JPH07304799A - Human influenza virus-resistant antibody - Google Patents
Human influenza virus-resistant antibodyInfo
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- JPH07304799A JPH07304799A JP6117466A JP11746694A JPH07304799A JP H07304799 A JPH07304799 A JP H07304799A JP 6117466 A JP6117466 A JP 6117466A JP 11746694 A JP11746694 A JP 11746694A JP H07304799 A JPH07304799 A JP H07304799A
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- Japan
- Prior art keywords
- virus
- subtype
- human influenza
- antibody
- influenza
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
- Peptides Or Proteins (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
Description
【０００１】[0001]
【産業上の利用分野】本発明は、ヒトインフルエンザウ
イルスの感染による発症の診断、予防に利用される抗ヒ
トインフルエンザウイルス抗体に関する。BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to an anti-human influenza virus antibody used for diagnosing and preventing the onset of human influenza virus infection.
【０００２】[0002]
【従来の技術】インフルエンザウイルスには３つの型
（Ａ、Ｂ及びＣ）があり、インフルエンザの世界的な流
行を起こし、多くの死者がでるのはＡ型のヒトインフル
エンザウイルスによるものである。インフルエンザＡ型
ウイルスは更にウイルス表面タンパク質であるヘマグル
チニン( haemagglutinin：以下、ＨＡと略す）及びノイ
ラミニダーゼ（以下、ＮＡと略す）の抗原性により多く
のサブタイプに分類され、ヒトインフルエンザＡ型ウイ
ルスとしてはＨ１Ｎ１サブクラス、Ｈ２Ｎ２サブクラ
ス、Ｈ３Ｎ２サブクラスの３種が現在知られている。こ
れらのサブクラス中、Ｈ１Ｎ１サブクラス、Ｈ３Ｎ２サ
ブクラスのウイルスが現在流行しているヒトインフルエ
ンザＡ型ウイルスであり、Ｈ２Ｎ２サブクラスのウイル
スは消滅し、１９６８年以降、日本においての分離例は
無い。このインフルエンザＡ型ウイルスのＨＡは、球状
部領域( head region ) と幹領域( stem region ) とい
う二つの構造の異なった領域で構成され、球状部領域
は、ウイルスが標的細胞に結合するための受容体結合部
位を含みＨＡの血球凝集活性に関与し、一方、幹領域
は、ウイルスのエンベロープと細胞のエンドソーム膜間
の膜融合に必要な融合ペプチドを含み、融合活性に関与
している〔ウイリー( Wiley ) ら、アニュアル レビュ
ー オブ バイオケミストリー( Ann. Rev.Biochem.
)、第５６巻、第３６５〜３９４頁（１９８７）〕。BACKGROUND OF THE INVENTION There are three types of influenza virus (A, B and C), which cause a worldwide pandemic of influenza, and many deaths are caused by human influenza A virus. Influenza A virus is further classified into many subtypes according to the antigenicity of viral surface proteins haemagglutinin (hereinafter abbreviated as HA) and neuraminidase (hereinafter abbreviated as NA), and H1N1 is a human influenza A virus. Three types are currently known: subclass, H2N2 subclass, and H3N2 subclass. Among these subclasses, the H1N1 subclass and H3N2 subclass of viruses are human influenza A viruses that are currently prevalent, and the H2N2 subclass of virus has disappeared, and since 1968 there is no isolated case in Japan. HA of this influenza A virus is composed of two regions having different structures, a globular region (head region) and a stem region (stem region), and the globular region is a receptor for binding the virus to target cells. It contains a body-binding site and is involved in HA's hemagglutination activity, while the stem region contains a fusion peptide required for membrane fusion between the viral envelope and the endosomal membrane of cells and is involved in fusion activity [Willy ( Wiley) et al., Annual Review of Biochemistry (Ann. Rev. Biochem.
), 56, pp. 365-394 (1987)].
【０００３】[0003]
【発明が解決しようとする課題】ヒトインフルエンザＡ
型ウイルスは周期的にＨＡとＮＡの型を変えて大流行を
引起こし、インフルエンザの流行期である冬期前にワク
チン接種を受けても、別の型のウイルスによるインフル
エンザが流行するためワクチンの効果が期待できないこ
とが多い。しかし、ＨＡ分子やＮＡ分子中の、ウイルス
のサブタイプに共通で、抗原変異の生じ難い抗原部位、
特に立体構造を認識する抗体を得ることができれば、こ
の抗体はＡ型ウイルスの感染による発症の診断、予防に
利用できる。本発明の目的は、ヒトインフルエンザＡ型
ウイルスのサブタイプを特異的に認識する抗体、該抗体
を用いるヒトインフルエンザウイルスの検出方法、及び
該方法に使用するキットを提供することにある。[Problems to be Solved by the Invention] Human influenza A
Type virus periodically changes the type of HA and NA, causing a pandemic, and even if vaccinated before the winter season, when the influenza is outbreak, another type of virus causes an influenza pandemic, so the effect of the vaccine Can often not be expected. However, in the HA molecule and NA molecule, an antigenic site that is common to the virus subtypes and is unlikely to cause antigenic mutation,
In particular, if an antibody that recognizes a three-dimensional structure can be obtained, this antibody can be used for diagnosing and preventing the onset of infection by type A virus. An object of the present invention is to provide an antibody that specifically recognizes a subtype of human influenza A virus, a method for detecting human influenza virus using the antibody, and a kit used for the method.
【０００４】[0004]
【課題を解決するための手段】本発明を概説すれば、本
発明の第１の発明は抗ヒトインフルエンザウイルス抗体
に関し、下記特性（ａ）及び（ｂ）を有することを特徴
とする。 （ａ）ヒトインフルエンザＡ型ウイルスのＨ３Ｎ２サブ
タイプのヘマグルチニン分子中の幹領域の配列表の配列
番号１で表されるＴＧＭＲＮポリペプチド配列と配列表
の配列番号２で表されるＱＩＮＧＫＬＮＲ（Ｌ／Ｖ）Ｉ
ＥＫポリペプチド配列とを認識する。 （ｂ）ヒトインフルエンザＡ型ウイルスのＨ１Ｎ１サブ
タイプとＨ２Ｎ２サブタイプのヘマグルチニン分子中の
幹領域の配列表の配列番号３で表されるＴＧＬＲＮポリ
ペプチド配列と、配列表の配列番号４で表されるＧＩＴ
ＮＫＶＮＳＶＩＥＫポリペプチド配列とを認識しない。 また本発明の第２の発明はヒトインフルエンザウイルス
の検出方法に関し、本発明の第１の発明の抗ヒトインフ
ルエンザウイルス抗体をＨ３Ｎ２サブタイプのヒトイン
フルエンザウイルスに結合させる工程を包含することを
特徴とする。更に本発明の第３の発明は、本発明の第２
の発明の方法を用いて検出を行うためのヒトインフルエ
ンザウイルス検出キットに関し、本発明の第１の発明の
抗ヒトインフルエンザウイルス抗体を含有していること
を特徴とする。Means for Solving the Problems To outline the present invention, the first invention of the present invention relates to an anti-human influenza virus antibody, which is characterized by having the following characteristics (a) and (b). (A) The TGMRN polypeptide sequence represented by SEQ ID NO: 1 in the sequence listing of the stem region in the hemagglutinin molecule of the H3N2 subtype of human influenza A virus and the QINGKLNR (L / V represented by SEQ ID NO: 2 in the sequence listing). ) I
Recognizes EK polypeptide sequences. (B) The TGLRN polypeptide sequence represented by SEQ ID NO: 3 in the sequence listing of the stem region in the hemagglutinin molecule of the H1N1 subtype and H2N2 subtype of human influenza A virus and represented by SEQ ID NO: 4 in the sequence listing GIT
It does not recognize the NKVNSVIEK polypeptide sequence. The second invention of the present invention relates to a method for detecting human influenza virus, which comprises a step of binding the anti-human influenza virus antibody of the first invention of the present invention to a human influenza virus of H3N2 subtype. . Further, the third invention of the present invention is the second invention of the present invention.
A human influenza virus detection kit for detection using the method of the present invention, which is characterized by containing the anti-human influenza virus antibody of the first invention of the present invention.
【０００５】本発明者らは鋭意研究の結果、ヒトインフ
ルエンザＡ型ウイルスのＨ３Ｎ２サブタイプの幹領域
に、共通に保存された抗原部位に対する抗体を得ること
に成功し、次に、該抗体がヒトインフルエンザＡ型ウイ
ルスのＨ１Ｎ１サブタイプ、Ｈ２Ｎ２サブタイプに全く
交さ認識性を示さないことより、Ｈ３Ｎ２サブタイプの
確定診断に有用であることを見出し、本発明を完成し
た。As a result of diligent research, the present inventors have succeeded in obtaining an antibody against an antigen site commonly conserved in the stem region of the H3N2 subtype of human influenza A virus. The present invention has been completed by finding that it is useful for definitive diagnosis of H3N2 subtype because it does not show cross-recognition to H1N1 subtype and H2N2 subtype of influenza A virus at all.
【０００６】ヒトインフルエンザＡ型ウイルスのＨ３Ｎ
２サブタイプのＨＡ分子幹領域中の共通部位を特異的に
認識する抗体は、モノクローナル抗体として、次の様に
調製することができる。例えばマウス、モルモット、ウ
サギのような、ほ乳動物を下記抗原で免疫する。抗原と
しては次のような物質を使用することができる。Ｈ３Ｎ
２サブタイプ、例えばＡ／Fukuoka ／Ｃ２９／８５、Ａ
／Sichuan ／２／８７、Ａ／Ibaraki ／１／９０、Ａ／
Suita ／１／９０（以上、大阪大学微生物病研究所保存
株）、Ａ／Port Chalmers ／１／７３〔インフルエンザ
Ａ（Ｈ３Ｎ２）、ＡＴＣＣ ＶＲ−８１０〕、Ａ２／Ai
chi ／２／６８〔インフルエンザＡ、ＡＴＣＣ ＶＲ−
５４７〕より選択されるウイルス粒子を使用することが
できる。あるいはこれらのウイルスから得られるＨＡ分
子、若しくは遺伝子組換え技術を用いて調製されるＨＡ
ポリペプチド、若しくは本発明の抗体の認識部位、すな
わちＨＡ分子中の幹領域の抗原部位を分子内に含有する
組換えポリペプチド、又はＨＡ分子中の幹領域の抗原部
位を分子内に含有する合成ポリペプチドで免疫すること
ができる。Human influenza A virus H3N
An antibody that specifically recognizes a common site in the HA subregions of two subtypes can be prepared as a monoclonal antibody as follows. Mammals such as mice, guinea pigs and rabbits are immunized with the following antigens. The following substances can be used as the antigen. H3N
2 subtypes, eg A / Fukuoka / C29 / 85, A
/ Sichuan / 2/87, A / Ibaraki / 1/90, A /
Suita / 1/90 (above, preserved strain of the Institute for Microbial Diseases, Osaka University), A / Port Chalmers / 1/73 [Influenza A (H3N2), ATCC VR-810], A2 / Ai
chi / 2/68 [Influenza A, ATCC VR-
547] can be used. Alternatively, HA molecules obtained from these viruses, or HA prepared using gene recombination technology
Recombinant polypeptide containing the polypeptide, or the recognition site of the antibody of the present invention, that is, the antigenic region of the stem region in the HA molecule in the molecule, or synthetic containing the antigenic region of the stem region in the HA molecule in the molecule It can be immunized with the polypeptide.
