JPH04221398A - Antibody to non-a, non-b hepatitis virus and immunochemically reactive peptide - Google Patents
Antibody to non-a, non-b hepatitis virus and immunochemically reactive peptideInfo
- Publication number
- JPH04221398A JPH04221398A JP9153991A JP9153991A JPH04221398A JP H04221398 A JPH04221398 A JP H04221398A JP 9153991 A JP9153991 A JP 9153991A JP 9153991 A JP9153991 A JP 9153991A JP H04221398 A JPH04221398 A JP H04221398A
- Authority
- JP
- Japan
- Prior art keywords
- nanbh
- peptide
- antibody
- arg
- test solution
- 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.)
- Pending
Links
- 108090000765 processed proteins & peptides Proteins 0.000 title claims abstract description 76
- 208000006454 hepatitis Diseases 0.000 title abstract description 10
- 231100000283 hepatitis Toxicity 0.000 title abstract description 10
- 241000700605 Viruses Species 0.000 title abstract description 8
- 208000005176 Hepatitis C Diseases 0.000 claims abstract description 39
- 208000010710 hepatitis C virus infection Diseases 0.000 claims abstract description 39
- 102000004196 processed proteins & peptides Human genes 0.000 claims abstract description 24
- 238000012360 testing method Methods 0.000 claims abstract description 15
- 230000000984 immunochemical effect Effects 0.000 claims abstract description 14
- 239000003153 chemical reaction reagent Substances 0.000 claims abstract description 13
- 229920001184 polypeptide Polymers 0.000 claims abstract description 4
- 125000003275 alpha amino acid group Chemical group 0.000 claims abstract 2
- 239000007788 liquid Substances 0.000 claims abstract 2
- 238000000034 method Methods 0.000 claims description 22
- 239000012085 test solution Substances 0.000 claims description 19
- 239000012634 fragment Substances 0.000 claims description 7
- 238000003018 immunoassay Methods 0.000 claims description 2
- 230000036039 immunity Effects 0.000 claims 1
- 238000001514 detection method Methods 0.000 abstract description 8
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- QOSSAOTZNIDXMA-UHFFFAOYSA-N Dicylcohexylcarbodiimide Chemical compound C1CCCCC1N=C=NC1CCCCC1 QOSSAOTZNIDXMA-UHFFFAOYSA-N 0.000 description 1
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Abstract
Description
【０００１】0001
【産業上の利用分野】本発明は非Ａ非Ｂ型肝炎ウイルス
（ＮＡＮＢＨウイルス）に対する抗体と免疫化学反応す
るペプチドに関する。FIELD OF THE INVENTION The present invention relates to a peptide that immunochemically reacts with antibodies against non-A, non-B hepatitis virus (NANBH virus).
【０００２】本発明は更に、試験液中のＮＡＮＢＨ又は
抗ＮＡＮＢＨの検出方法、並びにこの検出方法を適用す
るときに使用すべき免疫化学試薬及び試験用キットに関
する。The present invention further relates to a method for detecting NANBH or anti-NANBH in a test solution, and immunochemical reagents and test kits to be used when applying this detection method.
【０００３】0003
【従来の技術】Ｃ型肝炎ウイルス（ＨＣＶ）により引き
起こされ得る又は引き起こされ得ない非Ａ非Ｂ型肝炎は
伝達可能な疾病又はウイルス誘発性疾病群である。この
非Ａ非Ｂ型肝炎は公知の肝炎ウイルス、即ちＡ型肝炎ウ
イルス（ＨＡＶ）、Ｂ型肝炎ウイルス（ＨＢＶ）及びデ
ルタ肝炎ウイルス（ＨＤＶ）により引き起こされる疾患
並びにサイトメガロウイルス（ＣＭＶ）又はエプスタイ
ン−バールウイルス（ＥＢＶ）により誘発される肝炎を
含む他の形態のウイルス性肝臓疾患とは異なり得る。Ｎ
ＡＮＢＨは輸血を受けた個人で最初に同定された。ヒト
からチンパンジーに接種し、チンパンジーで連続継代し
たところ、ＮＡＮＢＨが単数もしくは複数の伝達可能な
感染要因に起因していることが判明した。BACKGROUND OF THE INVENTION Non-A, non-B hepatitis, which may or may not be caused by the hepatitis C virus (HCV), is a group of transmissible or virus-induced diseases. This non-A, non-B hepatitis is a disease caused by known hepatitis viruses, namely hepatitis A virus (HAV), hepatitis B virus (HBV) and hepatitis delta virus (HDV), as well as cytomegalovirus (CMV) or Epstein-mediated hepatitis. It may be different from other forms of viral liver disease, including hepatitis induced by Barr virus (EBV). N
ANBH was first identified in individuals who received blood transfusions. When chimpanzees were inoculated from humans and serially passaged in chimpanzees, NANBH was found to be caused by one or more transmissible infectious agents.
【０００４】ＮＡＮＢＨには３つの型、即ち水系感染流
行型、血液又は針を介して感染する型及び散発性（集団
後天性（ｃｏｍｍｕｎｉｔｙ ａｃｑｕｉｒｅｄ））型
があり得るという疫学的証拠は示唆的である。しかしな
がら、ＮＡＮＢＨの原因となり得る要因の多くは知られ
ていない。Epidemiological evidence is suggestive that NANBH may be of three types: waterborne epidemic, blood- or needle-borne, and sporadic (community acquired). . However, many of the factors that can cause NANBH are unknown.
【０００５】[0005]
【発明が解決しようとする課題】ＮＡＮＢＨの臨床診断
及び同定はまず、他のウイルスマーカーを排除して行わ
れた。推定上のＮＡＮＢＨ抗原及び抗体の検出に使用さ
れる方法には、寒天ゲル拡散法、カウンター免疫電気泳
動法、免疫蛍光顕微鏡検査法、免疫電子顕微鏡検査法、
ラジオイムノアッセイ及び酵素結合性免疫吸着剤検定法
がある。しかしながら、これらの検定のいずれも、ＮＡ
ＮＢＨ用診断試験として使用するには感受性、特異性及
び再現性が不十分であることが判明した。[Problems to be Solved by the Invention] Clinical diagnosis and identification of NANBH was first performed by excluding other viral markers. Methods used to detect putative NANBH antigens and antibodies include agar gel diffusion, counterimmunoelectrophoresis, immunofluorescence microscopy, immunoelectron microscopy,
There are radioimmunoassays and enzyme-linked immunosorbent assays. However, none of these tests
It was found that the sensitivity, specificity and reproducibility were insufficient for use as a diagnostic test for NBH.
