JP2013087069A - Monoclonal antibody specifically recognizing h5 subtype influenza virus - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide an antibody capable of specifically recognizing an H5 subtype influenza virus among type A influenza viruses.SOLUTION: The monoclonal antibody specifically recognizing an H5 subtype influenza virus among type A influenza viruses is produced by a hybridoma deposited under the accession number FERM BP-11229 or a hybridoma deposited under the accession number FERM BP-11230.

Description

  The present invention relates to a monoclonal antibody capable of specifically recognizing H5 subtype influenza virus among influenza A viruses, a method for detecting H5 subtype influenza virus using such an antibody, and a kit for the same.

  Influenza viruses are classified into types A, B, and C based on the difference in antigenicity between nucleoprotein (NP) and membrane protein (M). In particular, influenza A viruses are further classified into hemagglutinin (HA) and neuraminidase (HA). NA) is classified into a plurality of subtypes depending on the amino acid sequence or antigenic difference.

  Influenza A viruses include seasonal influenza viruses that are human influenza and avian influenza viruses that are avian influenza. There are several subtypes of avian influenza virus. Among these, the H5 subtype influenza virus is a subtype called “highly pathogenic influenza virus”, and there is a concern that the spread of infection in humans is feared.

  Currently, immunological diagnostic methods for influenza that are performed in the clinic detect all subtypes of type A (Non-Patent Documents 1 to 12). Therefore, when humans show signs of influenza, it is impossible to distinguish by immunodiagnostic method whether it is due to highly pathogenic avian influenza virus or seasonal influenza virus. Yes, they are distinguished by time-consuming and laborious genetic tests. Therefore, there is a need for a method for rapidly typifying highly pathogenic avian influenza viruses.

Hideyuki Ikematsu et al .: Journal of infectious diseases. 73: 1153-1158, 1999 Atsuo Goto et al .: Clinical and viruses. 28: 248-252, 2000 Masahiko Yamazaki et al .: Journal of infectious diseases. 73: 1064-1068, 1999 Keiko Mitamura et al .: Journal of infectious diseases. 73: 1069-1073, 1999 Toshimi Watanabe et al .: Journal of infectious diseases. 73: 1199-1204, 1999 Masahiko Yamazaki et al .: Journal of infectious diseases. 74: 1032-1037, 1999 Hideaki Shimizu et al .: Journal of infectious diseases. 74: 1038-1043, 1999 Keiko Mitamura et al .: Journal of infectious diseases. 74: 12-16,2000 Kawakami Chiharu et al .: Journal of infectious diseases. 75: 792-799, 2001 Masahiko Yamazaki et al .: Journal of infectious diseases. 75: 1047-1053, 2001 Keiko Mitamura et al .: Influenza. 3: 105-109, 2002 Okuno Yoshinobu et al .: Medicine and pharmacy. 48: 895-904, 2002

  The present invention has been made in view of the current state of the prior art, and its purpose is to obtain an antibody capable of specifically recognizing H5 subtype influenza virus among influenza A viruses, and such The purpose is to develop a specific, rapid and simple detection method of H5 subtype influenza virus using an antibody and a kit for the same.

  As a result of intensive studies to achieve the above object, the present inventors have produced a hybridoma that produces an anti-HA monoclonal antibody capable of specifically recognizing H5 subtype influenza virus among influenza A viruses. The present invention has been completed successfully.