【０００７】次に免疫動物より得られた脾臓細胞を、例
えばマウスのミエローマ細胞と融合させ、得られるハイ
ブリドーマから、下記（Ａ）〜（Ｃ）の性質を有する抗
体を産生する細胞を選択し、該細胞を培養することによ
って調製することができる。 （Ａ）Ｈ３Ｎ２のサブタイプウイルスに結合性を有す
る。 （Ｂ）Ｈ１Ｎ１サブタイプ及びＨ２Ｎ２サブタイプのウ
イルス、Ｈ１Ｎ１サブタイプとしては例えばＡ／Bangko
k ／１０／８３、Ａ／Yamagata／１２０／８６、Ａ／Os
aka ／９３０／８８、Ａ／Suita ／１／８９（以上、大
阪大学微生物病研究所保存株）、Ａ／ＰＲ／８／３４
〔インフルエンザ（Ｈ１Ｎ１）、ＡＴＣＣＶＲ−９
５〕、Ａ１／ＦＭ／１／４７〔インフルエンザＡ（Ｈ１
Ｎ１）、ＡＴＣＣ ＶＲ−９７〕、Ａ／New Jersey／８
／７６〔インフルエンザＡ（Ｈ１Ｎ１）、ＡＴＣＣ Ｖ
Ｒ−８９７〕、Ａ／ＮＷＳ／３３〔インフルエンザＡ
（Ｈ１Ｎ１）、ＡＴＣＣ ＶＲ−２１９〕、Ａ／Weiss
／４３〔インフルエンザＡ（Ｈ１Ｎ１）、ＡＴＣＣ Ｖ
Ｒ−９６〕、Ａ／ＷＳ／３３〔インフルエンザＡ（Ｈ１
Ｎ１）、ＡＴＣＣ ＶＲ−８２５〕、Ｈ２Ｎ２サブタイ
プとしては例えばＡ／Okuda／５７、Ａ／Adachi／２／
５７、Ａ／Kumamoto／1 ／６５、Ａ／Kaizuka ／２／６
５、Ａ／Izumi ／５／６５（以上、大阪大学微生物病研
究所保存株）、Ａ２／Japan ／３０５／５７〔インフル
エンザＡ（Ｈ２Ｎ２）、ＡＴＣＣ ＶＲ−１００〕、及
びＢ型のウイルス、例えばＢ／Nagasaki／１／８７（大
阪大学微生物病研究所保存株）、Ｂ／Allen ／４５〔イ
ンフルエンザＢ、ＡＴＣＣ ＶＲ−１０２〕には結合性
を有さない。Next, the spleen cells obtained from the immunized animal are fused with, for example, mouse myeloma cells, and cells producing antibodies having the following properties (A) to (C) are selected from the hybridomas obtained, It can be prepared by culturing the cells. (A) It has a binding property to a subtype virus of H3N2. (B) H1N1 subtype and H2N2 subtype viruses, examples of H1N1 subtype include A / Bangko
k / 10/83, A / Yamagata / 120/86, A / Os
aka / 930/88, A / Suita / 1/89 (above, Osaka University Research Institute for Microbial Diseases), A / PR / 8/34
[Influenza (H1N1), ATCCVR-9
5], A1 / FM / 1/47 [Influenza A (H1
N1), ATCC VR-97], A / New Jersey / 8
/ 76 [Influenza A (H1N1), ATCC V
R-897], A / NWS / 33 [Influenza A
(H1N1), ATCC VR-219], A / Weiss
/ 43 [Influenza A (H1N1), ATCC V
R-96], A / WS / 33 [Influenza A (H1
N1), ATCC VR-825], and H2N2 subtypes are, for example, A / Okuda / 57, A / Adachi / 2 /
57, A / Kumamoto / 1/65, A / Kaizuka / 2/6
5, A / Izumi / 5/65 (above, conserved strain of the Institute for Microbial Diseases of Osaka University), A2 / Japan / 305/57 [Influenza A (H2N2), ATCC VR-100], and B-type viruses such as B. / Nagasaki / 1/87 (Osaka University Research Institute for Microbial Diseases), B / Allen / 45 [Influenza B, ATCC VR-102] have no binding properties.
【０００８】ＨＡ分子幹領域中に配列表の配列番号１で
表されるアミノ酸配列、及び配列表の配列番号５又は６
で表されるアミノ酸配列を有するヒト以外のインフルエ
ンザウイルス、例えばＡ／duck／Czechoslovakia／１／
５６（Ｈ４Ｎ６）、Ａ／chiken／Germany “Ｎ”／４９
（Ｈ１０Ｎ７）（以上、大阪大学微生物病研究所保存
株）には結合性を有し、これらのアミノ酸配列を有さな
いヒト以外のインフルエンザウイルス、例えばＡ／whis
tling swan／Shimane ／４７６／８３（Ｈ５Ｎ３）、Ａ
／whistling swan／Shimane ／３７／８０（Ｈ６Ｎ
６）、Ａ／tufted duck ／Shimane ／１２４Ｒ／８０
（Ｈ７Ｎ７）、Ａ／turkey／Ontario ／６１１８／６８
（Ｈ８Ｎ４）、Ａ／turkey／Wisconsin ／６６（Ｈ９Ｎ
２）、Ａ／duck／England ／５６（Ｈ１１Ｎ６）（以
上、大阪大学微生物病研究所保存株）には結合性を有さ
ない。The amino acid sequence represented by SEQ ID NO: 1 in the sequence listing and the SEQ ID NO: 5 or 6 in the sequence listing in the HA molecular stem region
A non-human influenza virus having an amino acid sequence represented by, for example, A / duck / Czechoslovakia / 1 /
56 (H4N6), A / chiken / Germany “N” / 49
(H10N7) (above, conserved strain of the Institute for Microbial Diseases of Osaka University), which has binding properties and does not have these amino acid sequences, is a nonhuman influenza virus, such as A / whis.
tling swan / Shimane / 476/83 (H5N3), A
/ Whistling swan / Shimane / 37/80 (H6N
6), A / tufted duck / Shimane / 124R / 80
(H7N7), A / turkey / Ontario / 6118/68
(H8N4), A / turkey / Wisconsin / 66 (H9N
2), A / duck / England / 56 (H11N6) (above, preserved strain of the Institute for Microbial Diseases, Osaka University) does not have binding properties.
【０００９】（Ｃ）Ｈ３Ｎ２サブタイプのＨＡ分子は認
識するが、ＨＡ分子中の球状部領域が関与する血球凝集
活性は阻害しない。(C) The H3N2 subtype HA molecule is recognized, but the hemagglutination activity involving the globular region in the HA molecule is not inhibited.
【００１０】このハイブリドーマの調製に関しては、ネ
ーチャー( Nature )、第２５６巻、第４９５〜４９７頁
（１９７５）を基に行う。免疫用マウスとしては、Balb
／ｃ系マウス、Balb／ｃ系マウスと他系マウスとのＦ１
マウスなどが用いられる。免疫はマウス１匹に対して、
例えばウイルス粒子（１００〜１０００ＨＡ単位）を抗
原として用い、２〜５月間に例えば３回行う。なおマウ
スの飼育及び脾臓細胞の採取は常法に従う。The preparation of this hybridoma is carried out based on Nature, Vol. 256, pp. 495-497 (1975). Balb for immunization
/ C mouse, Balb / c mouse and other mouse F1
A mouse or the like is used. Immunization against one mouse
For example, virus particles (100 to 1000 HA units) are used as an antigen, and the treatment is performed, for example, three times in 2 to 5 months. The breeding of mice and the collection of splenocytes follow conventional methods.
【００１１】ミエローマ細胞としてはＳＰ２／０−Ａｇ
１４（ＡＴＣＣ ＣＲＬ１５８１）、ｐ３×６３Ａｇ８
Ｕ．１（ＡＴＣＣ ＣＲＬ１５９７）、ｐ３×６３Ａｇ
８（ＡＴＣＣ ＴＩＢ９）、ｐ３×６３−Ａｇ８．６５
３（ＡＴＣＣ ＣＲＬ１５８０）等が好適に用いられ
る。脾臓細胞とミエローマ細胞は１：１〜１０：１の割
合で混合し、融合はＮａＣｌ（約0.８５％）、ジメチル
スルホキシド〔１０〜２０％（ｖ／ｖ）〕及び分子量１
０００〜６０００のポリエチレングリコールを含有する
リン酸緩衝液（ｐＨ7.２〜7.４）中で、両細胞の混合物
を３５〜３７℃で１〜５分間保温することによって行
う。融合細胞の選択は、ＨＡＴ培地を用い、生育してく
る細胞として選択する。融合細胞のクローン化は限界希
釈法にて少なくとも３回繰返して行う。As the myeloma cell, SP2 / 0-Ag
14 (ATCC CRL1581), p3 x 63Ag8
U. 1 (ATCC CRL1597), p3 x 63Ag
8 (ATCC TIB9), p3 × 63-Ag8.65.
3 (ATCC CRL1580) and the like are preferably used. Spleen cells and myeloma cells were mixed at a ratio of 1: 1 to 10: 1, and fusion was performed with NaCl (about 0.85%), dimethyl sulfoxide [10 to 20% (v / v)] and molecular weight 1
It is carried out by incubating the mixture of both cells at 35 to 37 ° C for 1 to 5 minutes in a phosphate buffer (pH 7.2 to 7.4) containing 000 to 6000 polyethylene glycol. The fused cells are selected using HAT medium as the growing cells. Cloning of the fused cells is repeated at least 3 times by the limiting dilution method.
【００１２】ハイブリドーマを通常の動物細胞と同様に
して培養すれば、その結果培地中に本発明の抗体を得る
ことができる。また該ハイブリドーマをプリスタン処理
のヌードマウス、又はBalb／ｃマウスの腹腔内に移植し
て増殖させることにより腹水中に本発明の抗体を蓄積さ
せることができる。すなわち、これらのマウス腹腔内に
プリスタン0.５〜１mgを接種し、その後２〜３週目に腹
腔に５×１０6 〜１×１０7 個のハイブリドーマを移植
する。通常７〜１０日後に腹水が蓄積し、これを採取す
る。培養物及び腹水中のモノクローナル抗体は通常の手
段で精製される。When the hybridoma is cultured in the same manner as ordinary animal cells, the antibody of the present invention can be obtained in the medium as a result. In addition, the antibody of the present invention can be accumulated in ascites by transplanting the hybridoma into the abdominal cavity of a pristane-treated nude mouse or Balb / c mouse and proliferating it. That is, 0.5 to 1 mg of pristane was inoculated into the abdominal cavity of these mice, and then 5 to 10 6 to 1 × 10 7 hybridomas were transplanted to the abdominal cavity in the second to third weeks. Ascites usually accumulates after 7 to 10 days and is collected. Monoclonal antibodies in culture and ascites fluid are purified by conventional means.
【００１３】得られたモノクローナル抗体はＨ３Ｎ２サ
ブタイプのＨＡ分子の幹領域を認識し、詳細には以下の
性状を有している。The obtained monoclonal antibody recognizes the stem region of the HA molecule of the H3N2 subtype, and has the following properties in detail.