【０００６】しかしながらＮＡＮＢＨ感染の種々の段階
での信頼性のある診断の実施を可能とする特定の感受性
のある方法の開発のためには、この型の免疫優勢ウイル
スエピトープを同定することが非常に重要である。However, for the development of specific sensitive methods that would allow reliable diagnosis to be carried out at various stages of NANBH infection, it is very important to identify immunodominant viral epitopes of this type. is important.
【０００７】[0007]
【課題を解決するための手段】ＮＡＮＢＨ抗体と特に免
疫化学反応する１５個のアミノ酸からなるペプチドを発
見した。アミノ酸配列は配列表に示す。[Means for Solving the Problems] We have discovered a peptide consisting of 15 amino acids that is particularly immunochemically reactive with NANBH antibodies. The amino acid sequence is shown in the sequence listing.
【０００８】本発明は更に、ＮＡＮＢＨ抗体と免疫化学
反応する前記ペンタデカペプチドのフラグメント、及び
ペンタデカペプチドを主成分として含むポリペプチド又
はＮＡＮＢＨ抗体と免疫化学反応するそのフラグメント
に関する。The present invention further relates to a fragment of the pentadecapeptide that immunochemically reacts with a NANBH antibody, and a polypeptide containing the pentadecapeptide as a main component or a fragment thereof that immunochemically reacts with a NANBH antibody.
【０００９】アミノ酸配列がＡｒｇ−Ｌｙｓ−Ｔｈｒ−
Ｌｙｓ−Ａｒｇ−Ｓｅｒ−Ｔｈｒ−Ａｓｎ−Ａｒｇのノ
ナペプチドは、本発明のペンタデカペプチドのフラグメ
ントとして好ましい。[0009] The amino acid sequence is Arg-Lys-Thr-
The nonapeptide of Lys-Arg-Ser-Thr-Asn-Arg is preferred as the pentadecapeptide fragment of the invention.
【００１０】本発明は更に、本発明のペプチドの少なく
とも１種を含んでいる免疫化学試薬に関する。The invention further relates to immunochemical reagents containing at least one of the peptides of the invention.
【００１１】本発明は更に、本発明のペプチドの少なく
とも１種を使用して行う、試験液中のＮＡＮＢＨに対す
る抗体の検出方法に関する。The present invention further relates to a method for detecting antibodies against NANBH in a test solution using at least one of the peptides of the present invention.
【００１２】本発明は更に、本発明のペプチドの少なく
とも１種を使用して行う、試験液中のＮＡＮＢＨの検出
方法に関する。The present invention further relates to a method for detecting NANBH in a test solution using at least one of the peptides of the present invention.
【００１３】最後に本発明は、イムノアッセイを実施す
るために使用すべき試験用キットに関する。この試験用
キットは本発明の少なくとも１種の免疫化学試薬を含ん
でいる。Finally, the invention relates to a test kit to be used for carrying out immunoassays. This test kit contains at least one immunochemical reagent of the invention.
【００１４】前述したペプチドは、試験液中のＮＡＮＢ
Ｈ又はＮＡＮＢＨ抗体の存在の確定のための診断方法で
使用するのに特に適している。[0014] The above-mentioned peptide is the NANB in the test solution.
It is particularly suitable for use in diagnostic methods for determining the presence of H or NANBH antibodies.
【００１５】天然のＮＡＮＢＨとは対照的に、本発明の
ペプチドは、安全な非感染源であるという大きな利点を
有する。In contrast to natural NANBH, the peptides of the invention have the great advantage of being a safe, non-infectious source.
【００１６】本発明のペプチドの製造は、公知のペプチ
ド合成用有機化学法の１つによって行うか又は組換え体
ＤＮＡ技術を利用して行う。この組換え体ＤＮＡ技術は
、宿主として適切な微生物中で１種以上の問題のペプチ
ドをコードしているポリヌクレオチド配列で組換え体ポ
リヌクレオチドを発現させることにより所望のペプチド
を製造することからなる。The peptides of the invention are produced by one of the known organic chemical methods for peptide synthesis or by using recombinant DNA technology. This recombinant DNA technique consists of producing the desired peptide by expressing a recombinant polynucleotide with a polynucleotide sequence encoding one or more peptides in question in a suitable microorganism as host. .
【００１７】ペプチド合成有機化学法は、均質相又はい
わゆる固相における縮合反応を介しての必要なアミノ酸
の結合を含むことが考えられる。[0017] Organic chemical methods for peptide synthesis are believed to involve the attachment of the necessary amino acids via condensation reactions in a homogeneous phase or in the so-called solid phase.
【００１８】縮合反応は以下の如く実施し得る。The condensation reaction can be carried out as follows.
【００１９】ａ） 縮合剤の存在下で、遊離カルボキ
シル基と保護された他の反応基とを含む化合物（アミノ
酸、ペプチド）を、遊離アミノ基と保護された他の反応
基とを含む化合物（アミノ酸、ペプチド）と縮合する。a) In the presence of a condensing agent, a compound (amino acid, peptide) containing a free carboxyl group and a protected other reactive group is converted into a compound (amino acid, peptide) containing a free amino group and a protected other reactive group ( Condenses with amino acids, peptides).
【００２０】ｂ） 活性化カルボキシル基と遊離又は
保護された他の反応基とを含む化合物（アミノ酸、ペプ
チド）を、遊離アミノ基と遊離又は保護された他の反応
基とを含む化合物（アミノ酸、ペプチド）と縮合する。b) Compounds containing an activated carboxyl group and other free or protected reactive groups (amino acids, peptides) are converted into compounds containing free amino groups and other free or protected reactive groups (amino acids, peptides). peptide).
