JPH03135766A - Immunochemical measurement method of human il-6 and kit for this purpose - Google Patents
Immunochemical measurement method of human il-6 and kit for this purposeInfo
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- JPH03135766A JPH03135766A JP27186289A JP27186289A JPH03135766A JP H03135766 A JPH03135766 A JP H03135766A JP 27186289 A JP27186289 A JP 27186289A JP 27186289 A JP27186289 A JP 27186289A JP H03135766 A JPH03135766 A JP H03135766A
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- antibody
- human
- galactosidase
- biotin
- concentration
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Links
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- 238000000691 measurement method Methods 0.000 title description 4
- 102000004889 Interleukin-6 Human genes 0.000 claims abstract description 66
- 108090001005 Interleukin-6 Proteins 0.000 claims abstract description 66
- YBJHBAHKTGYVGT-ZKWXMUAHSA-N (+)-Biotin Chemical compound N1C(=O)N[C@@H]2[C@H](CCCCC(=O)O)SC[C@@H]21 YBJHBAHKTGYVGT-ZKWXMUAHSA-N 0.000 claims abstract description 20
- 102000005936 beta-Galactosidase Human genes 0.000 claims abstract description 13
- 108010005774 beta-Galactosidase Proteins 0.000 claims abstract description 13
- 101001076408 Homo sapiens Interleukin-6 Proteins 0.000 claims abstract description 10
- 108010090804 Streptavidin Proteins 0.000 claims abstract description 10
- 229960002685 biotin Drugs 0.000 claims abstract description 10
- 235000020958 biotin Nutrition 0.000 claims abstract description 10
- 239000011616 biotin Substances 0.000 claims abstract description 10
- 102000052611 human IL6 Human genes 0.000 claims description 9
- 239000000758 substrate Substances 0.000 claims description 8
- 238000005259 measurement Methods 0.000 claims description 7
- 238000001514 detection method Methods 0.000 claims description 5
- 238000003556 assay Methods 0.000 claims description 3
- 101710195316 Beta-galactosidase 6 Proteins 0.000 claims 1
- 230000035945 sensitivity Effects 0.000 abstract description 8
- 229940100601 interleukin-6 Drugs 0.000 abstract description 7
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- 229940116886 human interleukin-6 Drugs 0.000 abstract 1
- 238000000034 method Methods 0.000 description 25
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- 210000001744 T-lymphocyte Anatomy 0.000 description 4
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- 241000598436 Human T-cell lymphotropic virus Species 0.000 description 3
- 241000725303 Human immunodeficiency virus Species 0.000 description 3
- 238000004458 analytical method Methods 0.000 description 3
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- HSHNITRMYYLLCV-UHFFFAOYSA-N 4-methylumbelliferone Chemical compound C1=C(O)C=CC2=C1OC(=O)C=C2C HSHNITRMYYLLCV-UHFFFAOYSA-N 0.000 description 2
- YUDPTGPSBJVHCN-DZQJYWQESA-N 4-methylumbelliferyl beta-D-galactoside Chemical compound C1=CC=2C(C)=CC(=O)OC=2C=C1O[C@@H]1O[C@H](CO)[C@H](O)[C@H](O)[C@H]1O YUDPTGPSBJVHCN-DZQJYWQESA-N 0.000 description 2
- 102000002260 Alkaline Phosphatase Human genes 0.000 description 2
- 108020004774 Alkaline Phosphatase Proteins 0.000 description 2
- 206010061818 Disease progression Diseases 0.000 description 2
- 206010061598 Immunodeficiency Diseases 0.000 description 2
- CDBYLPFSWZWCQE-UHFFFAOYSA-L Sodium Carbonate Chemical compound [Na+].[Na+].[O-]C([O-])=O CDBYLPFSWZWCQE-UHFFFAOYSA-L 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- 230000002596 correlated effect Effects 0.000 description 2
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- 102000004169 proteins and genes Human genes 0.000 description 1
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- 229910000029 sodium carbonate Inorganic materials 0.000 description 1
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- RYFMWSXOAZQYPI-UHFFFAOYSA-K trisodium phosphate Chemical compound [Na+].[Na+].[Na+].[O-]P([O-])([O-])=O RYFMWSXOAZQYPI-UHFFFAOYSA-K 0.000 description 1
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- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
Description
【発明の詳細な説明】
〔産業上の利用分野〕
本発明はヒト ＩＬ−６の免疫化学的測定方法に関する
ものである。DETAILED DESCRIPTION OF THE INVENTION [Field of Industrial Application] The present invention relates to a method for immunochemical measurement of human IL-6.
