JPH01269488A - Monoclonal antibody-producing cell line and monoclonal antibody therefrom - Google Patents
Monoclonal antibody-producing cell line and monoclonal antibody therefromInfo
- Publication number
- JPH01269488A JPH01269488A JP63097044A JP9704488A JPH01269488A JP H01269488 A JPH01269488 A JP H01269488A JP 63097044 A JP63097044 A JP 63097044A JP 9704488 A JP9704488 A JP 9704488A JP H01269488 A JPH01269488 A JP H01269488A
- Authority
- JP
- Japan
- Prior art keywords
- cell line
- monoclonal antibody
- trinitrobenzene
- producing cell
- derivative
- 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
- 210000000628 antibody-producing cell Anatomy 0.000 title claims abstract description 12
- UATJOMSPNYCXIX-UHFFFAOYSA-N Trinitrobenzene Chemical compound [O-][N+](=O)C1=CC([N+]([O-])=O)=CC([N+]([O-])=O)=C1 UATJOMSPNYCXIX-UHFFFAOYSA-N 0.000 claims abstract description 17
- 210000004027 cell Anatomy 0.000 claims abstract description 16
- 150000005186 trinitrobenzenes Chemical class 0.000 claims abstract description 13
- 230000002163 immunogen Effects 0.000 claims abstract description 11
- 206010035226 Plasma cell myeloma Diseases 0.000 claims abstract description 8
- 201000000050 myeloid neoplasm Diseases 0.000 claims abstract description 8
- 210000004989 spleen cell Anatomy 0.000 claims abstract description 8
- 108060003951 Immunoglobulin Proteins 0.000 claims abstract description 4
- 238000010367 cloning Methods 0.000 claims abstract description 4
- 239000012228 culture supernatant Substances 0.000 claims abstract description 4
- 230000004927 fusion Effects 0.000 claims abstract description 4
- 102000018358 immunoglobulin Human genes 0.000 claims abstract description 4
- 239000002671 adjuvant Substances 0.000 claims description 6
- 239000000126 substance Substances 0.000 claims description 6
- SPSSULHKWOKEEL-UHFFFAOYSA-N 2,4,6-trinitrotoluene Chemical compound CC1=C([N+]([O-])=O)C=C([N+]([O-])=O)C=C1[N+]([O-])=O SPSSULHKWOKEEL-UHFFFAOYSA-N 0.000 claims description 4
- 239000000015 trinitrotoluene Substances 0.000 claims description 4
- 241000237988 Patellidae Species 0.000 claims description 2
- 108060003552 hemocyanin Proteins 0.000 claims description 2
- 241000187479 Mycobacterium tuberculosis Species 0.000 claims 1
- 238000002360 preparation method Methods 0.000 abstract description 6
- 238000012216 screening Methods 0.000 abstract description 4
- 238000010790 dilution Methods 0.000 abstract description 2
- 239000012895 dilution Substances 0.000 abstract description 2
- 238000003018 immunoassay Methods 0.000 abstract description 2
- 241000534475 Fissurellidae Species 0.000 abstract 2
- 239000003153 chemical reaction reagent Substances 0.000 abstract 1
- 150000001875 compounds Chemical class 0.000 abstract 1
- 239000000463 material Substances 0.000 abstract 1
- FNUFHLAIHWESDA-UHFFFAOYSA-M sodium;2,3,4-trinitrobenzenesulfonate Chemical compound [Na+].[O-][N+](=O)C1=CC=C(S([O-])(=O)=O)C([N+]([O-])=O)=C1[N+]([O-])=O FNUFHLAIHWESDA-UHFFFAOYSA-M 0.000 abstract 1
- 239000000243 solution Substances 0.000 description 20
- 241000699670 Mus sp. Species 0.000 description 8
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 7
- 241000699666 Mus <mouse, genus> Species 0.000 description 7
- 239000000427 antigen Substances 0.000 description 7
- 102000036639 antigens Human genes 0.000 description 7
- 108091007433 antigens Proteins 0.000 description 7
- 229940098773 bovine serum albumin Drugs 0.000 description 5