That is, the present invention has the following configurations (1) to (6).
(1) Specific to H5 subtype influenza virus among influenza A viruses characterized by being produced from a hybridoma deposited under accession number FERM BP-11229 or a hybridoma deposited under accession number FERM BP-11230 Monoclonal antibodies that recognize themselves.
(2) A method for detecting H5 subtype influenza virus in a sample, wherein the detection of H5 subtype influenza virus is performed by using the antibody according to (1).
(3) The method according to claim 1, wherein the method comprises at least one of an anti-H5 subtype influenza virus primary antibody bound to a carrier immobilization element and an anti-H5 subtype influenza virus secondary antibody bound to a label. Prepare using antibody, contact sample with primary antibody, secondary antibody and carrier, H5 subtype influenza virus in sample is sandwiched between primary antibody and secondary antibody and primary antibody is immobilized on carrier The method according to (2), comprising forming a structure fixed to the carrier via the element, and then detecting the label of the secondary antibody in the structure.
(4) Either one of the primary antibody and the secondary antibody is prepared using the antibody described in (1), and the other is an antibody produced from a hybridoma deposited under accession number FERM BP-10903. The method according to (3), wherein the method is prepared.
(5) A primary antibody is prepared using the antibody described in (1), and the secondary antibody uses an antibody that can recognize influenza A virus but cannot specifically recognize H5 subtype influenza virus. The method according to (3), which is prepared.
(6) The method according to claim 3, wherein both the primary antibody and the secondary antibody are prepared using the antibody according to (1).
(7) An H5 subtype influenza virus detection reagent comprising the antibody according to (1) as an active ingredient.
(8) An H5 subtype influenza virus detection kit comprising the detection reagent according to (7).

  The monoclonal antibody of the present invention can specifically recognize H5 subtype influenza virus among influenza A viruses. Therefore, according to the present invention, it is possible to easily distinguish whether a person who has shown signs of influenza has highly pathogenic avian influenza or seasonal influenza. In particular, since the method of the present invention is based on immunological measurement, it is quicker and simpler than conventional genetic testing methods, and is extremely advantageous in practice.

FIG. 1 shows the container used in Example 6. In FIG. 1, (a) is a perspective view of the container, and (b) is a cross-sectional view of the container.

  The monoclonal antibody of the present invention is produced from a hybridoma deposited under accession number FERM BP-11229 or a hybridoma deposited under accession number FERM BP-11230. Among influenza A viruses, H5 subtype influenza virus Has a feature that can be specifically recognized. There are at least 15 types of hemagglutinin (HA) possessed by influenza A virus, and the influenza A virus is classified into H1-H15 subtypes depending on the difference. Since the antibody of the present invention is directed against HA of H5 influenza virus, it can specifically recognize H5 influenza virus among influenza A viruses.

The antibody of the present invention comprises two hybridomas prepared by immunizing a mouse with H5 subtype influenza virus isolated from human as an antigen, and fusing the spleen cell of the immunized mouse with a myeloma cell-derived strain of the mouse. (Hybridoma Mouse-Mouse hybridoma OM-b (Accession No. FERM BP-11229) and AY-2C2 (Accession No. FERM BP-11230)). Hybridoma OM-b and AY-2C2 are deposited at the National Institute of Advanced Industrial Science and Technology, Patent Biological Deposit Center (1st, 1st East, 1st Street, Tsukuba City, Ibaraki). Specifically, the antibody of the present invention is produced by culturing these hybridomas in a medium containing 10% fetal serum or in a serum-free medium under conditions of 37 ° C. and 5% CO _2. It can manufacture by collect | recovering and refine | purifying b and AY-2C2 antibody by a well-known method.

  Monoclonal antibodies of the present invention also include enzymatically treated fragments thereof, and modified and mutated antibodies such as recombinant peptides, chimeric and humanized antibodies thereof. These fragments, modified antibodies or mutant antibodies also have the same specificity for H5 subtype influenza virus as the original antibody. These can be produced by means and methods known to those skilled in the art.

  According to the present invention, there is also provided a method for detecting H5 subtype influenza virus in a sample, wherein the detection of H5 subtype influenza virus is performed by using the antibody of the present invention. Is done. Samples include pharyngeal swabs, nasal swabs, nasal aspirates, virus cultures, feces, stool, and various tissues / organs. By detecting the H5 subtype influenza virus in such a sample using the method of the present invention, it is possible to quickly, easily and reliably determine whether the subject from which the sample is derived is infected with an H5 subtype influenza virus. Can be diagnosed.