インターロイキン−６（ＢＳＦ ２ ／以下ＩＬ−６と
略す）は、種々の重要な生理活性を有し、広く細胞の増
殖分化に関与しているタンパク質である。さらにＩＬ−
６の異常産生が種々の自己免疫疾患の病因因子である可
能性が報告されている（岸本、平野、Ａｎｎ。Interleukin-6 (BSF 2 /hereinafter abbreviated as IL-6) is a protein that has various important physiological activities and is widely involved in cell proliferation and differentiation. Furthermore, IL-
It has been reported that abnormal production of 6 may be an etiological factor for various autoimmune diseases (Kishimoto, Hirano, Ann.
Ｒｅｖ、Ｉｍｍｕｎｏｌ、、 ６． ｐ４Ｂ５．１９８
８年参照）。Rev. Immunol, 6. p4B5.198
(See Year 8).
生体内で多様な生理活性を強く発揮するＩＬ−６の生理
的濃度を知ることは、各種疾患の新しい診断マーカーと
して期待されている。ＩＬ−６の生物活性測定法として
は、１１００ｆ／ｍｉ’のＩＬ−６が測定可能な高感度
のものが報告されている（ＴｏＭａｔｓｕｄａら、Ｓｕ
ｒ。Knowing the physiological concentration of IL-6, which strongly exhibits various physiological activities in vivo, is expected to serve as a new diagnostic marker for various diseases. As a method for measuring the biological activity of IL-6, a highly sensitive method capable of measuring IL-6 at 1100 f/mi' has been reported (ToMatsuda et al., Su
r.
Ｊ、Ｉｍｍｕｎｏｌ、、 １ｇ、 ｐ９５１．１９８８
年参照）。しかしながら、この方法は、多数の検体を迅
速、且つ簡単に測定することができないという欠点を有
する。J. Immunol, 1g, p951.1988
(see year). However, this method has the disadvantage that a large number of analytes cannot be measured quickly and easily.
多数の検体を迅速、且つ簡単に測定する方法として一般
に種々の免疫測定法が知られている。ＩＬ−６の測定の
ために免疫測定法を応用した例として、前記Ｔ、 Ｍａ
ｔｓｕｄａらの報告によれば、検出系にアルカリホスフ
ァターゼを使用する方法によって、ヒト膀胱癌細胞由来
ＩＬ−６をヒト血清に稀釈したモデル検体系において５
０ｐｇ／ｍｊ！のＩＬ−６が測定可能である旨記載され
ている。しかしながら、この方法を大腸菌由来組換ＩＬ
−６をヒト血清に溶解して調製した検体モデル系におい
ては上記のごとき感度は得られなかった（第２図を参照
のこと）。Various immunoassay methods are generally known as methods for quickly and easily measuring a large number of specimens. As an example of applying an immunoassay method for the measurement of IL-6, the above-mentioned T, Ma
According to a report by Tsuda et al., in a model specimen system in which human bladder cancer cell-derived IL-6 was diluted with human serum, a method using alkaline phosphatase as a detection system detected
0pg/mj! It is stated that IL-6 can be measured. However, this method cannot be applied to E. coli-derived recombinant ILs.
In the sample model system prepared by dissolving -6 in human serum, the sensitivity described above was not obtained (see Figure 2).