【００１４】（ａ）染色試験により、Ｈ３Ｎ２サブタイ
プで感染したＭＤＣＫ細胞（ＡＴＣＣＣＣＬ３４）を認
識し、Ｈ１Ｎ１サブタイプ及びＨ２Ｎ２サブタイプで感
染したＭＤＣＫ細胞は認識しない。染色試験は４種類の
抗体（本発明のモノクローナル抗体、ウサギ抗マウスイ
ムノグロブリンＧ血清、ヤギ抗ウサギイムノグロブリン
Ｇ血清、ペルオキシダーゼ−ウサギ抗ペルオキシダーゼ
複合体）を用い、ジャーナル オブ クリニカル ミク
ロバイオロジー（Ｊ．Clin. Microbiol ) 、第２８巻、
第１３０８〜１３１３頁（１９９０）に記載の方法に準
じ行う。(A) The staining test recognizes MDCK cells (ATCCCCL34) infected with H3N2 subtype, but does not recognize MDCK cells infected with H1N1 subtype and H2N2 subtype. For the staining test, four kinds of antibodies (monoclonal antibody of the present invention, rabbit anti-mouse immunoglobulin G serum, goat anti-rabbit immunoglobulin G serum, peroxidase-rabbit antiperoxidase complex) were used, and Journal of Clinical Microbiology (J. Clin. Microbiol), Volume 28,
It is carried out according to the method described on pages 1308 to 1313 (1990).
【００１５】（ｂ）免疫沈降法により、Ｈ３Ｎ２サブタ
イプのＨＡ分子を認識し、Ｈ１Ｎ１サブタイプ及びＨ２
Ｎ２サブタイプのＨＡ分子は認識しない。(B) By the immunoprecipitation method, the HA molecule of the H3N2 subtype is recognized, and the H1N1 subtype and H2 are recognized.
It does not recognize HA molecules of the N2 subtype.
【００１６】（ｃ）血球凝集試験において、Ｈ１Ｎ１サ
ブタイプ、Ｈ２Ｎ２サブタイプ、Ｈ３Ｎ２サブタイプの
それぞれの血球凝集活性を阻害しない。(C) In the hemagglutination test, the hemagglutination activity of each of the H1N1 subtype, H2N2 subtype and H3N2 subtype is not inhibited.
【００１７】（ｄ）ＨＡ分子をコードする遺伝子解析に
より特定される、Ｈ３Ｎ２サブタイプのＨＡ分子中の幹
領域に特有の共通保存領域を認識し、Ｈ１Ｎ１サブタイ
プ及びＨ２Ｎ２サブタイプのＨＡ分子中の幹領域に特有
の共通保存領域は認識しない。(D) Recognizing the common conserved region unique to the stem region in the HA molecule of the H3N2 subtype, which is identified by the genetic analysis encoding the HA molecule, and is recognized in the HA molecules of the H1N1 subtype and the H2N2 subtype. It does not recognize the common storage area specific to the trunk area.
【００１８】Ｈ３Ｎ２サブタイプのＨＡ分子中の共通保
存領域としては、ジャーナル オブビロロジー（Ｊ．Vi
rology )、第６７巻、第２５５２〜２５５８頁（１９９
３）に記載のＨ３Ｎ２サブタイプのＨＡ分子中の幹領域
の配列表の配列番号１で表されるＴＧＭＲＮポリペプチ
ド配列と配列表の配列番号２で表されるＱＩＮＧＫＬＮ
Ｒ（Ｌ／Ｖ）ＩＥＫポリペプチド配列がある。図１にＨ
Ａ分子中の三次構造の模式図〔前出ウイリーら〕、及び
Ｈ３Ｎ２サブタイプのＨＡ分子中の共通保存領域の位置
を示す。図中Ａ領域、Ｂ領域で示す両ポリペプチド配列
は、図１に示す様にＨＡ分子中の幹領域の中央で互に近
接して位置しており、抗体の一例のHybridoma ＡＩ３Ｃ
（ＦＥＲＭ ＢＰ−４５１６）の生産するモノクローナ
ル抗体ＡＩ３Ｃは、該ＨＡ分子中の幹領域の配列表の配
列番号１で表されるＴＧＭＲＮポリペプチド配列と配列
表の配列番号２で表されるＱＩＮＧＫＬＮＲ（Ｌ／Ｖ）
ＩＥＫポリペプチド配列とを認識する。As a common conserved region in HA molecules of H3N2 subtype, Journal of Virology (J. Vi.
rology), 67, 2555-2558 (199)
3) The TGMRN polypeptide sequence represented by SEQ ID NO: 1 in the sequence listing and the QINGKLN represented by SEQ ID NO: 2 in the sequence listing of the stem region in the HA molecule of H3N2 subtype
There is an R (L / V) IEK polypeptide sequence. H in Figure 1
A schematic diagram of the tertiary structure in the A molecule [Willie et al., Supra] and the position of the common conserved region in the HA molecule of the H3N2 subtype is shown. Both polypeptide sequences shown in the regions A and B in the figure are located close to each other in the center of the stem region in the HA molecule as shown in FIG. 1, and hybridoma AI3C as an example of an antibody.
The monoclonal antibody AI3C produced by (FERM BP-4516) is the TGMRN polypeptide sequence represented by SEQ ID NO: 1 in the sequence listing of the stem region of the HA molecule and the QINGKLNR (L / V)
Recognizes the IEK polypeptide sequence.
【００１９】Ｈ１Ｎ１サブタイプ、及びＨ２Ｎ２サブタ
イプのＨＡ分子中の共通保存領域としては、前出ジャー
ナル オブ ビロロジーに記載の、Ｈ１Ｎ１サブタイプ
及びＨ２Ｎ２サブタイプのＨＡ分子中の幹領域の配列表
の配列番号３で表されるＴＧＬＲＮポリペプチド配列と
配列表の配列番号４で表されるＧＩＴＮＫＶＮＳＶＩＥ
Ｋポリペプチド配列があるが、本発明の抗体はこれらの
部位は認識しない。The common conserved region in the HA molecule of the H1N1 subtype and the H2N2 subtype is the sequence in the sequence list of the stem region in the HA molecule of the H1N1 subtype and H2N2 subtype described in the above-mentioned Journal of Birology. TGLRN polypeptide sequence represented by No. 3 and GITNKVNSVIE represented by SEQ ID No. 4 in the sequence listing
Although there are K polypeptide sequences, the antibodies of the invention do not recognize these sites.
【００２０】本発明により得られる抗体、又は抗体由来
のＦａｂ、抗体をコードする遺伝子より調製した抗体、
Ｆａｂ、Ｆｖ、ｓｃＦｖ等はヒトインフルエンザＡ型ウ
イルスのＨ３Ｎ２サブクラスのＨＡ分子中の幹領域に特
有の共通保存領域を特異的に認識する。したがって、球
状部領域の抗原変異に全く影響されることなく、ヒトイ
ンフルエンザＡ型ウイルスのＨ３Ｎ２サブクラスを特異
的に検出することができ、ヒトインフルエンザウイルス
による発症の診断や予防のための、ヒトインフルエンザ
ウイルスの検出、特に、流行中のウイルスをＨ１Ｎ１サ
ブクラス、Ｈ３Ｎ２サブクラス、Ｂ型ウイルスにタイピ
ングする際に有用である。The antibody obtained by the present invention, or Fab derived from the antibody, an antibody prepared from a gene encoding the antibody,
Fab, Fv, scFv and the like specifically recognize a common conserved region unique to the stem region in the HA molecule of the H3N2 subclass of human influenza A virus. Therefore, the H3N2 subclass of human influenza A virus can be specifically detected without being affected by antigen mutations in the globular region, and the human influenza virus for diagnosis and prevention of onset of human influenza virus can be obtained. Is particularly useful for typing a pandemic virus into H1N1 subclass, H3N2 subclass, and B virus.
【００２１】ヒトインフルエンザウイルスの検出に際し
ては、ヒトインフルエンザＡ型ウイルスのＨ１Ｎ１サブ
クラス、Ｈ２Ｎ２サブクラスは認識し、Ｈ３Ｎ２サブク
ラスは認識しない抗体、例えばHybridoma Ｃ１７９（Ｆ
ＥＲＭ ＢＰ−４５１７）より産生され（特願平４−２
７２５３８号）、前出ジャーナル オブ ビロロジーに
記載のモノクローナル抗体、Ｃ１７９と組合せ使用する
ことにより、検体中のＨ１Ｎ１サブクラス、Ｈ２Ｎ２サ
ブクラスとＨ３Ｎ２サブクラスの分別検出を簡便に行う
ことができる。更に抗Ｂ型ウイルス抗体を組合せること
により、流行中のヒトインフルエンザウイルス（Ｈ１Ｎ
１サブタイプ、Ｈ３Ｎ２サブタイプ、Ｂ型ウイルス）の
タイピングを正確、迅速、簡便に行うことができる。In detecting human influenza virus, an antibody that recognizes the H1N1 and H2N2 subclasses of the human influenza A virus but does not recognize the H3N2 subclass, such as Hybridoma C179 (F
ERM BP-4517) (Japanese Patent Application No. 4-2)
No. 72538), C179, a monoclonal antibody described in the above-mentioned Journal of Birology, can be used to conveniently perform differential detection of H1N1 subclass, H2N2 subclass, and H3N2 subclass in a sample. Furthermore, by combining an anti-B virus antibody, the human influenza virus (H1N)
1 subtype, H3N2 subtype, and B virus) can be typed accurately, quickly, and easily.
【００２２】本発明の抗体を用いるＨ３Ｎ２サブクラス
の検出方法としては、従来この分野でよく知られた免疫
測定法、すなわち酵素免疫測定法、ラジオイムノアッセ
イ免疫比濁法、ラテックス凝集法等が使用でき、中でも
酵素免疫測定法が、感度、簡便さにおいて最も実用的で
ある。As a method for detecting the H3N2 subclass using the antibody of the present invention, immunoassay methods well known in the art, that is, enzyme immunoassay method, radioimmunoassay immunoturbidimetric method, latex agglutination method and the like can be used. Among them, the enzyme immunoassay method is the most practical in terms of sensitivity and simplicity.
【００２３】検体としては、うがい液より調製したウイ
ルス液、鼻汁より調製したウイルス液、咽頭ぬぐい液よ
り調製したウイルス液等を使用するのが最も簡便であ
る。例えばウイルス液中のウイルスをヒトインフルエン
ザウイルス高感受性のＭＤＣＫ細胞に感染させ、モノク
ローナル抗体ＡＩ３Ｃを用い、前出の染色試験を行うこ
とにより、高感度、簡便にＨ３Ｎ２サブタイプを検出す
ることができる。この時、Ｃ１７９、抗Ｂ型ウイルス抗
体と併用すれば、検体中のヒトインフルエンザウイルス
の検出を効率よく行うことができる。また各ウイルスが
混合感染した場合の検体中からも、各型のウイルスを効
率よく分別、検出することができる。It is most convenient to use a virus solution prepared from a mouthwash, a virus solution prepared from nasal discharge, a virus solution prepared from a pharyngeal swab, etc., as a sample. For example, H3N2 subtype can be detected with high sensitivity by infecting MDCK cells highly sensitive to human influenza virus with a virus in a virus solution and performing the above-mentioned staining test using the monoclonal antibody AI3C. At this time, if C179 and an anti-B virus antibody are used in combination, the human influenza virus in the sample can be efficiently detected. In addition, each type of virus can be efficiently separated and detected from a sample obtained by mixed infection of the viruses.