  The detection method of the present invention can be performed by incubating a sample to be detected with the antibody of the present invention to form an antigen / antibody complex, and then detecting the complex. When performing such detection based on solid phase sandwich immunoassay, specifically, an anti-H5 subtype influenza virus primary antibody bound to a carrier immobilization element and an anti-H5 subtype influenza virus bound to a label. At least one of the secondary antibodies is prepared using the antibody of the present invention, and the sample is contacted with the primary antibody, the secondary antibody and a carrier so that the H5 subtype in the sample is between the primary antibody and the secondary antibody. A structure in which the influenza virus is held and the primary antibody is fixed to the carrier via the carrier fixing element may be formed, and then the label of the secondary antibody in this structure may be detected. The order in which the sample is contacted with the primary antibody, the secondary antibody, and the carrier is not particularly limited, and the sample is first contacted with the primary antibody and the secondary antibody, and the primary antibody, the secondary antibody, and the H5 subtype influenza virus in the sample The complex may then be contacted with a carrier to fix the primary antibody in the complex to the carrier, or only the primary antibody may be contacted with the sample first, and then the carrier The primary antibody may be immobilized on a carrier by contacting with the carrier, and then the secondary antibody may be contacted. Known carriers such as polystyrene particles, polystyrene plates, silica membranes, glass filters, and silica magnetic particles can be used as the carrier, and biotin can be used as the carrier fixing element. As the label, known substances such as a fluorescent chemical substance, a luminophore, an enzyme, a fluorescent protein, a photoprotein, a magnetic substance, and a conductive substance can be used.

  In the method of the present invention, both the primary antibody and the secondary antibody can be prepared using the antibody of the present invention, but only one of the primary antibody and the secondary antibody is the antibody described in the present invention. Can also be prepared using an antibody produced from a hybridoma deposited with the other accession number FERM BP-10903. This hybridoma was previously created by one of the applicants and deposited at the Patent Organism Depositary Center of the National Institute of Advanced Industrial Science and Technology (1st, 1st East, 1st Street, Tsukuba City, Ibaraki Prefecture). It is known that the produced antibody specifically recognizes the H5 subtype influenza virus among influenza A viruses in the same manner as the antibody of the present invention. Such a combination of two specific antibodies can further improve the detection sensitivity of H5 subtype influenza virus.

  Alternatively, instead of the above combination, a primary antibody is prepared using the antibody of the present invention, and the secondary antibody can recognize influenza A virus but cannot specifically recognize H5 subtype influenza virus (that is, , A common influenza A virus detection antibody). For the influenza A virus detection antibody, an efficient production method has already been established, and it can be obtained at low cost. Moreover, when the secondary antibody is an influenza A detection antibody, the detection sensitivity of the H5 subtype influenza virus is slightly lowered, but it is at a level that is not problematic in practice. Therefore, a combination of the antibody of the present invention and the influenza A virus detection antibody can provide a practical detection method with reduced costs.

  According to the present invention, there is also provided an H5 subtype influenza virus detection reagent comprising the antibody of the present invention as an active ingredient, and an H5 subtype influenza virus detection kit comprising such a detection reagent. . When detection is performed based on solid phase sandwich immunoassay, the detection reagent of the present invention specifically includes an anti-H5 subtype influenza virus primary antibody bound to a carrier immobilization element, and an anti-label bound to a label. H2 subtype influenza virus secondary antibody can be included, and the primary antibody and / or secondary antibody can be prepared using the antibody of the present invention. The detection kit of the present invention contains a carrier, a reaction reagent necessary for detecting a label, and the like in addition to the detection reagent.

  The effects of the present invention will be described below by examples, but these examples do not limit the present invention.