一般に、ヒト検体中のＩＬ−６を測定するには、分析系
が、ヒト検体中に自然に存在するＩＬ−６をそれが自然
に存在する程度の濃度レベルにおいて検出することがで
きるだけの感度を有すると共に、同レベルの濃度の組換
ＩＬ−６を検出できなければならない。なぜなら、そう
でなければ、分析系の標準試薬として組換ＩＬ−６を使
用することができないからである。Generally, to measure IL-6 in a human specimen, the analytical system must be sensitive enough to detect IL-6 naturally present in the human specimen at the concentration level at which it naturally occurs. It must be possible to detect similar concentrations of recombinant IL-6. This is because, otherwise, recombinant IL-6 cannot be used as a standard reagent in an analytical system.
従って、種々の一般的な免疫測定法が知られているにも
かかわらず、ヒト検体中のＩＬ−６をそれが自然に存在
するような低濃度レベルにおいて検出できる程の実用的
な感度を有する分析方法は知られていなかった。Therefore, although a variety of common immunoassays are known, none have practical sensitivity to detect IL-6 in human samples at the low concentration levels at which it naturally occurs. The method of analysis was unknown.
従って本発明は、ヒトの検体中に存在するＩＬ−６を検
出するのに十分な感度を有するＩＬ−６の免疫測定法を
提供しようとするものであり、その前提条件としてヒト
の検体中に存在するＩＬ−６及び組換ＩＬ−６の両者を
同程度の感度で検出することができる方法、及びその方
法に使用するためのキットを提供しようとするものであ
る。Therefore, the present invention aims to provide an immunoassay method for IL-6 that has sufficient sensitivity to detect IL-6 present in human samples, and the prerequisite for this is that IL-6 is present in human samples. It is an object of the present invention to provide a method capable of detecting both existing IL-6 and recombinant IL-6 with comparable sensitivity, and a kit for use in the method.
本発明者らは前記の課題を解決すべく種々検討した結果
、−次抗体として抗＝■シー６モノクローナル抗体を使
用し、二次抗体としてビオチンにより標識された抗−Ｉ
シー６ポリクローナル抗体を使用し、そしてストレプト
アビジン／β−ガラクトシダーゼ接合体とβ−ガラクト
シダーゼの基質とを用いるという特定の組合せにより、
数十ｐｇ／−の濃度のＩＬ−６を測定することができる
という全く新しい知見を得、これに基いて本発明を完成
した。As a result of various studies to solve the above-mentioned problems, the present inventors used an anti-C6 monoclonal antibody as a secondary antibody and an anti-I labeled with biotin as a secondary antibody.
By the specific combination of using the Sea6 polyclonal antibody and using a streptavidin/β-galactosidase conjugate and a substrate for β-galactosidase,
We obtained a completely new finding that it is possible to measure IL-6 at a concentration of several tens of pg/-, and based on this we completed the present invention.
従って本発明は、ヒトＩＬ−６に対するモノクローナル
抗体、ビオチン標識抗ヒトＩＬ−６ポリクローナル抗体
、並びに検出系としてのストレプトアビジン／β−ガラ
クトシダーゼ及びその基質を用いることを特徴とするヒ
ト ＩＬ−６の免疫学的測定法；並びにヒトＩＬ−６に
対するモノクローナル抗体、ビオチン標識抗ヒトＩＬ−
６ポリクローナル抗体、並びに検出系としてのストレプ
トアビジン／β−ガラクトシダーゼを含んで成るＩＬ−
６の免疫化学的測定キットを提供する。Therefore, the present invention provides a method for immunizing human IL-6, which is characterized by using a monoclonal antibody against human IL-6, a biotin-labeled anti-human IL-6 polyclonal antibody, and streptavidin/β-galactosidase and its substrate as a detection system. chemical measurement method; and monoclonal antibody against human IL-6, biotin-labeled anti-human IL-6
6 polyclonal antibody and streptavidin/β-galactosidase as a detection system.
6 immunochemical measurement kits are provided.