【００２１】中でも、カルボキシル基を酸ハロゲン化物
、酸アジド、酸無水物、酸イミダゾリド又は活性化エス
テル（例えばＮ−ヒドロキシスクシンイミド、Ｎ−ヒド
ロキシベンゾトリアゾール若しくはｐ−ニトロフェニル
エステル）に転換することによりカルボキシル基を活性
化し得る。Among others, the carboxyl group can be modified by converting the carboxyl group into an acid halide, an acid azide, an acid anhydride, an acid imidazolide or an activated ester (for example, N-hydroxysuccinimide, N-hydroxybenzotriazole or p-nitrophenyl ester). groups can be activated.
【００２２】前記縮合反応の最も一般的な方法は、カル
ボジイミド法、アジド法、混合無水物法及びＴｈｅ Ｐ
ｅｐｔｉｄｅｓ， Ａｎａｌｙｓｉｓ， Ｓｙｎｔｈｅ
ｓｉｓ， Ｂｉｏｌｏｇｙ，Ｖｏｌ． １−３（Ｅｄ．
Ｇｒｏｓｓ，Ｅ． ａｎｄ Ｍｅｉｅｎｈｏｆｅｒ，
Ｊ．）１９７９， １９８０， １９８１（Ａｃａｄ
ｅｍｉｃ Ｐｒｅｓｓ， Ｉｎｃ．）に記載の如き活性
化エステルを使用する方法である。The most common methods of the condensation reaction are the carbodiimide method, the azide method, the mixed anhydride method and the P
eptides, Analysis, Synthe
sis, Biology, Vol. 1-3 (Ed.
Gross, E. and Meienhofer,
J. ) 1979, 1980, 1981 (Acad
emic Press, Inc. This method uses an activated ester as described in ).
【００２３】上述した本発明のペプチドの“固相”を使
用しての製造は例えば、Ｊ． Ａｍｅｒ． Ｃｈｅｍ．
Ｓｏｃ． ８５， ２１４９ （１９６３）及びＩｎ
ｔ． Ｊ． Ｐｅｐｔｉｄｅ Ｐｒｏｔｅｉｎ Ｒｅｓ
． ３５， １６１−２１４（１９９０）に記載されて
いる。製造すべきペプチドのアミノ酸の結合は通常、カ
ルボキシル末端側から出発する。この方法では、その上
に反応基があるか又はこのような基を導入することので
きる固相が必要である。これは例えば反応性クロロメチ
ル基を含むベンゼンとジビニルベンゼンとのコポリマー
、又はヒドロキシメチル官能基若しくはアミン官能基と
反応させられるポリマー性固相であり得る。The production of the above-described peptide of the present invention using a "solid phase" is described, for example, in J. Amer. Chem.
Soc. 85, 2149 (1963) and In
t. J. Peptide Protein Res
．． 35, 161-214 (1990). The attachment of amino acids of the peptide to be produced usually starts from the carboxyl terminal side. This method requires a solid phase on which there are reactive groups or into which such groups can be introduced. This can be, for example, a copolymer of benzene and divinylbenzene containing reactive chloromethyl groups, or a polymeric solid phase which is reacted with hydroxymethyl or amine functions.
【００２４】特に適した固相は例えば、Ｗａｎｇにより
Ｊ． Ａｍ． Ｃｈｅｍ． Ｓｏｃ． ９５，１３２８
（１９７４）に記載されているｐ−アルコキシベンジル
アルコール樹脂（４−ヒドロキシメチルフェノキシメチ
ルコポリスチレン−１％ ジビニルベンゼン樹脂）で
ある。合成後に、穏和な条件下においてペプチドをこの
固相から分離することができる。Particularly suitable solid phases are described, for example, by Wang, J.; Am. Chem. Soc. 95,1328
(1974) is a p-alkoxybenzyl alcohol resin (4-hydroxymethylphenoxymethylcopolystyrene-1% divinylbenzene resin). After synthesis, the peptide can be separated from this solid phase under mild conditions.
【００２５】所望のアミノ酸配列の合成後に、ペプチド
を例えば、トリフルオロメタンスルホン酸で又はトリフ
ルオロ酢酸に溶解したメタンスルホン酸で樹脂から分離
する。低級アルコール、好ましくはメタノール又はエタ
ノールによるエステル交換反応によりペプチドを担体か
ら除去することもできる。この場合、ペプチドの低級ア
ルキルエステルは直接生成される。同様にアンモニアを
使用して分離すると、本発明のペプチドのアミドが生成
される。After synthesis of the desired amino acid sequence, the peptide is separated from the resin, for example with trifluoromethanesulfonic acid or with methanesulfonic acid dissolved in trifluoroacetic acid. The peptide can also be removed from the carrier by transesterification with lower alcohols, preferably methanol or ethanol. In this case, the lower alkyl ester of the peptide is produced directly. Similarly, separation using ammonia produces amides of the peptides of the invention.
【００２６】縮合反応に関与し得ない反応基は前述した
如く、酸、塩基又は還元による加水分解により再度非常
に簡単に除去され得る基により実質上保護する。従って
、カルボキシル基は例えばメタノール、エタノール、第
三ブタノール、ベンジルアルコール又はｐ−ニトロベン
ジルアルコールによるエステル化及び固形支持体に結合
したアミンにより実質上保護され得る。Reactive groups which cannot take part in the condensation reaction are, as mentioned above, substantially protected by groups which can be very easily removed again by acid, base or reductive hydrolysis. Thus, the carboxyl group may be substantially protected by esterification with, for example, methanol, ethanol, tert-butanol, benzyl alcohol or p-nitrobenzyl alcohol and an amine attached to a solid support.