Example 1
A. Production of monoclonal antibody-producing hybridoma specifically recognizing H5 subtype influenza virus Hemagglutinin (HA) gene and neuraminidase (N. Vietnam / 1194/2005) strain isolated from a Vietnamese H5N1 influenza-infected person N1) H5N1 influenza vaccine (NIBRG-14) produced by recombining the gene with an existing vaccine strain was used as an antigen, and Balb / c mice were immunized twice with this antigen. Three days later, the spleen of the immunized mouse was removed, and 1 × 10 ⁸ spleen cells were mixed with 3 × 10 ⁷ mouse myeloma cell-derived strain SP-2 / 0xxx cells. After centrifugation, the supernatant was aspirated and 1 ml of 50% polyethylene glycol solution was added at 37 ° C. over 1 minute. Next, 50 ml of RPMI 1640 medium was added over 10 minutes. After centrifugation, RMPI 1640 medium containing 10% fetal bovine serum was added and dispensed into a 96-well microplate. The next day, after adding HAT selection medium and culturing for about 10 days, the presence or absence of specific antibody in the supernatant was screened by the enzyme antibody method using inactivated virus particles. The procedure of the enzyme antibody method is as follows. A 96-well microplate was coated with inactivated H5 influenza virus particles or H1 influenza virus and blocked overnight in PBS containing 1% BSA. Next, the hybridoma supernatant was added and incubated for 1 hour at room temperature. After incubation, the plate was washed with PBS / 0.05% Tween 20, and then reacted with a peroxidase-labeled anti-mouse immunoglobulin antibody for 1 hour. After the reaction, it was washed with PBS / 0.05% Tween 20, and an OPD (orthophenylenediamine) solution was added to visualize the anti-mouse immunoglobulin antibody. Two antibody-producing hybridomas that respond to H5 subtype influenza virus but not H1 subtype influenza virus (Mouse-Mouse hybridoma OM-b (Accession No. FERM BP-11229) and Mouse-Mouse hybridoma AY-2C2 (Accession No.) FERM BP-11230)) was obtained.

B. Identification of recognition site of monoclonal antibody specifically recognizing H5 subtype influenza virus OM-b and AY2C2 antibodies are present in most clade H5 subtype influenza virus particles (clade 1, clade 2.2, 2.3). It was confirmed that there was no reaction and cross-reactivity to influenza viruses other than H5 subtype. These results are shown in Tables 1 and 2. In addition, when clade 1 recombinant HA (H5HA NIBRG14 recHA) was prepared with baculovirus and the enzyme antibody method (same as above) using it as an antigen was used, it was confirmed that it reacted specifically with HA. . From these results, it was determined that the recognition site of the monoclonal antibody produced by the obtained hybridoma is a clade-common H5HA epitope.

C. Purification of monoclonal antibody recognizing H5 subtype influenza virus The resulting hybridoma Mouse-Mouse hybridoma OM-b and AY2C2 were added to serum-free medium (Invitrogen, 12045-084) containing IL-6 (0.2 ng / ml). The culture supernatant was recovered after culturing for 5 days at 37 ° C. and 5% CO ₂ . The collected culture supernatant was purified using a protein A column to obtain anti-HA monoclonal antibodies OM-b and AY2C2 (hereinafter referred to as OM-b antibody and AY2C2 antibody, respectively).