１、ＩＬ−６の免疫化学的測定法
ＩＬ−６の免疫化学的測定法は、ヒト血清、尿、その他
例えば関節液等中に含まれるＩＬ−６の生理的濃度、ヒ
ト細胞を培養したときの培養上清中のＩＬ−６濃度、さ
らにはＩＬ−５を遺伝子工学的に生産したり、ＩＬ−６
を反応させたときに生じるサンプルのニジ−６濃度を正
確かつ迅速に測定することを目的としている。1. Immunochemical measurement method for IL-6 The immunochemical measurement method for IL-6 is based on the physiological concentration of IL-6 contained in human serum, urine, and other fluids such as synovial fluid, and when human cells are cultured. The concentration of IL-6 in the culture supernatant of
The purpose of this study is to accurately and quickly measure the concentration of Nizi-6 in a sample that is generated when reacting.
この方法においては、まず−次抗体としての抗−ＩＬ−
６モノクロ一ナル抗体を固体支持体に固定する。この固
体支持体としては、マイクロタイタープレート、各種の
ビーズ状の例えばポリスチレン、ポリプロピレン等のプ
ラスチック製、金属セラミックス等の無機物質製等の免
疫測定法において常用されている支持体を用いることが
できる。また、抗体の固定も常法に従って行うことがで
きる。次に、この固定された一次抗体と分析検体とを接
触せしめる。これにより検体中のＩＬ−６は特異的に該
抗体と結合する。次に二次抗体を加え、−次抗体を介し
て支持体に固定されたＩＬ−６と二次抗体とを結合せし
める。二次抗体には標識物質としてビオチンが結合して
いる。次に、検出系としてのストレプトアビジン／β−
ガラクトシダーゼを添加し、前記ビオチンとストレプト
アビジンとの結合を介してβ−ガラクトシダーゼが固定
される。検出系の成分としてβ−ガラクトシダーゼを使
用するのが本発明の特徴である。次に、β−ガラクトシ
ダーゼの基質を加えることにより発色せしめ、この発色
強度を常法、例えば光度計により読み取る。In this method, first, anti-IL-
6 monoclonal antibodies are immobilized on a solid support. As this solid support, supports commonly used in immunoassay methods such as microtiter plates, various bead-shaped supports made of plastics such as polystyrene and polypropylene, and inorganic materials such as metal ceramics can be used. Furthermore, immobilization of antibodies can also be carried out according to conventional methods. Next, this immobilized primary antibody is brought into contact with an analysis sample. Thereby, IL-6 in the sample specifically binds to the antibody. Next, a secondary antibody is added to allow IL-6 immobilized on the support to bind to the secondary antibody via the secondary antibody. Biotin is bound to the secondary antibody as a labeling substance. Next, streptavidin/β-
Galactosidase is added, and β-galactosidase is immobilized through the binding between the biotin and streptavidin. A feature of the present invention is the use of β-galactosidase as a component of the detection system. Next, a substrate for β-galactosidase is added to develop a color, and the intensity of the color is read by a conventional method, for example, using a photometer.
発色性のβ−ガラクトシダーゼ基質としては、免疫測定
において常用されている基質、例えば４−メチルウンベ
リフェリル−β−Ｄ−ガラクトシド、オルトニトロフェ
ニルガラクトピラノシド（ＯＰＮＧ）等を用いることが
できる。As the chromogenic β-galactosidase substrate, substrates commonly used in immunoassays, such as 4-methylumbelliferyl-β-D-galactoside, orthonitrophenylgalactopyranoside (OPNG), and the like can be used.
上記の方法を実施するための測定キットは、抗−ＩＬ−
６モノクロ一ナル抗体、ビオチン標識された抗−ＩＬ−
６ポリクロ一ナル抗体、及びストレプトアビジン／β−
ガラクトシダーゼ接合体を含んで成る。このキットはさ
らにβ−ガラクトシダーゼ基質を含むことができるが、
これは分析現場において別途調達することも容易である
。The measurement kit for carrying out the above method is anti-IL-
6 monoclonal antibody, biotinylated anti-IL-
6 polyclonal antibody, and streptavidin/β-
comprising a galactosidase conjugate. The kit can further include a β-galactosidase substrate,
This can also be easily procured separately at the analysis site.