【００２７】アミノ基を実質上保護し得る基はエトキシ
カルボニル基、ベンジルオキシカルボニル基、ｔ−ブト
キシカルボニル基、ｐ−メトキシベンジルオキシカルボ
ニル基又はスルホン酸から得られる酸基（例えばベンゼ
ンスルホニル基若しくはｐ−トルエンスルホニル基）で
あるが、他の基、例えば置換若しくは未置換アリール若
しくはアラルキル基（例えばベンジル及びトリフェニル
メチル）、又はオルトニトロフェニルスルフェニル及び
２−ベンゾイル−１−メチルビニルのような基を使用す
ることもできる。特に適したα−アミノ保護基は例えば
、塩基感受性９−フルオレニルメトキシカルボニル（Ｆ
ｍｏｃ）基［Ｃａｒｐｉｎｏ ＆ Ｈａｎ （１９７０
）Ｊ．Ａｍｅｒ． Ｃｈｅｍ． Ｓｏｃ．９２， ５７
４８］である。Groups capable of substantially protecting an amino group include an ethoxycarbonyl group, a benzyloxycarbonyl group, a t-butoxycarbonyl group, a p-methoxybenzyloxycarbonyl group, or an acid group obtained from a sulfonic acid (for example, a benzenesulfonyl group or a p-methoxycarbonyl group). -toluenesulfonyl) but also other groups, such as substituted or unsubstituted aryl or aralkyl groups (e.g. benzyl and triphenylmethyl), or orthonitrophenylsulfenyl and 2-benzoyl-1-methylvinyl. You can also use Particularly suitable α-amino protecting groups are, for example, base-sensitive 9-fluorenylmethoxycarbonyl (F
moc) group [Carpino & Han (1970
)J. Amer. Chem. Soc. 92, 57
48].
【００２８】Ｔｈｅ Ｐｅｐｔｉｄｅｓ， Ａｎａｌｙ
ｓｉｓ， Ｓｙｎｔｈｅｓｉｓ， Ｂｉｏｌｏｇｙ，
Ｖｏｌ． １−９（Ｅｄｓ． ＧｒｏｓｓＵｄｅｎｆｒ
ｉｅｎｄ ａｎｄ Ｍｅｉｅｎｈｏｆｅｒ）１９７９−
１９８７（Ａｃａｄｅｍｉｃ Ｐｒｅｓｓ， Ｉｎｃ．
）に、可能な保護基についてより広範な説明が行われて
いる。[0028] The Peptides, Analysis
sis, Synthesis, Biology,
Vol. 1-9 (Eds.
1979-
1987 (Academic Press, Inc.
) provides a more extensive explanation of possible protecting groups.
【００２９】リシンのε−アミノ基を保護することも必
要であり、アルギニンのグアニジル基にとって適切であ
る。この場合、慣用的な保護基はリシンの場合Ｂｏｃ基
であり、アルギニンの場合Ｐｍｃ基、Ｐｍｓ基、Ｍｂｓ
基又はＭｔｒ基である。It is also necessary to protect the ε-amino group of lysine, which is appropriate for the guanidyl group of arginine. In this case, the customary protecting groups are the Boc group for lysine, the Pmc group, Pms group, Mbs group for arginine.
group or Mtr group.
【００３０】特定基の種類に応じて、種々の従来方法に
より、例えばトリフルオロ酢酸を使用して、又は例えば
水素と触媒（例えばパラジウム）による、又は氷酢酸中
のＨＢｒによる弱い還元により、保護基を分離すること
ができる。Depending on the nature of the particular group, the protecting group is removed by various conventional methods, for example using trifluoroacetic acid, or by mild reduction, for example with hydrogen and a catalyst (eg palladium) or with HBr in glacial acetic acid. can be separated.
【００３１】既に前述した如く、組換え体ＤＮＡ技術を
使用して本発明のペプチドを同様に製造することができ
る。この可能性は、ペプチドを反復配列（”縦列”）に
組み込むとき又はペプチドを（遥かに大きい）タンパク
質又はポリペプチドの成分として製造し得るときに特に
重要である。従って、この型のペプチド製造も同様に本
発明の範囲内にある。このために本発明のペプチドをコ
ードするポリヌクレオチドを組み換えＤＮＡの構成成分
として使用し、更には天然のＮＡＮＢＨゲノムにおいて
、前述したポリヌクレオチド配列の側に位置するポリヌ
クレオチドセグメントを実質的に含まない。As already mentioned above, the peptides of the invention can likewise be produced using recombinant DNA technology. This possibility is particularly important when incorporating peptides into repeating sequences ("tandems") or when peptides can be produced as components of (much larger) proteins or polypeptides. Therefore, this type of peptide production is also within the scope of the invention. For this purpose, a polynucleotide encoding a peptide of the invention is used as a component of a recombinant DNA, which is furthermore substantially free of polynucleotide segments flanking the aforementioned polynucleotide sequences in the natural NANBH genome.
【００３２】本発明のペプチドをコードするこの型のポ
リヌクレオチド及びこのポリヌクレオチドが組み込まれ
ている組換えＤＮＡも同様に本発明の範囲内にある。Polynucleotides of this type encoding the peptides of the invention and recombinant DNA into which this polynucleotide has been incorporated are likewise within the scope of the invention.
【００３３】このことはクレームには特別明記されてい
ないが、本発明のペプチド中の１個以上のアミノ酸を他
のアミノ酸に置換することができることは自明である。Although this is not specifically stated in the claims, it is obvious that one or more amino acids in the peptides of the present invention can be substituted with other amino acids.
【００３４】概して、（ａ）ペプチドの酸付加塩、（ｂ
）ペプチドのアミド、特にＣ末端アミド、（ｃ）エステ
ル、特にＣ末端エステル及び（ｄ）Ｎ−アシル誘導体、
特にＮ−末端アシル誘導体、とりわけＮ−アセチル誘導
体を意味するこれらのペプチドの官能誘導体も本発明の
ペプチドとみなす。In general, (a) an acid addition salt of a peptide; (b) an acid addition salt of a peptide;
) amides, especially C-terminal amides, (c) esters, especially C-terminal esters, and (d) N-acyl derivatives of peptides,
Functional derivatives of these peptides, meaning in particular N-terminal acyl derivatives, especially N-acetyl derivatives, are also considered as peptides according to the invention.
【００３５】本発明の“免疫化学試薬”は通常、本発明
のペプチドの１種以上と、適切な支持体又は標識物質と
からなる。The "immunochemical reagent" of the present invention usually consists of one or more peptides of the present invention and a suitable support or labeling substance.
【００３６】使用し得る支持体は例えば、ミクロ試験用
ウェルの内壁若しくはキュベット、管若しくは毛細管、
膜、フィルター、試験用ストリップ、又は粒子表面（例
えばラテックス粒子、赤血球、染料ゾル、金属ゾル若し
くはゾル粒子としての金属化合物、担体タンパク質（例
えばＢＳＡ若しくはＫＬＨ）である。Supports that can be used are, for example, the inner walls of microtest wells or cuvettes, tubes or capillaries,
Membranes, filters, test strips, or particle surfaces such as latex particles, red blood cells, dye sols, metal compounds as metal sols or sol particles, carrier proteins such as BSA or KLH.