- 238000002474 experimental method Methods 0.000 description 5
- 238000002965 ELISA Methods 0.000 description 4
- PXIPVTKHYLBLMZ-UHFFFAOYSA-N Sodium azide Chemical compound [Na+].[N-]=[N+]=[N-] PXIPVTKHYLBLMZ-UHFFFAOYSA-N 0.000 description 4
- 238000006243 chemical reaction Methods 0.000 description 4
- 210000004408 hybridoma Anatomy 0.000 description 4
- 102000004169 proteins and genes Human genes 0.000 description 4
- 108090000623 proteins and genes Proteins 0.000 description 4
- 238000003756 stirring Methods 0.000 description 4
- LYCAIKOWRPUZTN-UHFFFAOYSA-N ethylene glycol Natural products OCCO LYCAIKOWRPUZTN-UHFFFAOYSA-N 0.000 description 3
- 239000002360 explosive Substances 0.000 description 3
- 230000003053 immunization Effects 0.000 description 3
- 238000002649 immunization Methods 0.000 description 3
- 238000000034 method Methods 0.000 description 3
- -1 polyoxyethylene Polymers 0.000 description 3
- 230000035755 proliferation Effects 0.000 description 3
- 210000002966 serum Anatomy 0.000 description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 3
- NHJVRSWLHSJWIN-UHFFFAOYSA-N 2,4,6-trinitrobenzenesulfonic acid Chemical compound OS(=O)(=O)C1=C([N+]([O-])=O)C=C([N+]([O-])=O)C=C1[N+]([O-])=O NHJVRSWLHSJWIN-UHFFFAOYSA-N 0.000 description 2
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- 241001465754 Metazoa Species 0.000 description 2
- 229920003171 Poly (ethylene oxide) Polymers 0.000 description 2
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 description 2
- 210000000683 abdominal cavity Anatomy 0.000 description 2
- 230000015572 biosynthetic process Effects 0.000 description 2
- 210000004369 blood Anatomy 0.000 description 2
- 239000008280 blood Substances 0.000 description 2
- 238000011156 evaluation Methods 0.000 description 2
- 239000012091 fetal bovine serum Substances 0.000 description 2
- WGCNASOHLSPBMP-UHFFFAOYSA-N hydroxyacetaldehyde Natural products OCC=O WGCNASOHLSPBMP-UHFFFAOYSA-N 0.000 description 2
- 238000002347 injection Methods 0.000 description 2
- 239000007924 injection Substances 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
- 238000005259 measurement Methods 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 229910052757 nitrogen Inorganic materials 0.000 description 2
- 239000000758 substrate Substances 0.000 description 2
- 239000006228 supernatant Substances 0.000 description 2
- 239000004094 surface-active agent Substances 0.000 description 2
- 238000003786 synthesis reaction Methods 0.000 description 2
- JNYAEWCLZODPBN-JGWLITMVSA-N (2r,3r,4s)-2-[(1r)-1,2-dihydroxyethyl]oxolane-3,4-diol Chemical compound OC[C@@H](O)[C@H]1OC[C@H](O)[C@H]1O JNYAEWCLZODPBN-JGWLITMVSA-N 0.000 description 1
- GHCZTIFQWKKGSB-UHFFFAOYSA-N 2-hydroxypropane-1,2,3-tricarboxylic acid;phosphoric acid Chemical compound OP(O)(O)=O.OC(=O)CC(O)(C(O)=O)CC(O)=O GHCZTIFQWKKGSB-UHFFFAOYSA-N 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- 241000283707 Capra Species 0.000 description 1
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 description 1
- 101001051524 Parabuthus granulatus Toxin PgKL2 Proteins 0.000 description 1
- 241000220317 Rosa Species 0.000 description 1
- BUGBHKTXTAQXES-UHFFFAOYSA-N Selenium Chemical compound [Se] BUGBHKTXTAQXES-UHFFFAOYSA-N 0.000 description 1
- BZHJMEDXRYGGRV-UHFFFAOYSA-N Vinyl chloride Chemical compound ClC=C BZHJMEDXRYGGRV-UHFFFAOYSA-N 0.000 description 1
- 238000002835 absorbance Methods 0.000 description 1