２、 ＩＬ−６に対するモノクローナル抗体本発明に
適当なＩＬ−６に対するモノクローナル抗体とは生体内
で産生されるＩＬ−６および標準品として使用する遺伝
子工学的に作製されたＩＬ−６に同程度の抗体価を有す
るモノクローナル抗体である。2. Monoclonal antibody against IL-6 The monoclonal antibody against IL-6 suitable for the present invention is a monoclonal antibody against IL-6 that is equivalent to IL-6 produced in vivo and genetically engineered IL-6 used as a standard product. It is a monoclonal antibody with an antibody titer.
該抗体は常法により作製することができる。すなわち遺
伝子工学的に作製した組換ＩＬ−６や培養細胞の上清等
から分離精製したＩＬ−６をマウス、例えばＢａ１ｂ／
ｃ等に免疫して、抗体価が上がったところで、肺細胞を
ミエローマ細胞と融合してハイブリドーマを作製する。The antibody can be produced by conventional methods. That is, recombinant IL-6 produced by genetic engineering or IL-6 separated and purified from the supernatant of cultured cells is injected into mice, such as Ba1b/
When the antibody titer increases after immunization with C. c., the lung cells are fused with myeloma cells to create a hybridoma.
次にＩＬ−６を特異的に認識するモノクローナル抗体を
産生ずるハイブリドーマをスクリーニングすればよい。Next, hybridomas that produce monoclonal antibodies that specifically recognize IL-6 may be screened.
３、 ＩＬ−６に対するポリクローナル抗体本発明に
適当なＩＬ−６に対するポリクローナル抗体とは、生体
内で産生されるＩＬ−６および標準品として使用する遺
伝子工学的に生産されるＩＬ−６に対して、同程度の抗
体価を有する抗体である。該抗体を生産する生物種は、
ウサギ、ヒツジ、ヤギなど種々の生物種を例示すること
ができる。該抗体を作製するための免疫原としては、遺
伝子工学的に作製された組換ＩＬ−６、例えば大腸菌由
来ＩＬ−６等を例示できる。3. Polyclonal antibody against IL-6 The polyclonal antibody against IL-6 suitable for the present invention refers to the polyclonal antibody against IL-6 produced in vivo and the IL-6 produced by genetic engineering used as a standard product. , are antibodies with similar antibody titers. The species that produces the antibody is
Examples include various species such as rabbits, sheep, and goats. An example of the immunogen for producing the antibody is recombinant IL-6 produced by genetic engineering, such as E. coli-derived IL-6.
以下本発明をさらに詳細に説明するために実施例を示す
が、本発明はこれら実施例に限定されるものではない。Examples will be shown below to explain the present invention in more detail, but the present invention is not limited to these Examples.
実施例１． ヒトＩＬ−６の免疫化学的測定法コート用
緩衝液（０，０５Ｍ炭酸ナトリウム、ｐＨ８〜９）に溶
解した２ｘ／ｍｊ！の抗−ＩＬ−６モノクロ一ナル抗体
ＭＨ１６６（ＴｏＭａｔｓｕｄａ ら、Ｂｕｒ、 Ｊ、
Ｉｍｍｕｎｏｌ、 。Example 1. Immunochemical assay of human IL-6 2x/mj! dissolved in coating buffer (0.05M sodium carbonate, pH 8-9). anti-IL-6 monoclonal antibody MH166 (ToMatsuda et al., Bur, J.
Immunol.
１８、 ｐ９５１．１９８８年参照）を９６穴プレート
（ＮＵＮＣ社製）にウェルあたり１００１１！入れ、４
℃で１晩放置した。翌日、溶液を捨て、ブロック用緩衝
液（ＰＢＳ。18, p. 951, 1988) in a 96-well plate (manufactured by NUNC) at 10,011 cells per well. Put in, 4
It was left overnight at °C. The next day, discard the solution and add blocking buffer (PBS).