【００３７】使用し得る標識物質は特に、放射性同位体
、蛍光化合物、酵素、染料ゾル、金属ゾル又はゾル粒子
である金属化合物である。Labeling substances that can be used are in particular radioisotopes, fluorescent compounds, enzymes, metal compounds which are dye sols, metal sols or sol particles.
【００３８】試験液中のＮＡＮＢＨに対する抗体の検出
方法では、本発明の免疫化学試薬を試験液と接触させる
。その後、ペプチドと試験液中の抗体との間で生成され
る免疫複合体の存在を検出し、この検出により試験液中
のＮＡＮＢＨ抗体の存在を確認し、また定量することが
できる。[0038] In the method for detecting antibodies against NANBH in a test solution, the immunochemical reagent of the present invention is brought into contact with the test solution. Thereafter, the presence of an immune complex formed between the peptide and the antibody in the test solution is detected, and by this detection, the presence of NANBH antibodies in the test solution can be confirmed and quantified.
【００３９】生じる免疫化学反応は、免疫化学試薬の種
類、更には特性に応じて、いわゆるサンドイッチ型反応
、凝集反応、競合反応又は阻害反応である。The immunochemical reaction that occurs is a so-called sandwich type reaction, agglutination reaction, competitive reaction or inhibition reaction, depending on the type and further characteristics of the immunochemical reagent.
【００４０】試験液中のＮＡＮＢＨの検出のために、本
発明の免疫化学試薬を試験液及び抗ＮＡＮＢＨに接触さ
せ、その後生成した免疫錯体の存在を検出し、それによ
り試験液中のＮＡＮＢＨの存在を確定することができる
。For the detection of NANBH in the test solution, the immunochemical reagent of the present invention is contacted with the test solution and anti-NANBH, and the presence of the formed immune complex is then detected, thereby detecting the presence of NANBH in the test solution. can be determined.
【００４１】特に適した試験液中のＮＡＮＢＨの検出方
法は、標識物質を備えた本発明のペプチドと（試験液中
に存在する）ＮＡＮＢＨ抗原との競合反応に基づいてお
り、それによってペプチドと抗原とが固形支持体に結合
したＮＡＮＢＨに対する抗体と競合している。A particularly suitable method for the detection of NANBH in a test solution is based on a competitive reaction between the peptide of the invention provided with a labeling substance and the NANBH antigen (present in the test solution), whereby the peptide and antigen are separated. and compete with an antibody against NANBH bound to a solid support.
【００４２】本発明の試験用キットは、必須構成要素と
して前述した如き免疫化学試薬を含んでいる。ＮＡＮＢ
Ｈ抗体の検出のためにサンドイッチ型反応を実施する場
合、試験用キットは例えば、固形支持体（例えばミクロ
試験用ウェルの内壁）に被覆した本発明のペプチド、及
び本発明の標識ペプチド又は標識抗体を含み得る。The test kit of the present invention contains the above-mentioned immunochemical reagents as essential components. NANB
When performing a sandwich reaction for detection of H antibody, the test kit includes, for example, the peptide of the present invention coated on a solid support (for example, the inner wall of a micro test well), and the labeled peptide or labeled antibody of the present invention. may include.
【００４３】競合反応を実施する場合、試験用キットは
固形支持体に被覆した本発明のペプチド、及びＮＡＮＢ
Ｈに対する標識抗体、好ましくは前記ペプチドに対する
モノクローナル抗体を含み得る。When carrying out a competitive reaction, the test kit contains the peptide of the invention coated on a solid support and NANB.
The peptide may contain a labeled antibody against H.H., preferably a monoclonal antibody against said peptide.
【００４４】凝集反応では、試験用キットは、粒子又は
ゾルに被覆した本発明のペプチドからなる免疫化学試薬
を含んでいる。For agglutination reactions, the test kit contains an immunochemical reagent consisting of a peptide of the invention coated onto particles or sols.
【００４５】ＮＡＮＢＨ抗原の検出のための試験用キッ
トは例えば、本発明の標識ペプチド及び固形支持体に被
覆されるＮＡＮＢＨに対する抗体を含んでいる。A test kit for the detection of NANBH antigen contains, for example, a labeled peptide of the invention and an antibody against NANBH coated on a solid support.
【００４６】[0046]
【実施例】実施例１配列表に示す如き配列からなるペン
タデカペプチドを段階的固相ペプチド合成により製造し
た。ＶＥＧＡ Ｃｏｕｐｌｅｒ２５０ Ｃ自動ペプ
チド合成器又はＬａｂｏｒｔｅｃ ＳＰ６４０半自動
ペプチド合成器を利用し、ｐ−ベンジルオキシベンジル
アルコール樹脂（Ｗａｎｇ樹脂；０．６−０．７ｍｍｏ
ｌｅｓ／ｇ， Ｂａｃｈｅｍ ＡＧ， Ｓｗｉｔ
ｚｅｒｌａｎｄ）及びＮ（α位）−Ｆｍｏｃで保護した
（Ｆｍｏｃとは９−フルオレニルメチルオキシカルボニ
ルのこと）アミノ酸を使用して、合成を実施した。EXAMPLES Example 1 A pentadecapeptide having the sequence shown in the sequence listing was produced by stepwise solid phase peptide synthesis. Using a VEGA Coupler 250 C automatic peptide synthesizer or a Labortec SP640 semi-automatic peptide synthesizer, p-benzyloxybenzyl alcohol resin (Wang resin; 0.6-0.7 mm
les/g, Bachem AG, Swit
The synthesis was carried out using an amino acid protected with N (α-position)-Fmoc (Fmoc refers to 9-fluorenylmethyloxycarbonyl).