- 238000001042 affinity chromatography Methods 0.000 description 1
- 230000000903 blocking effect Effects 0.000 description 1
- 238000004113 cell culture Methods 0.000 description 1
- 230000007910 cell fusion Effects 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 239000011248 coating agent Substances 0.000 description 1
- 238000000576 coating method Methods 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 238000007865 diluting Methods 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 239000000839 emulsion Substances 0.000 description 1
- 150000002148 esters Chemical class 0.000 description 1
- 230000008014 freezing Effects 0.000 description 1
- 238000007710 freezing Methods 0.000 description 1
- 125000000524 functional group Chemical group 0.000 description 1
- 238000005227 gel permeation chromatography Methods 0.000 description 1
- 239000003102 growth factor Substances 0.000 description 1
- 229910052739 hydrogen Inorganic materials 0.000 description 1
- 230000028993 immune response Effects 0.000 description 1
- 230000016784 immunoglobulin production Effects 0.000 description 1
- 239000003112 inhibitor Substances 0.000 description 1
- 230000005764 inhibitory process Effects 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 230000007774 longterm Effects 0.000 description 1
- 229920002521 macromolecule Polymers 0.000 description 1
- 102000013415 peroxidase activity proteins Human genes 0.000 description 1
- 108040007629 peroxidase activity proteins Proteins 0.000 description 1
- 239000008363 phosphate buffer Substances 0.000 description 1
- 229910052711 selenium Inorganic materials 0.000 description 1
- 239000011669 selenium Substances 0.000 description 1
- 238000011896 sensitive detection Methods 0.000 description 1
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 210000000952 spleen Anatomy 0.000 description 1
- 230000000638 stimulation Effects 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- 201000008827 tuberculosis Diseases 0.000 description 1
- 210000003462 vein Anatomy 0.000 description 1
- 210000001835 viscera Anatomy 0.000 description 1
Landscapes
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
Description
【発明の詳細な説明】
産業上の利用分野
本発明は新規なモノクローナル抗体産生細胞ラインに間
するものである。この細胞ラインより産生されるモノク
ローナル抗体は、大気中の極微量のトリニトロベンゼン
またはトリニトロベンゼンの誘導体を、免疫測定法によ
って高感度に検知するために有用である。DETAILED DESCRIPTION OF THE INVENTION Field of Industrial Application The present invention relates to a novel monoclonal antibody producing cell line. The monoclonal antibodies produced by this cell line are useful for highly sensitive detection of minute amounts of trinitrobenzene or trinitrobenzene derivatives in the air by immunoassay.
トリニトロベンゼンまたはトリニトロベンゼンの誘導体
は１、多くの場合爆発性の危険物である。Trinitrobenzene or derivatives of trinitrobenzene 1 are often explosive hazards.
従来の技術
トリニトロヘンセンまたはトリニトロベンゼンの誘導体
に対する抗体は、ジョン・ヴアンランドらによって報告
されている。 （例えは、ジョン・ヴアンラント アン
ト マーレン・テ゛ルカ、アナリティ力ル バイオケミ
ストリー、１２２．３８５〜３９３（１９８２）、
Ｊｏｎ Ｗａｎｎｌｕｎｄ ａｎｄ’Ｍａｒｌｅｎｅ’
Ｄｅ’１ｕｃａ’、Ａｎａｌｙｔｉｃａｌ Ｂｉｏｃｈ
ｅｍｉｓｔｒｙ、 １２２．３８５〜３９３（１９８２
））。Prior Art Antibodies against trinitrobenzene or derivatives of trinitrobenzene have been reported by John Vanland et al. (For example, see John Vanlandt and Maren Teelka, Analytical Power Biochemistry, 122.385-393 (1982),
Jon Wannlund and'Marlene'
De'1uca', Analytical Bioch
emistry, 122.385-393 (1982
)).
しかしながら、報告されている抗体は免疫したヤギの血
液を精製して得られるポリクローナル抗体である。However, the reported antibodies are polyclonal antibodies obtained by purifying the blood of immunized goats.