【００２４】また本発明の抗ヒトインフルエンザウイル
ス抗体を含有するキットを用いることにより、ヒトイン
フルエンザＡ型ウイルス、Ｈ３Ｎ２サブクラスの検出を
簡便に行うことができる。なおキット中には、Ｈ１Ｎ１
サブクラス検出用のＣ１７９、Ｂ型ウイルス検出用の抗
Ｂ血清を含有させることにより更に簡便に、ヒトインフ
ルエンザウイルスを検出することができる。なおキット
に用いる試薬は溶液状でも良いし、凍結乾燥物でも良
い。By using the kit containing the anti-human influenza virus antibody of the present invention, human influenza A virus and H3N2 subclass can be easily detected. In addition, in the kit, H1N1
By including C179 for detecting subclass and anti-B serum for detecting B virus, human influenza virus can be detected more easily. The reagent used in the kit may be in the form of a solution or a freeze-dried product.
【００２５】[0025]
【実施例】以下に本発明を実施例により説明するが、本
発明はこれらの実施例に限定されるものではない。EXAMPLES The present invention will be described below with reference to examples, but the present invention is not limited to these examples.
【００２６】実施例１ 1.ウイルスの調製 Ｈ１Ｎ１サブタイプとしてＡ／ＰＲ／８／３４、Ａ／Ba
ngkok ／１０／８３、Ａ／Yamagata／１２０／８６、Ａ
／Osaka ／９３０／８８、Ａ／Suita ／１／８９、Ｈ２
Ｎ２サブタイプとしてＡ／Okuda ／５７、Ａ／Adachi／
２／５７、Ａ／Kumamoto／１／６５、Ａ／Kaizuka ／２
／６５、Ａ／Izumi ／５／６５、Ｈ３Ｎ２サブタイプと
してＡ２／Aichi ／２／６８、Ａ／Fukuoka ／Ｃ２９／
８５、Ａ／Sichuan ／２／８７、Ａ／Ibaraki ／１／９
０、Ａ／Suita ／１／９０、Ｂ型ウイルスとしてＢ／Na
gasaki／１／８７を用い、ヒト以外のインフルエンザウ
イルスとして、Ｈ４Ｎ６サブタイプとしてはＡ／duck／
Czechoslovakia／１／５６、Ｈ５Ｎ３サブタイプとして
はＡ／whistling swan／Shimane ／４７６／８３、Ｈ６
Ｎ６サブタイプとしてはＡ／whistling swan／Shimane
／３７／８０、Ｈ７Ｎ７サブタイプとしてはＡ／turfte
d duck／Shimane ／１２４Ｒ／８０、Ｈ８Ｎ４サブタイ
プとしてはＡ／turkey／Ontario ／６１１８／６８、Ｈ
９Ｎ２サブタイプとしてはＡ／turkey／Wisconsin ／６
６、Ｈ１０Ｎ７サブタイプとしてはＡ／chicken ／Germ
any “Ｎ”／４９、Ｈ１１Ｎ６サブタイプとしてはＡ／
duck／England ／５６を用い、それぞれのウイルスを１
１日齢の発育鶏卵の尿膜腔内に接種し、３４℃で４日間
培養後、各ウイルス液を採取した。Example 1 1. Preparation of virus A / PR / 8/34 and A / Ba as H1N1 subtypes
ngkok / 10/83, A / Yamagata / 120/86, A
/ Osaka / 930/88, A / Suita / 1/89, H2
As N2 subtype, A / Okuda / 57, A / Adachi /
2/57, A / Kumamoto / 1/65, A / Kaizuka / 2
/ 65, A / Izumi / 5/65, A2 / Aichi / 2/68 as an H3N2 subtype, A / Fukuoka / C29 /
85, A / Sichuan / 2/87, A / Ibaraki / 1/9
0, A / Suita / 1/90, B / Na as type B virus
Using gasaki / 1/87, as a non-human influenza virus, the H4N6 subtype was A / duck /
Czechoslovakia / 1/56, A / whistling swan / Shimane / 476/83, H6 as H5N3 subtype
A / whistling swan / Shimane as N6 subtype
/ 37/80, A / turfte as H7N7 subtype
d duck / Shimane / 124R / 80, A / turkey / Ontario / 6118/68, H for H8N4 subtype
A / turkey / Wisconsin / 6 as 9N2 subtype
6, A / chicken / Germ as H10N7 subtype
any "N" / 49, A / for H11N6 subtype
Use duck / England / 56 to set each virus to 1
One-day-old embryonated chicken eggs were inoculated into the allantoic cavity and cultured at 34 ° C for 4 days, and then each virus solution was collected.
【００２７】2.モノクローナル抗体の調製 （１）実施例１−１で調製したＡ２／Aichi ２／６８の
ウイルス（３２０ＨＡ単位）をフロイント完全アジュバ
ントに使用前に懸濁し、１ヵ月間隔で、Balb／ｃマウス
腹腔内注射により、２度免疫し、その１ヵ月後に、同抗
原（３２０ＨＡ単位）のＰＢＳ懸濁液を用い腹腔内注射
しブーストした。その３日後に、マウスより脾臓を摘出
し、脾細胞を調製した。マウスミエローマとしてはｐ３
×６３Ａｇ８を１０％牛胎児血清添加ＤＭＥ培地で継代
後２日間培養したものを、細胞融合前に生理食塩水で洗
浄し、調製した。次に脾細胞とミエローマ細胞を細胞数
１：５の割合で混合し、遠心分離して上清を除き、沈殿
した細胞塊を充分ほぐした後、かくはんしながら、１ml
の混合液〔ポリエチレングリコール−４０００（２
ｇ）、ＭＥＭ（２ml）、ジメチルスルホキシド〕に加
え、５分間３７℃に保温した後、液の全量が１０mlにな
るようにゆっくりＭＥＭを加えた。次に、遠心分離後、
上清を除き、ゆるやかに細胞をほぐした。これに正常培
地〔ＰＲＭＩ−１６４０に牛胎児血清１０％を加えたも
の〕３０mlを加え、メスピペットを用いてゆるやかに細
胞を懸濁した。2. Preparation of Monoclonal Antibody (1) The A2 / Aichi 2/68 virus (320 HA units) prepared in Example 1-1 was suspended in Freund's complete adjuvant before use, and at 1 month intervals, Balb / The mice were immunized twice by intraperitoneal injection, and one month later, they were boosted by intraperitoneal injection using a PBS suspension of the same antigen (320 HA units). Three days later, the spleen was removed from the mouse to prepare splenocytes. P3 for mouse myeloma
X63Ag8 was cultured in a DME medium supplemented with 10% fetal bovine serum for 2 days after passage, and washed with physiological saline before cell fusion to prepare. Next, splenocytes and myeloma cells were mixed at a cell ratio of 1: 5, centrifuged to remove the supernatant, and the precipitated cell mass was thoroughly loosened, and then stirred to give 1 ml.
Mixed solution of [polyethylene glycol-4000 (2
g), MEM (2 ml), dimethylsulfoxide] and kept at 37 ° C. for 5 minutes, and then MEM was slowly added so that the total amount of the solution was 10 ml. Then, after centrifugation,
The supernatant was removed and the cells were loosely loosened. To this, 30 ml of a normal medium [PRMI-1640 plus 10% fetal bovine serum] was added, and the cells were gently suspended using a measuring pipette.
【００２８】懸濁液を９６穴の培養プレートに分注し、
５％のＣＯ2 を含む培養器中で、３７℃で２４時間培養
した。次にＨＡＴ培地を加え、１０〜１４日間培養し
た。続いて培養上清の一部を採り、ハイブリドーマのス
クリーニングを行った。Dispense the suspension into a 96-well culture plate,
The cells were cultured at 37 ° C. for 24 hours in an incubator containing 5% CO 2 . Next, HAT medium was added and the cells were cultured for 10 to 14 days. Subsequently, a part of the culture supernatant was collected and a hybridoma was screened.
【００２９】（２）インフルエンザＡ型ウイルスのＨ３
Ｎ２サブタイプに保存されているＡ領域、Ｂ領域に反応
するモノクローナル抗体を得るために、希釈していな
い、上記培養上清を１次抗体として用い、３種のサブタ
イプ（Ｈ３Ｎ２、Ｈ１０Ｎ７、Ｈ１Ｎ１）のそれぞれに
感染したＭＤＣＫ細胞の染色試験を行った。染色試験は
前出のジャーナル オブ クリニカル ミクロバイオロ
ジーに記載の方法に準じて行った。すなわち９６穴マイ
クロタイタープレート（ファルコン３０７２：ベクトン
ディキンソン社製）上で、ヒトインフルエンザＡ型ウ
イルスの各サブタイプ株（Ｈ３Ｎ２：Ａ２／Aichi ／２
／６８、Ｈ１０Ｎ７：Ａ／chicken ／Germany “Ｎ”／
４９、Ｈ１Ｎ１：Ａ／ＰＲ／８／３４）に感染させたＭ
ＤＣＫ細胞を、ＰＢＳ（ｐＨ7.４）で洗浄後、無水エタ
ノールで室温下、１０分間固定した。次にこれらの細胞
を４種類の抗体〔モノクローナル抗体を含有する前記培
養上清、ウサギ抗マウスイムノグロブリンＧ血清（オル
ガノテクニカ社製）の１０００倍希釈液、ヤギ抗ウサギ
イムノグロブリンＧ血清（オルガノテクニカ社製）の５
００倍希釈液、ペルオキシダーゼ−ウサギ抗ペルオキシ
ダーゼ複合体（オルガノテクニカ社製）の１０００倍希
釈液〕で連続的に、３７℃、３０分間ずつ反応させ、処
理細胞をＰＢＳで洗浄した。最後にペルオキシダーゼ反
応を、0.０１％のＨ2 Ｏ2 と0.３mg／mlの３，３′−ジ
アミノベンジジン四塩酸のＰＢＳ溶液を用い、グラハ
ム、カルノフスキー( Graham、Karnovsky ) の方法で行
った。染色された細胞は通常の光学顕微鏡で観察し、Ｈ
３Ｎ２サブタイプ感染のＭＤＣＫ細胞及びＨ１０Ｎ７サ
ブタイプ感染のＭＤＣＫ細胞をそれぞれ認識する抗体を
選抜した。次に該抗体産生の確認された細胞が増殖して
いる穴の細胞を取り出し、限界希釈法を３回行い、目的
の細胞をクローニングし、クローニングされたハイブリ
ドーマ株をHybridoma ＡＩ３Ｃ、該ハイブリドーマが産
生するモノクローナル抗体をモノクローナル抗体ＡＩ３
Ｃと命名した。なお、該モノクローナル抗体ＡＩ３Ｃの
開発名はモノクロナール抗体Ｆ４９である。(2) H3 of influenza A virus
In order to obtain a monoclonal antibody that reacts with the A region and B region conserved in the N2 subtype, the undiluted culture supernatant was used as the primary antibody, and three subtypes (H3N2, H10N7, H1N1) were used. The staining test of MDCK cells infected with each of the above was performed. The dyeing test was performed according to the method described in the above-mentioned Journal of Clinical Microbiology. That is, on a 96-well microtiter plate (Falcon 3072: Becton Dickinson), each subtype strain of human influenza A virus (H3N2: A2 / Aichi / 2)
/ 68, H10N7: A / chicken / Germany “N” /
49, H1N1: A / PR / 8/34) infected with M
DCK cells were washed with PBS (pH 7.4) and then fixed with absolute ethanol at room temperature for 10 minutes. Next, these cells were treated with four kinds of antibodies [the above-mentioned culture supernatant containing a monoclonal antibody, a 1000-fold dilution of rabbit anti-mouse immunoglobulin G serum (manufactured by Organotechnica), goat anti-rabbit immunoglobulin G serum (organotechnica). 5)
A 00-fold diluted solution and a 1000-fold diluted solution of peroxidase-rabbit anti-peroxidase complex (manufactured by Organotechnica) were continuously reacted at 37 ° C. for 30 minutes, and the treated cells were washed with PBS. Finally, the peroxidase reaction was performed by the method of Graham, Karnovsky using 0.01% H 2 O 2 and 0.3 mg / ml 3,3′-diaminobenzidine tetrahydrochloride in PBS. It was The stained cells are observed under a normal light microscope and
Antibodies that recognize MDCK cells infected with 3N2 subtype and MDCK cells infected with H10N7 subtype were selected. Next, the cells in the hole in which the cells confirmed to produce the antibody are taken out, and the limiting dilution method is performed three times to clone the target cells, and the cloned hybridoma strain is Hybridoma AI3C, which the hybridoma produces. Monoclonal antibody to monoclonal antibody AI3
It was named C. The development name of the monoclonal antibody AI3C is the monoclonal antibody F49.