０．１％ＢＳＡ、 ０．２％ｇｅｌａｔｉｎ、 ０．
０５％アジ化ナトリウム）をウェルあたり１５０Ｊｌｌ
加え、３０分以上室温で放置した。次に溶液を捨て、リ
ンス用緩衝液（ＰＢＳ、 ０．１％Ｔｗｅｅｎ ２０
）で３回以上洗った後、測定すべきサンプルを加え、室
温で２〜３時間放置した。溶液を捨て、リンス用緩衝液
で３回以上洗った後、リンス用緩衝液に溶解した５ｘ／
ｍｊ！のビオチン化抗−Ｉ Ｌ−６ポリクロ一ナル抗体
を加え、３７℃で１時間放置した。さらに溶液を捨て、
リンス用緩衝液で３回以上洗った後、１ ｍＭ ＭｇＣ
ｌ２を含むリンス用緩衝液で１０００倍に稀釈したスト
レプトアビジン／β−ガラクトシダーゼを加え、３７℃
で３０分放置した。その後、溶液を捨て、リンス用緩衝
液で３回以上洗った。それから基質用緩衝液（０，０１
Ｍリン酸ナトリウム、０．１ Ｍ ＮａＣ１，１ｍＭＭ
ｇＣｌ、、０．１％アジ化ナトリウム、ｐＨ７，０）に
溶解した０、１ｍｇ／ｍｊ！の４−メチルウンベリフェ
リル−β−Ｄ−ガラクトシド（シグマ社）をウェルあた
り１００Ｉ加え、遊離した４−メチルウンベリフェロン
の螢光強度を螢光イムノリーダーで測定した。0.1% BSA, 0.2% gelatin, 0.
05% sodium azide) at 150 Jll per well.
The mixture was then left at room temperature for 30 minutes or more. Next, discard the solution and rinse with rinsing buffer (PBS, 0.1% Tween 20
) at least three times, the sample to be measured was added and left at room temperature for 2-3 hours. Discard the solution and wash with rinsing buffer at least 3 times, then add 5x/ml dissolved in rinsing buffer.
mj! A biotinylated anti-IL-6 polyclonal antibody was added thereto, and the mixture was left at 37°C for 1 hour. Then discard the solution and
After washing at least three times with rinsing buffer, 1 mM MgC
Add streptavidin/β-galactosidase diluted 1000 times with a rinsing buffer containing l2, and incubate at 37°C.
I left it for 30 minutes. Thereafter, the solution was discarded, and the plate was washed three times or more with a rinsing buffer. Then substrate buffer (0,01
M Sodium Phosphate, 0.1 M NaCl, 1mM
gCl, 0.1 mg/mj dissolved in 0.1% sodium azide, pH 7.0)! 4-methylumbelliferyl-β-D-galactoside (Sigma) was added at 100 I per well, and the fluorescence intensity of liberated 4-methylumbelliferone was measured using a fluorescence immunoreader.
第１図は、本方法により、血清に溶解した組換ＩＬ−５
（Ｙ、Ａｓａｇｏｅら、ｂｉｏｔｅｃｈｎｏｌｏｇｙ、
６． ｐ８０６． １９８８年参照）が数十ｐｇ
／ｍｌまで測定できることを示している。第２図は、二
次抗体としてアルカリフォスファターゼ結合抗−ウサギ
イムノグロプリン及びその基質を用いて発色させたとき
と本方法との比較を示す。第１図及び第２図の結果から
、本方法のＩＬ−６の測定感度が優れることが示された
。Figure 1 shows recombinant IL-5 dissolved in serum by this method.
(Y, Asagoe et al., biotechnology,
6. p806. (see 1988) is several tens of pg.
This shows that it is possible to measure up to 1/ml. FIG. 2 shows a comparison between the present method and the case where color was developed using alkaline phosphatase-conjugated anti-rabbit immunoglobulin and its substrate as a secondary antibody. The results shown in FIGS. 1 and 2 show that the sensitivity of this method for measuring IL-6 is excellent.