発明が解決しようとする課題
ポリクローナル抗体は、これを得るまでの全体、の製造
行程が簡単である長所を有する。反面、得られる抗体の
特性は被免疫動物の各個体に依存するため再現性のある
抗体を提供することが困難であった。また、ポリクロー
ナル抗体は抗原に対するアフィニティーが様々な抗体の
混合物であるため、平均としてのアフィニティーが低く
、高感度測定には用いることができなかった。Problems to be Solved by the Invention Polyclonal antibodies have the advantage that the entire manufacturing process to obtain them is simple. On the other hand, it has been difficult to provide reproducible antibodies because the characteristics of the obtained antibodies depend on each individual immunized animal. Furthermore, since polyclonal antibodies are a mixture of antibodies with various affinities for antigens, their average affinities are low, and they cannot be used for high-sensitivity measurements.
課題を解決するための手段
トリニトロベンゼンまたはトリニトロベンゼンの誘導体
と、スカシ貝由来のヘモシアニンの結合物質からなる免
疫原で感作されたマウスの脾臓細胞と、骨髄腫由来の細
胞ラインと暮融合後、り１コーニングして得られ、培ｔ
、ｈ清中に産生されるイムノグロブリンが、トリニトロ
ベンゼンまたはトリニトロベンゼンの誘導体に特異的に
結合するモノクローナル抗体産生細胞ラインを構成する
。Means for Solving the Problem After fusion of mouse spleen cells sensitized with an immunogen consisting of trinitrobenzene or a derivative of trinitrobenzene and a binding substance of keyhole keyhole hemocyanin with a myeloma-derived cell line, obtained by 1 corning, cultured
, the immunoglobulin produced in the serum constitutes a monoclonal antibody-producing cell line that specifically binds to trinitrobenzene or derivatives of trinitrobenzene.
作用
モノクローナル抗体は同一種の抗体であるため、ポリク
ローナル抗体に比較して高アフィニティーが得られる。Since working monoclonal antibodies are antibodies of the same species, higher affinity can be obtained compared to polyclonal antibodies.
またハイブリトーマ細胞ラインを培養する限り一定の特
性のモノクローナル抗体を提供することができる。Furthermore, as long as hybridoma cell lines are cultured, monoclonal antibodies with certain characteristics can be provided.
実施例
免疫グロブリンを産生ずる細胞はＩ！！、臓内に蓄積さ
れる。脾臓細胞はそれ自体増殖能力を持たないか骨髄腫
細胞ラインと融合することによって、増殖しながら抗体
を産生ずるハイブリトーマ細胞ラインを作製することが
できる。もっとも優れたアフィニティーを有する抗体を
産生じ、かつ高い増殖能力を有するハイブリドーマ細物
１個を選択（クローニング）し、これを培養すると高ア
フィニティーのモノクローナル抗体が産生される。Example Cells producing immunoglobulin are I! ! , is accumulated in the internal organs. Spleen cells themselves do not have the ability to proliferate, or can be fused with myeloma cell lines to create hybridoma cell lines that proliferate and produce antibodies. A single hybridoma that produces an antibody with the best affinity and has a high proliferation ability is selected (cloned) and cultured to produce a high-affinity monoclonal antibody.
本発明乙こおいて、目的抗原はトリニトロベンゼンまた
はトリニトロペンセンの誘導体である。In the present invention, the antigen of interest is trinitrobenzene or a derivative of trinitropenzene.
ここで、トリニトロベンゼンの誘導体とは、例えは以十
のような構造を持つ物質を指し、多くの場合、トリニト
ロベンゼンと同様爆発性の危険物である。Here, the term "trinitrobenzene derivative" refers to a substance having a structure as shown below, and in many cases, it is an explosive and dangerous substance like trinitrobenzene.
Ｘは、ＣＩ（３、Ｓ０３、ＣＯＯ，Ｓ Ｈ，Ｎ Ｈ２，
０１−１を含Ｃ官能基を示す。X is CI(3, S03, COO, S H, N H2,
01-1 represents a C-containing functional group.
これらの物質は何れも低分子であるため、蛋白質等の高
分子に結合さ伊ることにより、初めて免疫原として作用
する。その際の蛋白質は、被免疫動物と動物学上なるべ
く遠く分類される生物由来の物が、免疫刺激を高める上
で望ましい。この意味において、スカシ貝由来のへモジ
アニン（ＫＬＨ）は好適な蛋白質である。Since these substances are all low molecules, they only act as immunogens by binding to macromolecules such as proteins. In order to enhance immune stimulation, it is desirable that the protein used be derived from an organism that is zoologically classified as far away from the immunized animal as possible. In this sense, keyhole limpet-derived hemodyanin (KLH) is a suitable protein.