【００３０】このHybridoma ＡＩ３Ｃは、Hybridoma Ａ
Ｉ３Ｃと表示して、工業技術院生命工学工業技術研究所
に、ＦＥＲＭ ＢＰ−４５１６として寄託されている。This Hybridoma AI3C is a Hybridoma A
It has been deposited as FERM BP-4516 at the Institute of Biotechnology, Institute of Biotechnology, designated as I3C.
【００３１】（３）プリスタン処理したBalb／ｃマウス
に上記ハイブリドーマ株を５×１０6個／匹マウスの腹
腔内に投与した。１０〜２１日後に、腹水ガンが誘発さ
れたマウスから腹水を採り、３０００rpm ／５分の遠心
処理により固型成分を除去し、腹水液を調製した。腹水
液１ml中には約５mgのモノクローナル抗体ＡＩ３Ｃ（以
下、単にＡＩ３Ｃと略す）が含有されていた。ＡＩ３Ｃ
はプロテインＡ−セファロース４Ｂ（ファルマシア社
製）で精製された。(3) Balb / c mice treated with pristane were intraperitoneally administered with the above hybridoma strain at 5 × 10 6 mice / mouse. After 10 to 21 days, ascites was collected from the mouse in which ascites cancer was induced, and solid components were removed by centrifugation at 3000 rpm for 5 minutes to prepare an ascites fluid. 1 ml of ascites fluid contained about 5 mg of monoclonal antibody AI3C (hereinafter, simply abbreviated as AI3C). AI3C
Was purified with Protein A-Sepharose 4B (Pharmacia).
【００３２】3.モノクローナル抗体の性状 （１）実施例１−２−（３）記載の腹水液の１００倍希
釈液を段階希釈し、実施例２−（２）記載の染色試験を
行い、ＡＩ３Ｃの抗原認識性を検討した。Ｈ１Ｎ１サブ
タイプとしてはＡ／ＰＲ／８／３４、Ａ／Bangkok ／１
０／８３、Ａ／Yamagata／１２０／８６、Ａ／Osaka ／
９３０／８８、Ａ／Suita ／１／８９、Ａ１／ＦＭ／１
／４７、Ｈ２Ｎ２サブタイプとしてはＡ／Okuda ／５
７、Ａ／Adachi／２／５７、Ａ／Kumamoto／１／６５、
Ａ／Kaizuka ／２／６５、Ａ／Izumi／５／６５、Ｈ３
Ｎ２サブタイプとしてＡ／Aichi ／２／６８、Ａ／Fuku
oka ／Ｃ２９／８５、Ａ／Sichuan ／２／８７、Ａ／Ib
araki ／１／９０、Ａ／Suita／１／９０、Ａ／Kitakyu
shu／１５９／９３、更にインフルエンザＢ型ウイルス
としてＢ／Nagasaki／１／８７を用い、ヒトインフルエ
ンザウイルス以外の実施例１−１記載のウイルスも用い
た。その結果を表１、表２に示す。3. Properties of Monoclonal Antibody (1) A 100-fold dilution of the ascites fluid described in Example 1-2- (3) was serially diluted, and the staining test described in Example 2- (2) was performed. The antigen recognizability of was examined. As H1N1 subtype, A / PR / 8/34, A / Bangkok / 1
0/83, A / Yamagata / 120/86, A / Osaka /
930/88, A / Suita / 1/89, A1 / FM / 1
/ 47, A / Okuda / 5 as H2N2 subtype
7, A / Adachi / 2/57, A / Kumamoto / 1/65,
A / Kaizuka / 2/65, A / Izumi / 5/65, H3
A / Aichi / 2/68, A / Fuku as N2 subtype
oka / C29 / 85, A / Sichuan / 2/87, A / Ib
araki / 1/90, A / Suita / 1/90, A / Kitakyu
shu / 159/93, B / Nagasaki / 1/87 was used as the influenza B virus, and viruses described in Example 1-1 other than the human influenza virus were also used. The results are shown in Tables 1 and 2.
【００３３】[0033]
【表１】 表 １ ─────────────────────────────── ウ イ ル ス 染 色 力 価 ─────────────────────────────── Ｈ１Ｎ１サブタイプ Ａ／ＰＲ／８／３４ ＜４００ Ａ／Bangkok ／１０／８３ ＜４００ Ａ／Yamagata／１２０／８６ ＜４００ Ａ／Osaka ／９３０／８８ ＜４００ Ａ／Suita ／１／８９ ＜４００ Ｈ２Ｎ２サブタイプ Ａ／Okuda ／５７ ＜４００ Ａ／Adachi／２／５７ ＜４００ Ａ／Kumamoto／１／６５ ＜４００ Ａ／Kaizuka ２／６５ ＜４００ Ａ／Izumi ５／６５ ＜４００ Ｈ３Ｎ２サブタイプ Ａ２／Aichi ／２／６８ ４０９６００ Ａ／Fukuoka ／Ｃ２９／８５ １０２４００ Ａ／Sichuan ／２／８７ １０２４００ Ａ／Ibaraki ／１／９０ １０２４００ Ａ／Suita ／１／９０ １０２４００ Ｂ Ｂ／Nagasaki／１／８７ ＜４００ ───────────────────────────────[Table 1] Table 1 ─────────────────────────────── Virus dyeing color strength ───── ────────────────────────── H1N1 Subtype A / PR / 8/34 <400 A / Bangkok / 10/83 <400 A / Yamagata / 120/86 <400 A / Osaka / 930/88 <400 A / Suita / 1/89 <400 H2N2 subtype A / Okuda / 57 <400 A / Adachi / 2/57 <400 A / Kumamoto / 1/65 <400 A / Kaizuka 2/65 <400 A / Izumi 5/65 <400 H3N2 subtype A2 / Aichi / 2/68 409600 A / Fukuoka / C29 / 85 102400 A / Sichuan / 2/87 102400 A / Ibaraki / 1 / 90 102400 A / Suita / 1/90 102400 B B / Nagasaki / 1/87 <400 ────────────────────────────────
【００３４】[0034]
【表２】 [Table 2]
【００３５】表１、表２中の数字は実施例１−２−
（３）の腹水液の希釈倍数であり、染色力価は染色試験
で細胞を染色可能な該腹水液の最大希釈倍数を示す。ま
た表２中のＡ領域アミノ酸配列、Ｂ領域アミノ酸配列と
は各サブタイプにおいてＨ３Ｎ２サブタイプのＡ領域、
Ｂ領域にそれぞれ対応する領域のアミノ酸配列を配列表
の配列番号で示したものであり、配列表の配列番号１、
及び配列番号５又は６で示すアミノ酸配列を有するサブ
タイプのウイルスをＡＩ３Ｃは認識する。The numbers in Tables 1 and 2 are those of Example 1-2.
It is the dilution factor of the ascites fluid of (3), and the staining titer indicates the maximum dilution factor of the ascites fluid capable of staining the cells in the staining test. Further, the A region amino acid sequence and the B region amino acid sequence in Table 2 mean the A region of the H3N2 subtype in each subtype,
The amino acid sequences of the regions respectively corresponding to the B regions are shown by SEQ ID NOs in the sequence listing, and SEQ ID NO: 1 in the sequence listings,
And AI3C recognizes a subtype of virus having the amino acid sequence shown in SEQ ID NO: 5 or 6.
【００３６】以上、ＡＩ３ＣはすべてのＨ３Ｎ２サブタ
イプ及びＡ／duck／Czechoslovakia／１／５６、Ａ／ch
icken ／Gevmany “Ｎ”／４９を認識し、他のサブタイ
プ、Ｂ型ウイルスは認識しなかった。As described above, AI3C is all H3N2 subtypes and A / duck / Czechoslovakia / 1/56, A / ch
icken / Gevmany "N" / 49 was recognized, and other subtypes, type B virus were not recognized.
【００３７】（２）抗体の血球凝集阻害活性（ＨＩ）は
次の様に行った。抗体としては実施例１−（３）の腹水
液を用い、該抗体は使用前に３倍容のレセプター分解酵
素（ＲＤＥ：武田薬品工業社製）溶液を加え、３７℃、
１８時間反応後、５６℃、４５分間の加熱処理でＲＤＥ
を失活させ、最終的に腹水液の１６倍希釈液として調製
し、被検液とし、その段階希釈液を調製し、次に実施例
１−１記載の各ウイルス（１６ＨＡ単位）と混合し、室
温で３０分間反応させた。その後、ニワトリ赤血球を加
えよく混和し、各ウイルスで赤血球凝集活性に及ぼす抗
体の影響を検討した。ＡＩ３Ｃはすべてのサブタイプの
ウイルスの血球凝集活性に影響を与えなかった。(2) The hemagglutination inhibitory activity (HI) of the antibody was measured as follows. The ascites fluid of Example 1- (3) was used as the antibody, and the antibody was added with a 3-fold volume of a receptor-degrading enzyme (RDE: Takeda Pharmaceutical Co., Ltd.) solution before use, and the temperature was adjusted to 37 ° C.
After reacting for 18 hours, heat treatment at 56 ° C for 45 minutes causes RDE
Was inactivated, and finally prepared as a 16-fold dilution of ascites fluid to prepare a test solution, and its serial dilution was prepared and then mixed with each virus (16 HA units) described in Example 1-1. The mixture was reacted at room temperature for 30 minutes. Then, chicken red blood cells were added and mixed well, and the effect of the antibody on the hemagglutination activity of each virus was examined. AI3C did not affect the hemagglutinating activity of all subtype viruses.