実施例２． エイズ患者血清中のヒト ＩＬ−６濃度の
測健常人及びエイズウィルス保持者（症状により３段階
に区別）の血清を実施例１に示す方法でＩＬ−６濃度を
測定した結果を第３図に示す。第３図はエイズウィルス
保持者では疾患の進行と血清中のＩＬ−６濃度が相関し
ていることを示す。Example 2. Measurement of human IL-6 concentration in the serum of AIDS patients. Figure 3 shows the results of measuring the IL-6 concentration in the serum of healthy individuals and AIDS virus carriers (differentiated into three levels depending on symptoms) using the method shown in Example 1. show. FIG. 3 shows that in AIDS virus carriers, disease progression is correlated with serum IL-6 concentration.
実施例３． ＡＴＬ患者血清中のヒＨＬ−６濃度の測定
健常人及びＨＴＬＶウィルス保持者（症状により３段階
に区別）の血清を実施例１に示す方法でＩＬ−６濃度を
測定した結果を第４図に示す。第４図はＨＴＬＶウィル
ス保持者では疾患の進行と血清中のＩＬ−６濃度が相関
していることを示す。Example 3. Measurement of human HL-6 concentration in the serum of ATL patients Figure 4 shows the results of measuring the IL-6 concentration in the serum of healthy subjects and HTLV virus carriers (differentiated into three stages depending on symptoms) using the method shown in Example 1. show. FIG. 4 shows that in HTLV virus carriers, disease progression is correlated with serum IL-6 concentration.
ＡＴＬ患者からＨＴＬＶ感染Ｔ細胞を採取し、これより
自立的に増殖するＴ細胞株を樹立し、ＴＬ−Ｍａｒと名
付けた。ＴＬ−Ｍａｒを１０％牛脂児血清を含むＲＰＭ
１１６４０培地でＩ ＸＩＯ’／ｍｌの濃度で３日間培
養し、土浦中のＩＬ−６濃度を測定した結果を第５図に
示す。第５図は、ＨＴＬＶ感染Ｔ細胞はＩＬ−６を産生
じていることを示している。HTLV-infected T cells were collected from ATL patients, and a T cell line that proliferated autonomously was established and named TL-Mar. RPM containing TL-Mar with 10% tallow serum
The cells were cultured in 11640 medium at a concentration of IXIO'/ml for 3 days, and the IL-6 concentration in Tsuchiura was measured. The results are shown in FIG. Figure 5 shows that HTLV-infected T cells produce IL-6.
本発明で提供されるＩＬ−６の免疫化学的測定法はＩシ
ー６濃度を高感度で迅速に、しかも多数の検体を同時に
測定することを可能にした。この方法は、各疾患でのＩ
Ｌ−６の生理的濃度を調べたり、ＩＬ−６の疾患におけ
る役割を解明したり、他の薬剤の効果を調べるのに有用
である。The immunochemical assay method for IL-6 provided by the present invention has made it possible to measure IL-6 concentration quickly and with high sensitivity, and in addition, to simultaneously measure a large number of specimens. This method is suitable for each disease.
It is useful for investigating the physiological concentration of L-6, elucidating the role of IL-6 in diseases, and investigating the effects of other drugs.
第１図は、血清に溶解した大腸菌由来組換ＩＬ−６濃度
を実施例１に示す方法で測定したときの、各種ＩＬ−６
濃度に対する発色を示す。
第２図は、血清に溶解した大腸菌由来組換ＩＬ−６濃度
をＴ、 Ｍ、Ｉｔｓｕｄａ らの方法（Ｂｕｒ、Ｊ、Ｉ
ｍｍｕｎｏｌ、、 １３゜ｐ９５１．１９８８年参照）
により測定した場合と、本方法により測定した場合の各
種ＩＬ−６濃度に対する基質の発色を示す。
第３図は、健常人（ＮＩＩＳ）及びエイズウィルス感染
者（ＡＳＹＭＰＴＯＭＭＡＴＩＣＣＡＲＲＩＥＲは症状
のでていない感染者、＾ＲＣは免疫不全様の患者、ＡＩ
ＤＳは免疫不全患者）の血清中のＩＬ−６濃度を実施例
１に示す方法で測定した結果（６〜１０検体の平均値）
を示す。
第４図は、健常人（ＮＨ３）及びＨＴＬＶ感染者（ＨＴ
ＬＶ−ＩＨＣは症状のでていない感染者、ＴＵＭＯＲＴ
ＹＰＢは皮膚に腫瘍をもつ患者、ＮＯＮ−ＴＵＭＯＲＴ
ＹＰＥは白血病性の患者）の血清中のＩＬ−６濃度を実
施例１に示す方法で測定した結果（６〜１０検体の平均
値）を示す。
第５図は、培養液及びＨＴＬＶ感染Ｔ細胞の培養上清中
のＩＬ−６濃度を実施例１に示す方法で測定した結果を
示す。
ＩＬ−６濃Ｉｆ （ｎｇ／ｍｌ ）
第１回FIG. 1 shows the concentration of various IL-6 derived from E. coli dissolved in serum as measured by the method shown in Example 1.