【００４７】ＤＤＣ（ジシクロヘキシルカルボジイミド
、１当量）、ＨＯＢｔ（１−ヒドロキシベンゾトリアゾ
ール、２当量）及びＤＭＡＰ（Ｎ，Ｎ−ジメチルアミノ
ピリジン、１当量）をＤＭＦ−ジクロロメタン（１：１
、ｖｏｌ／ｖｏｌ）中４℃で１８時間使用してＦｍｏｃ
−Ａｒｇ（Ｐｍｃ）−ＯＨを樹脂に結合することにより
、合成を開始した。次いで、ベンゾイルクロライド−ピ
リジンで２時間ベンゾイル化することにより、樹脂上の
未反応アルコール官能基をブロックした。DDC (dicyclohexylcarbodiimide, 1 eq.), HOBt (1-hydroxybenzotriazole, 2 eq.) and DMAP (N,N-dimethylaminopyridine, 1 eq.) were dissolved in DMF-dichloromethane (1:1).
, vol/vol) for 18 h at 4°C.
The synthesis was initiated by coupling -Arg(Pmc)-OH to the resin. Unreacted alcohol functionality on the resin was then blocked by benzoylation with benzoyl chloride-pyridine for 2 hours.
【００４８】得られたＦｍｏｃ−Ａｒｇ（Ｐｍｃ）−樹
脂（０．３８ｍｍｏｌ／ｇ）をＤＭＦ中２０％ピペリジ
ンで６分間３回連続処理して、Ｆｍｏｃ基を除去した。
次いで、アミノ酸配列により示されているように、保護
されたペンタデカペプチドをＨ−Ａｒｇ（Ｐｍｃ）−樹
脂上でＦｍｏｃ−アミノ酸の連続結合ステップにより製
造した。以下の側鎖の保護基を使用した。即ちＡｒｇの
場合は−Ｐｍｃ（２，２，５，７，８−ペンタメチルク
ロマン−６−スルホニル−）を、Ｔｈｒ及びＳｅｒの場
合は−ｔＢｕ（ｔ−ブチル−）を、Ｌｙｓの場合はＢｏ
ｃ（ｔ−ブチルオキシカルボニル）を、Ａｓｎ及びＧｌ
ｎの場合はＴｒｔ（トリチル）を使用した。The resulting Fmoc-Arg(Pmc)-resin (0.38 mmol/g) was treated with 20% piperidine in DMF three times for 6 minutes successively to remove the Fmoc group. The protected pentadecapeptide was then prepared by successive coupling steps of Fmoc-amino acids on H-Arg(Pmc)-resin, as indicated by the amino acid sequence. The following side chain protecting groups were used. That is, in the case of Arg, -Pmc (2,2,5,7,8-pentamethylchroman-6-sulfonyl-), in the case of Thr and Ser, -tBu (t-butyl-), and in the case of Lys, Bo
c (t-butyloxycarbonyl), Asn and Gl
In the case of n, Trt (trityl) was used.
【００４９】それぞれが３当量のＦｍｏｃ−アミノ酸、
ＢＯＰ（＝ベンゾトリアゾリルオキシ−トリス（ジメチ
ルアミノ）−ホスホニウムヘキサフルオロホスフェート
）及びＨＯＢｔと、４．５当量のＤＩＰＥＡ（＝Ｎ，Ｎ
−ジイソプロピルエチルアミン）とを含んでいる、樹脂
１グラム当たり１２〜１５ミリリットルのＤＭＦを使用
して、各結合段階を１５分間実施し、その後ＤＭＦ及び
エタノールで３サイクルの洗浄（１サイクルが１分）を
実施した。結合反応の完全性をＫａｉｓｅｒのニンヒド
リン試験で監視した（Ａｎａｌ． Ｂｉｏｃｈｅｍ．
３４， ５９５−５９８，１９７０）。Ｓｅｒ１０及び
Ａｒｇ５の結合の後に、陽性ニンヒドリン反応を観察し
た。それぞれの場合において、１当量の対応Ｆｍｏｃ−
アミノ酸、、ＢＯＰ、ＨＯＢｔ及びＤＩＰＥＡを使用す
る結合反応を再度３０分間繰り返した。次いで、無水酢
酸−ＤＭＦ（５：５９；ｖｏｌ／ｖｏｌ）で１０分間ア
セチル化し、その後ＤＭＦ及びエタノールで洗浄（それ
ぞれ１分間）して、任意の残留遊離アミノ基をブロック
した。3 equivalents of each Fmoc-amino acid,
BOP (=benzotriazolyloxy-tris(dimethylamino)-phosphonium hexafluorophosphate) and HOBt and 4.5 equivalents of DIPEA (=N,N
-diisopropylethylamine) for 15 minutes using 12-15 milliliters of DMF per gram of resin, followed by three cycles of washing with DMF and ethanol (each cycle is 1 minute). was carried out. The completeness of the binding reaction was monitored by Kaiser's ninhydrin test (Anal. Biochem.
34, 595-598, 1970). A positive ninhydrin reaction was observed after binding of Ser10 and Arg5. In each case, one equivalent of the corresponding Fmoc-
The coupling reaction using amino acids, BOP, HOBt and DIPEA was repeated again for 30 minutes. It was then acetylated with acetic anhydride-DMF (5:59; vol/vol) for 10 min, followed by washing with DMF and ethanol (1 min each) to block any remaining free amino groups.
【００５０】各合成サイクルの後に、前述した如くＤＭ
Ｆ中２５％ピペリジンで処理してＮ（α位）−Ｆｍｏｃ
保護基を除去した。After each synthesis cycle, DM
N(α-position)-Fmoc by treatment with 25% piperidine in F
Protecting groups were removed.
【００５１】合成終了後、得られた完全に保護されたペ
ンタデカペプチド樹脂を、トリフルオロ酢酸／水／フェ
ノール／チオアニソール／エタンジチオール（８２：５
：５：５：２．５ｖｏｌ／ｖｏｌ）混合物中にて、室温
で１８時間処理することにより、ペプチドを樹脂から分
離すると同時に全ての保護基を除去した。反応混合物を
ジエチルエーテルに加えて沈澱させた後に、粗ペプチド
を単離した。ｐＨ２．１の０．１Ｍリン酸塩緩衝液中の
アセトニトリル勾配を利用して、Ｃ１８シリカのＨＰＬ
Ｃによりペンタデカペプチドを精製した。After completion of the synthesis, the obtained fully protected pentadecapeptide resin was mixed with trifluoroacetic acid/water/phenol/thioanisole/ethanedithiol (82:5
:5:5:2.5vol/vol) mixture at room temperature for 18 hours to separate the peptide from the resin and simultaneously remove all protecting groups. The crude peptide was isolated after precipitation of the reaction mixture with diethyl ether. HPL of C18 silica using an acetonitrile gradient in 0.1 M phosphate buffer at pH 2.1
The pentadecapeptide was purified by C.