【００３８】4.エピトープの決定 ＡＩ３Ｃの認識タンパク質がＨＡ分子であることを免疫
沈降反応によって決定した。すなわち、Ｈ３Ｎ２サブタ
イプのＡ２／Aichi ／２／６８を３０分間ＭＤＣＫ細胞
に吸着、感染させた後、培地中のメチオニンを１０μCi
の「35Ｓ〕メチオニンで置換したＭＥＭで２４時間培養
し、感染細胞を標識した。次に該細胞を集め、次にＲＩ
ＰＡ緩衝液〔５０mMトリス（ｐＨ7.４）、１５０mM Ｎ
ａＣｌ、１mM ＥＤＴＡ、１％ノニデットＰ−４０、１
％デオキシコール酸、0.１％ＳＤＳ〕に再懸濁した。続
いて不溶物を遠心除去した後、上清を得た。次に該上清
をＡＩ３Ｃと混合し、１時間、４℃で保温した後、プロ
テインＡ−セファロースＣＬ４Ｂビーズを添加し、室温
で２時間保持し、免疫沈降物をビーズに吸着させた。次
に該ビーズを集め、ＲＩＰＡ緩衝液で５回洗浄した後、
沸騰し、ＡＩ３Ｃの結合タンパク質を遊離させた。次に
該タンパク質のＳＤＳ−１2.５％ポリアクリルアミドゲ
ル電気泳動を行い、ゲルを固定し、１Ｍサリチル酸ナト
リウムに浸してから乾かし、オートラジオグラフィーを
行った。ＡＩ３Ｃの結合する標識タンパク質は、その電
気泳動パターンより、Ａ２／Aichi ／２／６８のＨＡ分
子と同定された。同様の試験をＨ１Ｎ１サブタイプ、Ｈ
２Ｎ２サブタイプ、他のＨ３Ｎ２サブタイプ及び実施例
１−１記載のＢ型ウイルス、ヒト以外のウイルスをそれ
ぞれ用い行った。ＡＩ３ＣはすべてのＨ３Ｎ２サブタイ
プ、Ａ／duck／Czechoslovakia／１／５６、及びＡ／ch
icken ／Germany " Ｎ "／４９のＨＡ分子と特異的に免
疫沈降した。これらのウイルスのＨＡ分子の幹領域に共
通に保存されているアミノ酸配列は前出の配列番号１で
表されるアミノ酸配列、及び配列番号２、５、６で表さ
れるアミノ酸配列であり、ＡＩ３Ｃのエピトープはこの
両アミノ酸配列と決定した。4. Determination of Epitope It was determined by immunoprecipitation that the AI3C recognition protein was the HA molecule. That is, H3N2 subtype A2 / Aichi / 2/68 was adsorbed and infected with MDCK cells for 30 minutes, and then 10 μCi of methionine in the medium was added.
The infected cells were labeled by culturing for 24 hours in MEM substituted with " 35 S] methionine.
PA buffer [50 mM Tris (pH 7.4), 150 mM N
aCl, 1 mM EDTA, 1% nonidet P-40, 1
% Deoxycholic acid, 0.1% SDS]. Subsequently, the insoluble matter was removed by centrifugation to obtain a supernatant. Next, the supernatant was mixed with AI3C, incubated at 4 ° C. for 1 hour, Protein A-Sepharose CL4B beads were added, and the mixture was kept at room temperature for 2 hours to adsorb the immunoprecipitate to the beads. The beads were then collected and washed 5 times with RIPA buffer,
Boil to release AI3C binding protein. Next, the protein was subjected to SDS-12.5% polyacrylamide gel electrophoresis, the gel was fixed, immersed in 1 M sodium salicylate, dried, and autoradiographed. The labeled protein to which AI3C binds was identified as an HA molecule of A2 / Aichi / 2/68 from its electrophoretic pattern. Similar tests for H1N1 subtype, H
A 2N2 subtype, another H3N2 subtype, the B virus described in Example 1-1, and a nonhuman virus were used. AI3C is for all H3N2 subtypes, A / duck / Czechoslovakia / 1/56, and A / ch
icken / Germany "N" / 49 HA molecules were specifically immunoprecipitated. The amino acid sequences commonly conserved in the stem regions of HA molecules of these viruses are the amino acid sequence represented by SEQ ID NO: 1 and the amino acid sequences represented by SEQ ID NOs: 2, 5, and 6 described above. The epitope was determined for both amino acid sequences.
【００３９】実施例２ 1.Ｃ１７９の調製 プリスタン処理したBalb／ｃマウスにHybridoma Ｃ１７
９（ＦＥＲＭ ＢＰ−４５１７）を５×１０6 個／匹マ
ウスの腹腔内に投与した。１０〜２１日後に、腹水ガン
が誘発されたマウスから腹水を採り、３０００rpm ／５
分の遠心処理により固型成分を除去し、腹水液を調製し
た。腹水液１ml中には約５mgのモノクローナル抗体Ｃ１
７９（以下、単にＣ１７９と略す）が含有されていた。
Ｃ１７９はプロテインＡ−セファロース４Ｂ（ファルマ
シア社製）で精製した。Example 2 1. Preparation of C179 Hybridoma C17 was added to Balb / c mice treated with pristane.
9 (FERM BP-4517) was intraperitoneally administered to 5 × 10 6 mice / mouse. After 10 to 21 days, ascites was collected from the mice in which ascites cancer was induced, and 3000 rpm / 5
The solid component was removed by centrifugation for minutes to prepare an ascites fluid. About 1 mg of monoclonal antibody C1 in 1 ml of ascites fluid
79 (hereinafter simply abbreviated as C179) was contained.
C179 was purified with Protein A-Sepharose 4B (Pharmacia).
【００４０】2.抗Ｂ型ウイルス抗体の調製 実施例１−１に記載のヒトインフルエンザＢ型ウイル
ス、Ｂ／Nagasaki／１／８７の５０００ＨＡ単位をフロ
イント完全アジュバントに使用前に懸濁し、１ヵ月間隔
で、ウサギ筋肉内に注射し、二度免疫した。二度目の免
疫の１０日後に、心臓より全採血し、抗Ｂ型ウイルス血
清を調製した。2. Preparation of anti-type B virus antibody Human HA type B virus described in Example 1-1, 5000 HA units of B / Nagasaki / 1/87, was suspended in Freund's complete adjuvant before use, and at 1 month intervals. Then, the rabbit was injected intramuscularly and immunized twice. Ten days after the second immunization, whole blood was collected from the heart to prepare anti-B virus serum.
【００４１】3.ヒトインフルエンザウイルス検出キット
の作製 実施例１−２−（３）記載のＡＩ３ＣのＰＢＳ溶液（１
mg／ml）１ml、実施例２−１記載のＣ１７９のＰＢＳ溶
液（１mg／ml）１ml、及び実施例２−２記載の抗Ｂ型ウ
イルス血清１mlをそれぞれ５ml容のバイアルに分注し、
凍結乾燥を行い、それぞれの凍結乾燥標品を得た。次に
これらの標品と、抗体溶解、希釈用の１％ブロックエー
ス（雪印社製）含有ＰＢＳ１００ml入バイアルを組合
せ、ヒトインフルエンザウイルス検出キットを作製し
た。3. Preparation of Human Influenza Virus Detection Kit AI3C PBS solution (1) described in Example 1-2- (3)
1 ml of the C179 PBS solution (1 mg / ml) described in Example 2-1 and 1 ml of the anti-B virus serum described in Example 2-2 were dispensed into 5 ml vials, respectively.
Lyophilization was performed to obtain each freeze-dried sample. Next, these preparations were combined with a 100 ml PBS containing 1% Block Ace (manufactured by Snow Brand) for dissolving and diluting antibodies to prepare a human influenza virus detection kit.
【００４２】実施例３ 1.臨床分離株のタイピング １９６８年以降に患者より分離された分離株８０株（大
阪府立公衆衛生研究所保存株）のタイピングを行った。
すなわち、９６穴マイクロプレート（コーニング２６８
６０）の各穴にＭＤＣＫ細胞を１×１０4 個ずつ分注
し、翌日、各分離株のウイルス液２５μｌを１株につき
３穴分注し、３５℃、４５分間保温し、ウイルスを感染
させた。各穴をＰＢＳで洗浄後、0.５％トラガカントゴ
ム（和光純薬社製）、及び５μｇ／mlのトリプシンを含
有するイーグルＭＥＭ１００μｌを添加した。これを３
５℃で１６時間保温した後、添加液を除去し、各穴をＰ
ＢＳで洗浄した。次に各穴の細胞を、室温下、無水エタ
ノールで１０分間固定した。次に実施例２で作製したキ
ットを用い、ＡＩ３Ｃ溶液（１mg／ml）の１０００倍希
釈液、Ｃ１７９溶液（１mg／ml）の１０００倍希釈液、
抗Ｂ型血清（原血清濃度）の１０００倍希釈液を調製
し、これらの抗体希釈液を用い、各細胞の染色試験を行
った。すなわち、各分離株が感染した３穴のうち、１穴
にはＡＩ３Ｃ希釈液、他の１穴にはＣ１７９希釈液、残
りの１穴には抗Ｂ型ウイルス血清希釈液の各１００μｌ
をそれぞれの穴で反応させた後、ウサギ抗マウスイムノ
グロブリンＧ血清の１０００倍希釈液１００μｌ、ヤギ
抗ウサギイムノグロブリン血清の５００倍希釈液１００
μｌ、ペルオキシダーゼ−ウサギ抗ペルオキシダーゼ複
合体の１０００倍希釈液１００μｌで連続的に、３７
℃、３０分間ずつ反応させ、処理細胞をＰＢＳで洗浄し
た。最後にペルオキシダーゼ反応を、0.０１％のＨ2 Ｏ
2 と0.３mg／mlの３，３′−ジアミノベンジジン四塩酸
のＰＢＳ溶液１００μｌを用い、各細胞を染色し、各穴
を水道水で洗浄し、乾燥させた。染色させた細胞はフォ
ーカスとして３穴のうち、いずれか１穴分に形成され、
可視化できた。可視化が困難な場合は通常の光学顕微鏡
で観察し、Ｈ１Ｎ１サブタイプ、Ｈ３Ｎ２サブタイプ及
びＢ型のいずれかであるかのタイピングを行った。該方
法に従って行われたタイピングの結果を、従来法の血清
学的に赤血球凝集抑制反応でタイピングされた結果と比
較した。その結果を下記表３に示す。Example 3 1. Typing of clinical isolates 80 isolates isolated from patients after 1968 (Osaka Prefectural Institute of Public Health) were typed.
That is, a 96-well microplate (Corning 268
60), 1 × 10 4 MDCK cells were dispensed into each well, and the next day, 25 μl of the virus solution of each isolate was dispensed into 3 wells of each well and kept at 35 ° C. for 45 minutes to infect the virus. It was After washing each well with PBS, 0.5% tragacanth gum (manufactured by Wako Pure Chemical Industries, Ltd.) and 100 μl of Eagle MEM containing 5 μg / ml trypsin were added. This 3
After incubating at 5 ° C for 16 hours, remove the added solution and P
Washed with BS. Next, the cells in each well were fixed with absolute ethanol for 10 minutes at room temperature. Next, using the kit prepared in Example 2, a 1000-fold dilution of AI3C solution (1 mg / ml), a 1000-fold dilution of C179 solution (1 mg / ml),
A 1000-fold diluted solution of anti-B-type serum (original serum concentration) was prepared, and a staining test was performed on each cell using these antibody diluted solutions. That is, of the 3 wells infected with each isolate, 100 μl each of AI3C dilution in one well, C179 dilution in the other well, and anti-B virus serum dilution in the remaining one well.
After reacting in each well, 100 μl of 1000-fold dilution of rabbit anti-mouse immunoglobulin G serum and 100-fold dilution of goat anti-rabbit immunoglobulin serum 100 times
37 μl, serially with 100 μl of a 1000-fold dilution of peroxidase-rabbit anti-peroxidase complex, 37
The cells were reacted at 30 ° C. for 30 minutes each, and the treated cells were washed with PBS. Finally, the peroxidase reaction was performed with 0.01% H 2 O.
2 and 0.3 mg / ml of 3,3'-diaminobenzidine tetrahydrochloride in 100 µl of PBS were used to stain each cell, each well was washed with tap water and dried. The stained cells are formed in one of the three holes as a focus,
I was able to visualize it. When it was difficult to visualize, it was observed with a normal optical microscope, and typing was carried out as to which of the H1N1 subtype, H3N2 subtype and B type. The results of typing performed according to the method were compared with the results of typing by the conventional serological hemagglutination inhibition reaction. The results are shown in Table 3 below.