Example 2
Detection of H5 subtype influenza virus using OM-b and AY2C2 antibodies (1)
OM-b or AY2C2 antibody is used as a primary antibody, and an antibody that can recognize influenza A virus but not H5 subtype influenza virus (anti-influenza A HA antibody) is used as a secondary antibody. Subtype influenza virus was detected.
The OM-b or AY-2C2 antibody was labeled with Biotin Labeling Kit-NH2 (Dojindo Laboratories) to prepare a primary antibody. This primary antibody is mixed with H5N1 influenza vaccine (NIBRG-14) or saline as a negative control, incubated at 37 ° C. for 15 minutes, dropped onto an anti-biotin antibody-conjugated glass fiber filter, and incubated at 37 ° C. for 5 minutes. did. To this was added 25 μl of a secondary antibody prepared by labeling anti-influenza A HA antibody with Alkaline Phosphatase Labeling Kit-NH2 (Dojindo Laboratories), incubated at 37 ° C. for 5 minutes, and 200 μl of washing solution (10 mM PBS 150 mM NaCl). After adding 0.1% Tween 20), the luminescent substrate CDPSstar was added, and the amount of luminescence was measured after a certain time. A value exceeding the blank fluctuation 4SD was defined as a cutoff value, and a sample exceeding the value was determined as positive. The results when the OM-b antibody is the primary antibody are shown in Table 3, and the results when the AY2C2 antibody is the primary antibody are shown in Table 4.

  As can be seen from Tables 3 and 4, when either the OM-b antibody or the AY2C2 antibody was used as the primary antibody, a positive result was obtained and the H5 subtype influenza virus could be detected.

Example 3
Detection of H5 subtype influenza virus using OM-b and AY2C2 antibodies (2)
H5 subtype influenza virus was detected in the same manner as in Example 2 except that the OM-b antibody was the primary antibody and the AY-2C2 antibody was the secondary antibody. The results are shown in Table 5.

  As can be seen from Table 5, H5 subtype influenza virus could be detected by combining OM-b antibody and AY2C2 antibody.

Example 4
Evaluation of sensitivity Using the serially diluted H5N1 influenza vaccine (NIBRG-14), the minimum detectable virus titer was determined in the same manner as in Example 3, and the OM-b antibody and AY-2C2 antibody were used. The sensitivity of the chemiluminescence detection method was evaluated. Distilled water (DW) was used as a negative control. As a result, the minimum detectable virus titer is 5 × 10 ⁴ FFU / ml (1 FFU (Focus Forming Unit) means 1 infectious particle) for both OM-b antibody and AY2C2 antibody, and OM-b It was found that the chemiluminescence detection method using the antibody and the AY-2C2 antibody has sufficient sensitivity.

Example 5
Specificity Evaluation Specificity of chemiluminescence detection method using OM-b antibody and AY-2C2 antibody in the same manner as in Example 3 using a virus other than influenza virus (virus titer 1 × 10 ⁶ TCID 50 ml or more) Evaluated. As a control, H5N1 influenza vaccine (NIBRG-14) was used. The results are shown in Table 6.

  As can be seen from Table 6, the results for all viruses other than influenza virus were negative, and the chemiluminescence detection method using the OM-b antibody and the AY2C2 antibody had high specificity.

Example 6
Detection of H5 subtype influenza virus using antibody produced from hybridoma deposited with OM-b antibody and accession number FERM BP-10903 OM-b antibody was deposited as accession number FERM BP-10903 with primary antibody The H5 subtype influenza virus was detected using an antibody produced from the hybridoma (hereinafter referred to as 1C10 antibody) as a secondary antibody.
A. Preparation of carrier Glass fiber filter paper (manufactured by Advantech Toyo Co., Ltd.) is rounded out, laminated on an absorption plug, and stored in a polyethylene liquid impervious container as shown in FIG. ) 100 mM citrate buffer: 100 μl of pH 3.0, (b) 50 μl of goat anti-biotin polyclonal antibody 47 μg / mL (100 mM citrate buffer: pH 3.0), (c) 5% glycerol / 1% bovine serum albumin 100 μl of the (BSA) solution (10 mM phosphate-saline buffer solution: pH 7.4) was sequentially supplied while waiting for the solution supplied immediately before to be aspirated, and then lyophilized.

B. Preparation of Reagent (1) Preparation of Primary Antibody Solution OM-b antibody and biotinamide caproic acid-N-hydroxysuccinimide ester are reacted at 25 ° C. for 4 hours and fractionated using NAP-5 (Amersham Biosciences). And it prepared so that it might become 10.0 microgram / mL, and it was set as the primary antibody solution.