【００５２】実施例２
配列表に示す如きアミノ酸配列からなるペンタデカペプ
チドを、ｐＨ８．０の１００ｍＭリン酸塩緩衝液７．５
μｇ／ミリリットルに溶解した。マイクロタイタープレ
ートを、ｐＨ５．０のリン酸塩緩衝液中０．２％グルタ
ルアルデヒドの１３５μｌ／ウェルで室温で４時間、連
続振とうしながら予備処理した。次いでプレートを空に
して、１３５μｌの上記ペプチド溶液を各ウェルに与え
た。３７℃で３時間放置してペプチドをマイクロタイタ
ープレートに結合させた。プレートを冷凍し、−２０℃
で一晩貯蔵した。Example 2 A pentadecapeptide consisting of an amino acid sequence as shown in the sequence listing was added to a 100mM phosphate buffer solution with a pH of 8.0 (7.5%).
Dissolved at μg/ml. Microtiter plates were pretreated with 135 μl/well of 0.2% glutaraldehyde in pH 5.0 phosphate buffer for 4 hours at room temperature with continuous shaking. The plate was then emptied and 135 μl of the above peptide solution was applied to each well. The peptide was allowed to bind to the microtiter plate by standing at 37°C for 3 hours. Freeze the plate at -20°C
It was stored overnight.
【００５３】その後、プレートを解凍して空にし、残留
結合部位を０．２ＭトリスｐＨ７．４／０．２Ｍ−Ｎａ
Ｃｌ中０．０５％Ｔｗｅｅｎ２０（登録商標）溶液で５
分間室温にてブロックした。次いで、０．２Ｍトリス（
ｐＨ７．４）／０．２Ｍ ＮａＣｌで１回洗浄、０．
０４Ｍトリス（ｐＨ７．４）で２回プレートを洗浄した
（ウェル当たり２５０μｌ）。非Ａ非Ｂ型肝炎に特異な
抗体の同定のために、血清試料をウェルにピペットで注
入した（容器当たり１００μｌ）試料稀釈剤（リン酸塩
緩衝生理食塩水（ＰＢＳ）／２０％正常ヤギ血清／１％
Ｔｒｉｔｏｎ Ｘ１００）で稀釈して、３７℃で１時
間インキュベートした。ウェルをＰＢＳ／０．０５％Ｔ
ｗｅｅｎ２０（登録商標）で洗浄した後に、試料稀釈剤
中で稀釈したペルオキシターゼ（ウェル当たり１００μ
ｌ、３７℃で１時間）で標識したヤギ抗ヒトイムノグロ
ブリンにより、結合したヒトの抗体を検出した。プレー
トをＰＢＳ／０．０５％Ｔｗｅｅｎ２０（登録商標）で
４回洗浄した。ペルオキシダーゼ酵素用基質としてＴＭ
Ｂを加えると（ウェル当たり１００μｌ）、室温で３０
分間反応を進ませた。１００μｌの２Ｍ Ｈ２ＳＯ４
を各ウェルに加えて反応を停止した。Ｏｒｇａｎｏｎ
Ｔｅｋｎｉｋａ ｍｉｃｒｏｅｌｉｓａ読取器では
４５０ｎｍで黄色を読取った。The plate was then thawed and emptied, and the remaining binding sites were removed with 0.2M Tris pH 7.4/0.2M Na
5 in 0.05% Tween20® solution in Cl.
Blocked for minutes at room temperature. Then, 0.2M Tris (
pH 7.4)/washed once with 0.2M NaCl, 0.
The plates were washed twice with 0.04M Tris (pH 7.4) (250 μl per well). For identification of antibodies specific for non-A, non-B hepatitis, serum samples were pipetted into wells (100 μl per container) using a sample diluent (phosphate-buffered saline (PBS)/20% normal goat serum). /1%
Triton X100) and incubated at 37°C for 1 hour. Fill wells with PBS/0.05%T
After washing with ween20®, peroxidase (100μ per well) diluted in sample diluent was added.
Bound human antibodies were detected by goat anti-human immunoglobulin labeled with 1 hour at 37°C. Plates were washed four times with PBS/0.05% Tween20®. TM as a substrate for peroxidase enzymes
When adding B (100 μl per well), 30 μl at room temperature
The reaction was allowed to proceed for minutes. 100μl 2M H2SO4
was added to each well to stop the reaction. Organon
The yellow color was read at 450 nm on a Teknika microelisa reader.
【００５４】非Ａ非Ｂ型肝炎の患者の血清を使用して、
１．８〜３．０の範囲の吸光係数を測定した。２０個の
正常なヒトの血清の平均吸光係数は０．３０１であった
（標準偏差０．０７）。対照として、２つの無関係のペ
ンタデカペプチドで手順を繰り返した。いずれの場合も
、正常なヒトの血清とＮＡＮＢＨの患者の血清試料とか
ら得られた吸光係数にそれほどの差は認められなかった
。[0054] Using serum from patients with non-A, non-B hepatitis,
Extinction coefficients ranging from 1.8 to 3.0 were measured. The average extinction coefficient of 20 normal human sera was 0.301 (standard deviation 0.07). As a control, the procedure was repeated with two unrelated pentadecapeptides. In all cases, no significant difference was observed in the extinction coefficients obtained from normal human serum and NANBH patient serum samples.
【００５５】実施例３
配列表に示すペンタデカペプチドのフラグメントをＮＡ
ＮＢＨの患者の血清との反応性についても再度検査した
。Example 3 The pentadecapeptide fragment shown in the sequence listing was
Reactivity with serum from patients with NBH was also tested again.