【００４３】[0043]
【表３】 表 ３ ──────────────────────────────────── Ｈ１Ｎ１ Ｈ３Ｎ２ Ｈ１Ｎ１、Ｈ３Ｎ２混合 Ｂ型 ──────────────────────────────────── 従来法 ２４ ３５ − ２１ 本発明方法 ２２ ３５ ２ ２１ ────────────────────────────────────[Table 3] Table 3 ──────────────────────────────────── H1N1 H3N2 H1N1, H3N2 mixture B type ──────────────────────────────────── Conventional method 24 35-21 Inventive method 22 35 2 21 ─ ───────────────────────────────────
【００４４】表３に示す様に従来法ではＨ１Ｎ１サブタ
イプと判定されていた２分離株が、Ｈ１Ｎ１サブタイプ
とＨ３Ｎ２サブタイプのウイルスの混合物であることも
判明し、本発明により各分離株のタイピングを正確に行
うことができた。As shown in Table 3, it was also found that the two isolates that were determined to be H1N1 subtypes by the conventional method were a mixture of H1N1 subtype and H3N2 subtype viruses. I was able to type accurately.
【００４５】なおフェレット免疫血清としては、Ａ／Ku
mamoto／３７／７９（Ｈ１Ｎ１）、Ａ／Bangkok ／１０
／８３（Ｈ１Ｎ１）、Ａ／Yamagata／１２０／８６（Ｈ
１Ｎ１）でそれぞれフェレットを免疫して得たＨ１Ｎ１
サブタイプ判定用血清、Ａ／Ishikawa／７／３２（Ｈ３
Ｎ２）、Ａ／Phillippin／２／８２（Ｈ３Ｎ２）、Ａ／
Fukuoka ／Ｃ２９／８５（Ｈ３Ｎ２）、Ａ／Sichuan ／
２／８７（Ｈ３Ｎ２）でそれぞれフェレットを免疫して
得たＨ３Ｎ２サブタイプ判定用血清、Ｂ／Singapore ／
２２２／７９、Ｂ／Ibaraki ／２／８５、Ｂ／Yamagata
／１６／８８、Ｂ／Aichi ／５／８８でそれぞれフェレ
ットを免疫して得たＢ型ウイルス判定用血清（以上、大
阪府立公衆衛生研究所保有）を用いた。As the ferret immune serum, A / Ku was used.
mamoto / 37/79 (H1N1), A / Bangkok / 10
/ 83 (H1N1), A / Yamagata / 120/86 (H
H1N1 obtained by immunizing each ferret with 1N1)
Subtype determination serum, A / Ishikawa / 7/32 (H3
N2), A / Phillippin / 2/82 (H3N2), A /
Fukuoka / C29 / 85 (H3N2), A / Sichuan /
Sera for H3N2 subtype determination obtained by immunizing ferrets with 2/87 (H3N2), B / Singapore /
222/79, B / Ibaraki / 2/85, B / Yamagata
Serums for judging B virus obtained by immunizing ferrets with / 16/16/88 and B / Aichi / 5/88 (above, owned by Osaka Prefectural Institute of Public Health) were used.
【００４６】2.臨床検体中のウイルスのタイピング １９９３〜１９９４年発症の患者のうがい液１０mlを４
℃、３０００回転、３０分間の遠心分離を行い、上澄液
を調製した。次にこの上澄液をウイルス液検体とし、実
施例３−１に従い、ウイルス感染細胞の染色試験を行っ
た。すなわち、２４穴マイクロプレート（コーニング２
５８２０１）の各穴にＭＤＣＫ細胞を１×１０4 個ずつ
分注し、翌日、ウイルス液検体0.８mlを１検体につき３
穴分注し、３５℃、４５分間保温し、ウイルスを感染さ
せた。次に各穴をＰＢＳで洗浄後、５μｇ／mlのトリプ
シンを含有するイーグルＭＥＭ１mlを添加した。これを
３５℃で４０時間保温後、添加液を除去し、各穴をＰＢ
Ｓで洗浄し、以下、ＡＩ３Ｃ、Ｃ１７９、抗Ｂ型ウイル
ス血清を用い、各細胞の染色試験を行った。また、ウイ
ルス感染後、３５℃で４日間保温した各細胞について
も、同様に染色試験を行った。なお、従来法として、２
４穴マイクロプレートの各穴にＭＤＣＫ細胞を１×１０
4 個ずつ分注し、翌日、ウイルス液検体0.８mlを１検体
につき４穴分注し、３５℃、４５分間保温し、ウイルス
を感染させた。2. Typing of virus in clinical specimens 4 ml of 10 ml of mouthwash from a patient who developed 1993-1994
Centrifugation was performed at 3,000 rpm for 30 minutes to prepare a supernatant. Next, this supernatant was used as a virus liquid sample, and a staining test of virus-infected cells was performed according to Example 3-1. That is, 24-well microplate (Corning 2
MDCK cells were dispensed into each well of 58201) at a dose of 1 × 10 4 , and the next day, 0.8 ml of the virus solution sample was used for 3 samples per sample.
The cells were dispensed into holes and kept at 35 ° C. for 45 minutes to infect the virus. After washing each well with PBS, 1 ml of Eagle MEM containing 5 μg / ml trypsin was added. After incubating this at 35 ° C for 40 hours, remove the added solution and PB each hole.
After washing with S, a staining test was performed on each cell using AI3C, C179 and anti-B virus serum. In addition, a staining test was similarly performed on each cell which was kept at 35 ° C. for 4 days after virus infection. As a conventional method, 2
1 x 10 MDCK cells in each well of a 4-well microplate
Four samples were dispensed, and the next day, 0.8 ml of the virus solution sample was dispensed in four wells per sample and kept warm at 35 ° C for 45 minutes to infect the virus.
【００４７】各穴をＰＢＳで洗浄後、５μｇ／mlのトリ
プシンを含有するイーグルＭＥＭ１mlを添加し、３５
℃、４日間培養した。培養４日目に細胞変性を観察し、
変性のみられた検体につき当該穴の細胞培養液の血球凝
集活性を測定した。また変性のみられなかった検体につ
いては再度、ウイルス液によるＭＤＣＫ細胞感染を行
い、４日後に再度細胞変性の有無を確認し、細胞変性の
みられた検体は当該穴の細胞培養液の血球凝集活性を測
定した。すなわち、0.５％ヒヨコ赤血球５０μｌに細胞
培養液５０μｌを混合し、３０分後の凝集の有無より、
ヒトインフルエンザウイルスの有無を判定した。ヒトイ
ンフルエンザウイルスの存在が確認された細胞培養液に
ついては、Ｈ１Ｎ１サブタイプ、Ｈ３Ｎ２サブタイプ、
Ｂ型ウイルス判定用のフェレット免疫血清、すなわち、
Ａ／Yamagata／３２／８９（Ｈ１Ｎ１）でフェレットを
免疫して得たＨ１Ｎ１サブタイプ判定用血清、Ａ／Osak
a ／１０８９／９３（Ｈ３Ｎ２）、及びＡ／Kitakyushu
／１５９／９３（Ｈ３Ｎ２）でそれぞれフェレットを免
疫して得たＨ３Ｎ２サブタイプ判定用血清、Ｂ／Bangko
k ／６３／９０及びＢ／Mie ／１／９３でそれぞれフェ
レットを免疫し得たＢ型ウイルス判定用血清（以上、大
阪府立公衆衛生研究所保有）を用い、該各血清の赤血球
凝集抑制反応でタイピングを行った。すなわち各血清２
５μｌごとに、細胞培養液２５μｌを添加し、３７℃、
１時間保温した後、0.５％ヒヨコ赤血球５０μｌを混合
し、３０分後の凝集の有無より、ヒトインフルエンザウ
イルスのタイピングを行った。本発明の方法と、従来法
の結果を表４に示す。After washing each well with PBS, 1 ml of Eagle MEM containing 5 μg / ml of trypsin was added, and 35
Culturing was performed at 4 ° C for 4 days. On the 4th day of culture, observe the cell degeneration,
The hemagglutination activity of the cell culture solution in the hole was measured for the denatured specimen. In addition, for samples that were not denatured, MDCK cells were again infected with the virus solution, and after 4 days, the presence or absence of cell degeneration was confirmed again, and for the samples that were only denatured, the hemagglutination activity of the cell culture solution in the hole was confirmed. It was measured. That is, 50 μl of 0.5% chick red blood cells was mixed with 50 μl of cell culture solution, and after 30 minutes, the presence or absence of aggregation
The presence or absence of human influenza virus was determined. Regarding the cell culture medium in which the presence of human influenza virus was confirmed, H1N1 subtype, H3N2 subtype,
Ferret immune serum for determining B virus, that is,
A / Osak for H1N1 subtype determination obtained by immunizing ferrets with A / Yamagata / 32/89 (H1N1)
a / 1089/93 (H3N2), and A / Kitakyushu
B / Bangko for H3N2 subtype determination obtained by immunizing ferrets with / 159/93 (H3N2)
Using the B-type virus determination sera that were immunized with ferrets at k / 63/90 and B / Mie / 1/93 respectively (above, owned by Osaka Prefectural Public Health Research Institute), I typed. That is, each serum 2
Add 25 μl of cell culture medium to every 5 μl,
After incubating for 1 hour, 50 μl of 0.5% chick red blood cells were mixed, and after 30 minutes, human influenza virus was typed based on the presence or absence of aggregation. Table 4 shows the results of the method of the present invention and the conventional method.
【００４８】[0048]
【表４】 表 ４ ──────────────────────────────────── Ｈ１Ｎ１ Ｈ３Ｎ２ Ｂ型 インフルエンザ ウイルス非検出 ──────────────────────────────────── 従来法 ０ ３３ ０ ３９ 本発明方法 保温４０時間 ０ ２５ ０ ４７ 保温４日間 ０ ３３ ０ ３９ ────────────────────────────────────[Table 4] Table 4 ──────────────────────────────────── H1N1 H3N2 Influenza B virus non-detection ──────────────────────────────────── Conventional method 0 33 0 39 Inventive method Insulation 40 hours 0 25 0 47 Insulation for 4 days 0 33 0 39 ─────────────────────────────────────
【００４９】本発明方法ではうがい液より調製したウイ
ルス液検体を細胞に感染後、２日後には、２５／３３
（７６％）をタイピングすることができ、更に保温を続
けた場合、従来法のタイピングと結果が完全に一致し、
１９９３〜１９９４年に流行のウイルスはＨ３Ｎ２サブ
タイプと決定した。In the method of the present invention, 25/33 was obtained 2 days after the cells were infected with the virus liquid sample prepared from the mouthwash.
(76%) can be typed, and when the heat retention is continued, the result is completely the same as the conventional method,
The epidemic virus from 1993 to 1994 was determined to be the H3N2 subtype.
【００５０】従来法において使用するフェレット血清
は、流行中のヒトインフルエンザウイルス株を分離し、
フェレットを免疫し、調製して得ているため、ヒトイン
フルエンザウイルスの抗原性の変化により、その結合活
性消失が生じること、よって常に、最新、複数の抗血清
を準備する必要がある。また、最近、フェレットが入手
困難であり、自家繁殖の結果、高力価血清の調製が困難
となっている。The ferret serum used in the conventional method isolates a human influenza virus strain in epidemics,
Since the ferret has been obtained by immunization and preparation, the binding activity is lost due to the change in the antigenicity of the human influenza virus. Therefore, it is always necessary to prepare a plurality of antisera at the latest. Further, recently, it has been difficult to obtain ferrets, and as a result of self-propagation, preparation of high titer serum has become difficult.