Shows color development relative to density. Figure 2 shows the concentration of E. coli-derived recombinant IL-6 dissolved in serum using the method of T, M, Itsuda et al.
mmunol, 13゜p951.1988)
3 shows the color development of the substrate for various IL-6 concentrations when measured by the method and by the present method. Figure 3 shows healthy people (NIIS) and people infected with the AIDS virus (ASYMPTOMMATICCARRIER is an infected person who has no symptoms, ^RC is an immunocompromised patient, AI
Results of measuring IL-6 concentration in serum of DS (immunocompromised patient) using the method shown in Example 1 (average value of 6 to 10 samples)
shows. Figure 4 shows healthy subjects (NH3) and HTLV infected subjects (HT
LV-IHC is an infected person without symptoms, TUMORT
YPB is a patient with a tumor on the skin, NON-TUMORT
YPE indicates the result (average value of 6 to 10 samples) of the IL-6 concentration in the serum of leukemic patients measured by the method shown in Example 1. FIG. 5 shows the results of measuring the IL-6 concentration in the culture medium and the culture supernatant of HTLV-infected T cells by the method shown in Example 1. IL-6 Concentration If (ng/ml) 1st
Claims (1)
ン標識抗ヒトＩＬ−６ポリクローナル抗体、並びに検出
系としてのストレプトアビジン／β−ガラクトシダーゼ
及びその基質を用いることを特徴とするヒトＩＬ−６の
免疫化学的測定法。 ２、ヒトＩＬ−６に対するモノクローナル抗体、ビオチ
ン標識抗ヒトＩＬ−６ポリクローナル抗体、ストレプト
アビジン／β−ガラクトシダーゼを含んで成るヒトＩＬ
−６の免疫化学的測定用キット。[Scope of Claims] 1. Human IL-6 characterized by using a monoclonal antibody against human IL-6, a biotin-labeled anti-human IL-6 polyclonal antibody, and streptavidin/β-galactosidase and its substrate as a detection system. 6. Immunochemical assay method. 2. Human IL comprising a monoclonal antibody against human IL-6, a biotin-labeled anti-human IL-6 polyclonal antibody, and streptavidin/β-galactosidase
-6 immunochemical measurement kit.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP27186289A JP2884628B2 (en) | 1989-10-20 | 1989-10-20 | Immunochemical assay for human IL-6 and kit therefor |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP27186289A JP2884628B2 (en) | 1989-10-20 | 1989-10-20 | Immunochemical assay for human IL-6 and kit therefor |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPH03135766A true JPH03135766A (en) | 1991-06-10 |
| JP2884628B2 JP2884628B2 (en) | 1999-04-19 |
Family
ID=17505931
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP27186289A Expired - Lifetime JP2884628B2 (en) | 1989-10-20 | 1989-10-20 | Immunochemical assay for human IL-6 and kit therefor |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JP2884628B2 (en) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103969438B (en) * | 2014-04-29 | 2016-03-30 | 北京普恩光德生物科技开发有限公司 | interleukin 6 detection kit |
-
1989
- 1989-10-20 JP JP27186289A patent/JP2884628B2/en not_active Expired - Lifetime
Also Published As
| Publication number | Publication date |
|---|---|
| JP2884628B2 (en) | 1999-04-19 |
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