine, alanine or serine; or (c) substitution of leucine at position 530 with arginine, lysine or histidine;
[II] The amino acid sequence shown in SEQ ID NO: 3, or the amino acid sequence in which at least one of the following substitutions (d), (e), and (f) has occurred in the amino acid sequence shown in SEQ ID NO: 3;
(D) replacement of isoleucine at position 425 with leucine;
(E) substitution of aspartic acid at position 438 with glycine; or (f) substitution of isoleucine at position 532 with arginine;
[III] An amino acid sequence represented by SEQ ID NO: 5, or an amino acid sequence in which at least one of the following substitutions (g), (h) and (i) has occurred in the amino acid sequence represented by SEQ ID NO: 5;
(G) replacement of isoleucine at position 425 with leucine;
(H) Replacement of aspartic acid at position 438 with glycine; or (i) Replacement of isoleucine at position 532 with arginine. A first luminescent reagent comprising N-acetylneuraminic acid-luciferin, luciferase and a first neuraminidase activity inhibitor; a control reagent comprising N-acetylneuraminic acid-luciferin and luciferase; and N-acetyl A second luminescent reagent comprising neuraminic acid-luciferin, luciferase and a second neuraminidase activity inhibitor;
The first neuraminidase activity inhibitor is selected from the group consisting of zanamivir, oseltamivir, peramivir, and laninamivir; the second neuraminidase activity inhibitor is selected from the group consisting of zanamivir, oseltamivir, peramivir, and laninamivir; and The neuraminidase activity inhibitor is different from the first neuraminidase activity inhibitor, and it is determined whether or not the subject sample contains influenza virus, and when it is determined that the influenza virus is contained, neuraminidase for the influenza virus is determined. Kit for determining the effectiveness of an activity inhibitor :
Here, with the kit for judging the effectiveness of the neuraminidase activity inhibitor against the influenza virus,
(1) Whether there is an influenza virus resistant to either the first neuraminidase activity inhibitor or the second neuraminidase activity inhibitor in the subject sample, or
(2) A kit for determining whether an influenza virus having sensitivity to both the first inhibitor and the second inhibitor is present in the subject sample.   The kit according to claim 4, further comprising an anti-influenza antibody. A first luminescent reagent comprising N-acetylneuraminic acid-luciferin, luciferase and a first neuraminidase activity inhibitor, comprising N-acetylneuraminic acid-luciferin, luciferase and a second neuraminidase activity inhibitor Incubating a second luminescent reagent and a control reagent comprising N-acetylneuraminic acid-luciferin and luciferase, respectively, with the subject sample;
Measuring a first luminescence amount from the first luminescence reagent after incubation, a second luminescence amount from the second luminescence reagent after incubation, and a control luminescence amount from the control reagent after incubation; And comparing each of the first luminescence level, the second luminescence level and the control luminescence level, wherein the first neuraminidase activity inhibitor is selected from the group consisting of zanamivir, oseltamivir, peramivir and laninamivir, A method for detecting an influenza virus, wherein the second neuraminidase activity inhibitor is selected from the group consisting of zanamivir, oseltamivir, peramivir and laninamivir, and is different from the first neuraminidase activity inhibitor.