【００５１】一方、本発明方法は、検体調製後、ウイル
スの増殖力が強い場合は２日後に、タイピングが可能な
こと、Ｈ１Ｎ１サブタイプ検出用抗体、Ｈ３Ｎ２サブタ
イプ検出用抗体が、それぞれのサブタイプの抗原変化の
影響を受けないこと、高力価の抗体がモノクローナル抗
体として常に安定して供給されること等で特に優れてい
る。On the other hand, in the method of the present invention, after preparation of the sample, two days after the preparation of the sample, if the virus has a strong proliferative activity, typing is possible, and the H1N1 subtype detection antibody and the H3N2 subtype detection antibody are It is particularly excellent in that it is not affected by changes in the type of antigen and that a high titer antibody is always stably supplied as a monoclonal antibody.
【００５２】[0052]
【発明の効果】本発明の抗ヒトインフルエンザウイルス
抗体、その検出方法及び本発明のキットを用いることに
より、インフルエンザウイルス分離株だけでなく、患者
のうがい液からも迅速に高感度でかつ正確にインフルエ
ンザウイルスを検出でき、しかも、同時にタイピングを
行うことができる。その結果、短時間に大量の検体のタ
イピングが可能となり、その年におけるインフルエンザ
の流行予測が迅速に行えることになる。INDUSTRIAL APPLICABILITY By using the anti-human influenza virus antibody of the present invention, the method for detecting the same, and the kit of the present invention, influenza can be rapidly, highly sensitively and accurately detected not only from the influenza virus isolate but also from the mouthwash of the patient. Viruses can be detected and typing can be performed at the same time. As a result, a large number of specimens can be typed in a short time, and the influenza epidemic in that year can be predicted quickly.
【００５３】[0053]
【００５４】配列番号：1 配列の長さ：5 配列の型：アミノ酸 鎖の数：一本鎖 トポロジー：直鎖状 配列の種類：ペプチド フラグメント型：中間部フラグメント SEQ ID NO: 1 Sequence length: 5 Sequence type: Amino acid Number of chains: Single chain Topology: Linear Sequence type: Peptide Fragment type: Intermediate fragment
【００５５】配列番号：2 配列の長さ：12 配列の型：アミノ酸 鎖の数：一本鎖 トポロジー：直鎖状 配列の種類：ペプチド フラグメント型：中間部フラグメント 配列の特徴：９番目のXaa はVal 又はLeu である。 SEQ ID NO: 2 Sequence length: 12 Sequence type: Amino acid Number of chains: Single chain Topology: Linear Sequence type: Peptide Fragment type: Intermediate fragment Sequence characteristics: Xaa at position 9 Val or Leu.
【００５６】配列番号：3 配列の長さ：5 配列の型：アミノ酸 鎖の数：一本鎖 トポロジー：直鎖状 配列の種類：ペプチド フラグメント型：中間部フラグメント SEQ ID NO: 3 Sequence length: 5 Sequence type: Amino acid Number of chains: Single chain Topology: Linear Sequence type: Peptide Fragment type: Intermediate fragment
【００５７】配列番号：4 配列の長さ：12 配列の型：アミノ酸 鎖の数：一本鎖 トポロジー：直鎖状 配列の種類：ペプチド フラグメント型：中間部フラグメント SEQ ID NO: 4 Sequence length: 12 Sequence type: Amino acid Number of chains: Single chain Topology: Linear Sequence type: Peptide Fragment type: Intermediate fragment
【００５８】配列番号：5 配列の長さ：12 配列の型：アミノ酸 鎖の数：一本鎖 トポロジー：直鎖状 配列の種類：ペプチド フラグメント型：中間部フラグメント SEQ ID NO: 5 Sequence length: 12 Sequence type: Amino acid Number of chains: Single chain Topology: Linear Sequence type: Peptide Fragment type: Intermediate fragment
【００５９】配列番号：6 配列の長さ：12 配列の型：アミノ酸 鎖の数：一本鎖 トポロジー：直鎖状 配列の種類：ペプチド フラグメント型：中間部フラグメント SEQ ID NO: 6 Sequence length: 12 Sequence type: Amino acid Number of chains: Single chain Topology: Linear Sequence type: Peptide Fragment type: Intermediate fragment
【００６０】配列番号：7 配列の長さ：5 配列の型：アミノ酸 鎖の数：一本鎖 トポロジー：直鎖状 配列の種類：ペプチド フラグメント型：中間部フラグメント SEQ ID NO: 7 Sequence length: 5 Sequence type: Amino acid Number of chains: Single chain Topology: Linear Sequence type: Peptide Fragment type: Intermediate fragment
【００６１】配列番号：8 配列の長さ：5 配列の型：アミノ酸 鎖の数：一本鎖 トポロジー：直鎖状 配列の種類：ペプチド フラグメント型：中間部フラグメント SEQ ID NO: 8 Sequence length: 5 Sequence type: Amino acid Number of chains: Single chain Topology: Linear Sequence type: Peptide Fragment type: Intermediate fragment
【００６２】配列番号：9 配列の長さ：5 配列の型：アミノ酸 鎖の数：一本鎖 トポロジー：直鎖状 配列の種類：ペプチド フラグメント型：中間部フラグメント SEQ ID NO: 9 Sequence length: 5 Sequence type: Amino acid Number of chains: Single chain Topology: Linear Sequence type: Peptide Fragment type: Intermediate fragment
【００６３】配列番号：10 配列の長さ：12 配列の型：アミノ酸 鎖の数：一本鎖 トポロジー：直鎖状 配列の種類：ペプチド フラグメント型：中間部フラグメント SEQ ID NO: 10 Sequence length: 12 Sequence type: Amino acid Number of chains: Single chain Topology: Linear Sequence type: Peptide Fragment type: Intermediate fragment
【００６４】配列番号：11 配列の長さ：12 配列の型：アミノ酸 鎖の数：一本鎖 トポロジー：直鎖状 配列の種類：ペプチド フラグメント型：中間部フラグメント SEQ ID NO: 11 Sequence length: 12 Sequence type: Amino acid Number of chains: Single chain Topology: Linear Sequence type: Peptide Fragment type: Intermediate fragment
【００６５】配列番号：12 配列の長さ：12 配列の型：アミノ酸 鎖の数：一本鎖 トポロジー：直鎖状 配列の種類：ペプチド フラグメント型：中間部フラグメント SEQ ID NO: 12 Sequence length: 12 Sequence type: Amino acid Number of chains: Single chain Topology: Linear Sequence type: Peptide Fragment type: Intermediate fragment
【００６６】配列番号：13 配列の長さ：12 配列の型：アミノ酸 鎖の数：一本鎖 トポロジー：直鎖状 配列の種類：ペプチド フラグメント型：中間部フラグメント SEQ ID NO: 13 Sequence length: 12 Sequence type: Amino acid Number of chains: Single chain Topology: Linear Sequence type: Peptide Fragment type: Intermediate fragment
【図１】ヘマグルチニン分子の三次構造の模式図であ
る。FIG. 1 is a schematic diagram of the tertiary structure of a hemagglutinin molecule.
───────────────────────────────────────────────────── フロントページの続き (51)Int.Cl.6 識別記号 庁内整理番号 ＦＩ 技術表示箇所 Ｃ１２Ｑ 1/70 9453−4Ｂ Ｇ０１Ｎ 33/53 Ｄ 33/569 Ｌ //(Ｃ１２Ｐ 21/08 Ｃ１２Ｒ 1:91） (72)発明者 吉岡 広文 滋賀県大津市瀬田３丁目４番１号 寳酒造 株式会社中央研究所内 (72)発明者 大島 淳 滋賀県大津市瀬田３丁目４番１号 寳酒造 株式会社中央研究所内 (72)発明者 加藤 郁之進 滋賀県大津市瀬田３丁目４番１号 寳酒造 株式会社中央研究所内─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. 6 Identification code Internal reference number FI Technical indication C12Q 1/70 9453-4B G01N 33/53 D 33/569 L // (C12P 21/08 C12R 1 : 91) (72) Hirofumi Yoshioka 3-4-1 Seta, Otsu City, Shiga Prefecture, Central Research Laboratory, Minami Shuzo Co., Ltd. (72) Inventor Jun Oshima, Otsu City, Shiga Prefecture 3-4-1 Seta Corporation Central Research Institute (72) Inventor Ikuno Susumu 3-4-1 Seta, Otsu City, Shiga Prefecture
Claims (3)
トインフルエンザウイルス抗体。 （ａ）ヒトインフルエンザＡ型ウイルスのＨ３Ｎ２サブ
タイプのヘマグルチニン分子中の幹領域の配列表の配列
番号１で表されるＴＧＭＲＮポリペプチド配列と配列表
の配列番号２で表されるＱＩＮＧＫＬＮＲ（Ｌ／Ｖ）Ｉ
ＥＫポリペプチド配列とを認識する。 （ｂ）ヒトインフルエンザＡ型ウイルスのＨ１Ｎ１サブ
タイプとＨ２Ｎ２サブタイプのヘマグルチニン分子中の
幹領域の配列表の配列番号３で表されるＴＧＬＲＮポリ
ペプチド配列と、配列表の配列番号４で表されるＧＩＴ
ＮＫＶＮＳＶＩＥＫポリペプチド配列とを認識しない。1. An anti-human influenza virus antibody having the following characteristics (a) and (b): (A) The TGMRN polypeptide sequence represented by SEQ ID NO: 1 in the sequence listing of the stem region in the hemagglutinin molecule of the H3N2 subtype of human influenza A virus and the QINGKLNR (L / V represented by SEQ ID NO: 2 in the sequence listing). ) I
Recognizes EK polypeptide sequences. (B) The TGLRN polypeptide sequence represented by SEQ ID NO: 3 in the sequence listing of the stem region in the hemagglutinin molecule of the H1N1 subtype and H2N2 subtype of human influenza A virus and represented by SEQ ID NO: 4 in the sequence listing GIT
It does not recognize the NKVNSVIEK polypeptide sequence.
イルス抗体をＨ３Ｎ２サブタイプのヒトインフルエンザ
Ａ型ウイルスに結合させる工程を包含することを特徴と
するヒトインフルエンザウイルスの検出方法。2. A method for detecting a human influenza virus, which comprises a step of binding the anti-human influenza virus antibody according to claim 1 to a human influenza A virus of H3N2 subtype.
ための検出キットであって、請求項１記載の抗体を含有
していることを特徴とするヒトインフルエンザウイルス
検出キット。3. A detection kit for detection using the method according to claim 2, wherein the detection kit contains the antibody according to claim 1.
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|---|---|---|---|
| JP11746694A JP3584990B2 (en) | 1994-05-09 | 1994-05-09 | Anti-human influenza virus antibody |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP11746694A JP3584990B2 (en) | 1994-05-09 | 1994-05-09 | Anti-human influenza virus antibody |
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| JPH07304799A true JPH07304799A (en) | 1995-11-21 |
| JP3584990B2 JP3584990B2 (en) | 2004-11-04 |
Family
ID=14712388
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|---|---|---|---|
| JP11746694A Expired - Fee Related JP3584990B2 (en) | 1994-05-09 | 1994-05-09 | Anti-human influenza virus antibody |
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