(2) Preparation of secondary antibody solution Using 1C10 antibody and bovine small intestine-derived alkaline phosphatase, label with EZ-Link Maleimide Activated Alkaline Phosphatase Kit (PIERCE), and prepare to 2.0 μg / mL, A secondary antibody solution was obtained.

C. As detection samples, three types of H5 subtype influenza viruses (A / Vietnam / 1194/2004 (NIBRG-14) (H5N1), A / Anhui / 01/2005 (PR8-IBCDC RG-5) (H5N1), A / turkey / 1/2005 (NIBRG-23) (H5N1)) was used. A reaction solution was prepared by mixing 50 μl of the sample, 20 μl of the primary antibody solution and 20 μl of the secondary antibody solution, and incubated at 40 ° C. for 10 minutes. 50 μl of physiological saline was added to the reaction vessel prepared in A, and after 5 minutes, 70 μl of the reaction solution was added and incubated at 40 ° C. Two minutes later, 100 μl of physiological saline containing 0.05% Tween 20 was added twice, and two more minutes later, 30 μl of APS-5 (Lumigen) was added as a luminescent substrate. One minute after the addition of the luminescent substrate, the amount of luminescence was read with a photomultiplier tube (manufactured by Hamamatsu Photonics), and the average + 4SD of the negative sample population was set as a negative and positive cutoff to evaluate specificity and sensitivity. went. The results are shown in Table 7.

  As can be seen from Table 7, it was confirmed that this method can detect all three clades of influenza H5N1.

Example 7
By the method of Example 6, the reactivity with other subtypes of influenza virus was evaluated in the same procedure as in Example 5. The results are shown in Table 8.

  As can be seen from the results in Table 8, it was confirmed that this method did not detect other influenza subtypes.

Example 8
By the method of Example 6, the reactivity with other viruses and bacteria other than influenza virus was evaluated in the same procedure as in Example 5. The results are shown in Tables 9 and 10.

  As can be seen from the results in Tables 9 and 10, it was confirmed that this method did not detect other viruses and bacteria other than influenza virus.

  Since the antibody of the present invention can specifically recognize H5 subtype influenza virus among influenza A viruses, it is extremely useful for diagnosing H5 subtype influenza virus quickly and easily.

Claims (8)

  A hybridoma deposited under accession number FERM BP-11229 or a hybridoma deposited under accession number FERM BP-11230, specifically recognizing H5 subtype influenza virus among influenza A viruses Monoclonal antibody to do.   A method for detecting an H5 subtype influenza virus in a sample, wherein the detection of the H5 subtype influenza virus is performed by using the antibody according to claim 1.   The method uses at least one of an anti-H5 subtype influenza virus primary antibody bound to a carrier-immobilizing element and an anti-H5 subtype influenza virus secondary antibody bound to a label, using the antibody according to claim 1. The sample is contacted with the primary antibody, the secondary antibody and the carrier, the H5 subtype influenza virus in the sample is sandwiched between the primary antibody and the secondary antibody, and the primary antibody is interposed via the carrier fixing element. The method according to claim 2, further comprising: forming a structure fixed to the carrier, and then detecting the label of the secondary antibody in the structure.   Either a primary antibody or a secondary antibody is prepared using the antibody of claim 1 and the other is prepared using an antibody produced from a hybridoma deposited with accession number FERM BP-10903. The method according to claim 3.   A primary antibody is prepared using the antibody of claim 1, and a secondary antibody is prepared using an antibody that can recognize influenza A virus but cannot specifically recognize H5 influenza virus. The method according to claim 3.   4. The method of claim 3, wherein both primary and secondary antibodies are prepared using the antibody of claim 1.   An H5 subtype influenza virus detection reagent comprising the antibody according to claim 1 as an active ingredient.   An H5 subtype influenza virus detection kit comprising the detection reagent according to claim 7.