【００５６】以下に示すノナペプチド２〜８を合成し、
本質的に実施例１及び実施例２に記載の如く固形支持体
に結合させた。ＮＡＮＢＨの患者から得られた血清試料
との免疫反応性を実施例２に記載の如く調べた。ノナペ
プチド５，６，７はＮＡＮＢＨ患者の血清中の抗体と特
異反応を示した。これらの結果を表１に示す。[0056] The following nonapeptides 2 to 8 were synthesized,
It was attached to a solid support essentially as described in Examples 1 and 2. Immunoreactivity with serum samples obtained from patients with NANBH was investigated as described in Example 2. Nonapeptides 5, 6, and 7 showed specific reactions with antibodies in the serum of NANBH patients. These results are shown in Table 1.
【００５７】[0057]
【表１】 ペプチド２：配列番号２のアミノ酸配列。[Table 1] Peptide 2: Amino acid sequence of SEQ ID NO: 2.
【００５８】ペプチド３：配列番号３のアミノ酸配列。Peptide 3: Amino acid sequence of SEQ ID NO:3.
【００５９】ペプチド４：配列番号４のアミノ酸配列。Peptide 4: Amino acid sequence of SEQ ID NO: 4.
【００６０】ペプチド５：配列番号５のアミノ酸配列。Peptide 5: Amino acid sequence of SEQ ID NO: 5.
【００６１】ペプチド６：配列番号６のアミノ酸配列。Peptide 6: Amino acid sequence of SEQ ID NO: 6.
【００６２】ペプチド７：配列番号７のアミノ酸配列。Peptide 7: Amino acid sequence of SEQ ID NO: 7.
【００６３】ペプチド８：配列番号８のアミノ酸配列。Peptide 8: Amino acid sequence of SEQ ID NO: 8.
【００６４】[0064]
【配列表】配列番号：１
配列の長さ：１５
配列の型：アミノ酸
配列の種類：ペプチド
配列
配列番号：２
配列の長さ：９
配列の型：アミノ酸
配列の種類：ペプチド
配列
配列番号：３
配列の長さ：９
配列の型：アミノ酸
配列の種類：ペプチド
配列
配列番号：４
配列の長さ：９
配列の型：アミノ酸
配列の種類：ペプチド
配列
配列番号：５
配列の長さ：９
配列の型：アミノ酸
配列の種類：ペプチド
配列
配列番号：６
配列の長さ：９
配列の型：アミノ酸
配列の種類：ペプチド
配列Ａｒｇ−Ｌｙｓ−Ｔｈｒ−Ｌｙｓ−Ａｒｇ−Ｓｅｒ
−Ｔｈｒ−Ａｓｎ−Ａｒｇ配列番号：７配列の長さ：９
配列の型：アミノ酸
配列の種類：ペプチド
配列
配列番号：８
配列の長さ：９
配列の型：アミノ酸
配列の種類：ペプチド
配列[Sequence list] Sequence number: 1 Sequence length: 15 Sequence type: Amino acid sequence type: Peptide sequence SEQ ID NO: 2 Sequence length: 9 Sequence type: Amino acid sequence type: Peptide sequence SEQ ID NO: 3 Sequence length: 9 Sequence type: Amino acid sequence type: Peptide sequence SEQ ID NO: 4 Sequence length: 9 Sequence type: Amino acid sequence type: Peptide sequence SEQ ID NO: 5 Sequence length: 9 Type: Amino acid sequence type: Peptide sequence SEQ ID NO: 6 Sequence length: 9 Sequence type: Amino acid sequence type: Peptide sequence Arg-Lys-Thr-Lys-Arg-Ser
-Thr-Asn-Arg Sequence number: 7 Sequence length: 9 Sequence type: Amino acid sequence type: Peptide sequence Sequence number: 8 Sequence length: 9 Sequence type: Amino acid sequence type: Peptide sequence
Claims (5)
らなるペンタデカペプチド及びＮＡＮＢＨ抗体と免疫化
学反応するそのフラグメント、並びに該ペンタデカペプ
チドを主成分として含むポリペプチド又はＮＡＮＢＨ抗
体と免疫化学反応するそのフラグメント。Claim 1: A pentadecapeptide consisting of an amino acid sequence as shown in SEQ ID NO: 1, a fragment thereof that immunochemically reacts with a NANBH antibody, and a polypeptide containing the pentadecapeptide as a main component or a fragment thereof that immunochemically reacts with a NANBH antibody. fragment.
る免疫化学試薬。2. An immunochemical reagent comprising the peptide according to claim 1.
検出方法であって、請求項２に記載の免疫化学試薬を試
験液と接触させ、ペプチドと試験液中の抗体との間で生
成される免疫錯体の存在を検出し、これが試験液中のＮ
ＡＮＢＨ抗体の存在の測定方法であることを特徴とする
方法。3. A method for detecting antibodies against NANBH in a test solution, comprising: contacting the immunochemical reagent according to claim 2 with the test solution, and detecting the immunity generated between the peptide and the antibody in the test solution; The presence of the complex is detected, which indicates that N in the test solution
A method characterized in that it is a method for measuring the presence of ANBH antibodies.
って、請求項２に記載の免疫化学試薬を試験液及び抗Ｎ
ＡＮＢＨに接触させ、その後生成した免疫錯体の存在を
検出し、それにより試験液中のＮＡＮＢＨの存在を確定
することを特徴とする方法。4. A method for detecting NANBH in a test solution, comprising adding the immunochemical reagent according to claim 2 to the test solution and anti-N
A method characterized in that the presence of NANBH in the test liquid is determined by contacting with ANBH and subsequently detecting the presence of the generated immune complex.
イで使用すべき試験用キット。5. A test kit to be used in the immunoassay according to claim 3 or 4.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| NL9020077 | 1990-03-30 | ||
| NL90.200775.6 | 1990-03-30 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| JPH04221398A true JPH04221398A (en) | 1992-08-11 |
Family
ID=19858360
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP9153991A Pending JPH04221398A (en) | 1990-03-30 | 1991-03-29 | Antibody to non-a, non-b hepatitis virus and immunochemically reactive peptide |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPH04221398A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH06500796A (en) * | 1991-06-13 | 1994-01-27 | デイド、インターナショナル、インコーポレイテッド | Immunoassay for non-A, non-B hepatitis |
-
1991
- 1991-03-29 JP JP9153991A patent/JPH04221398A/en active Pending
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH06500796A (en) * | 1991-06-13 | 1994-01-27 | デイド、インターナショナル、インコーポレイテッド | Immunoassay for non-A, non-B hepatitis |
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