また、Ｂａｌｂ／Ｃ系統またはＡ／Ｊ系統のマウスは良
好な免疫反応を示すため、本発明に使用するのに適して
いる。Furthermore, Balb/C strain or A/J strain mice exhibit good immune responses and are therefore suitable for use in the present invention.
これらを踏まえて、発明者らは免疫したマウスの脾臓細
胞とマウス骨髄腫由来細胞ラインを融合−トリニトロベ
ンゼンまたはトリニトロベンゼンの誘導体に対して高い
アフィニ讐イーを有するモノらローナル抗体を産生ずる
細組ラインを作製することに成功したものである。Based on these findings, the inventors fused the spleen cells of immunized mice with a mouse myeloma-derived cell line - a cell line that produced monolonal antibodies with high affinity for trinitrobenzene or derivatives of trinitrobenzene. The line was successfully created.
本発明を実施するに当たり、融合細胞の評価、モノクロ
ーナル抗体の評価、トリニトロベンゼンまたはトリニト
ロベンゼンの誘導体の検出実験等は、すべて抗原を固定
したＥＬＩＳＡ法を用いて行った。In carrying out the present invention, evaluations of fused cells, evaluations of monoclonal antibodies, experiments for detecting trinitrobenzene or trinitrobenzene derivatives, etc. were all performed using ELISA methods in which antigens were immobilized.
またトリニトロベンゼンの誘導体としては、爆発性の危
険物であるトリニトロトルエン（以下、ＴＮＴ）を用い
た。Further, as a derivative of trinitrobenzene, trinitrotoluene (hereinafter referred to as TNT), which is an explosive and dangerous substance, was used.
まず、ＥＬＩＳＡ法の実験手順を説明する。また、以下
の文章中て、Ｐｈｏｓｐｈａｔｅ−Ｂｕｆｆｅｒｅｄ
５ａｌｉｎｅをＰｕｓと略す。First, the experimental procedure of the ELISA method will be explained. Also, in the following text, Phosphate-Buffered
5aline is abbreviated as Pus.
（Ａ）抗原のコーティング
ウシ血清アルブミン（ＢＳＡ） １分子あたりトリニト
ロベンゼンが帆５分子結合したコンジュゲート（ＴＮＰ
［＋ ５− ＢＳＡ）を０．０４％のアジ化ナトリウム
を含む、ＰＢＳ （ＰＢＳ−Ａｚ）で希釈してＢＳＡの
濃度として０．１ｍｇ／ｍＬの抗原溶液を調製した。(A) Antigen coating Bovine serum albumin (BSA) Conjugate (TNP) in which 5 molecules of trinitrobenzene are bound per molecule
[+5-BSA) was diluted with PBS (PBS-Az) containing 0.04% sodium azide to prepare an antigen solution with a BSA concentration of 0.1 mg/mL.
マイクロプレート（塩化ビニル製９６ウエルプレート、
ダイナチック ラボラトリーズ社製、ＤＹＮＡＴＥＣＨ
ＬＡＢＯＲＡＴＯＲＩＥＳ社製）に抗原溶液を１００μ
Ｌ／ウエル注入し、２０℃で１夜保存した。アスピレー
タで抗原溶液を除去した後、０．０５χの界面活性剤（
ポリオキシエチレングリコールソルビタンアルキルエス
テル類）および帆０牡のアジ化ナトリウムを含むＰＢＳ
（ＰＢＳ、ＡＺ−Ｔ２Ｏ）で３回洗浄し、アスピレー
タで残存するＰＢＳ、ＡＺ−Ｔ２Ｏを除去した。Microplate (96-well plate made of vinyl chloride,
Manufactured by Dynatic Laboratories, DYNATECH
Add 100μ of antigen solution to LABORATORIES).
L/well was injected and stored at 20°C overnight. After removing the antigen solution with an aspirator, add 0.05χ surfactant (
PBS containing polyoxyethylene glycol sorbitan alkyl esters) and sodium azide
The plate was washed three times with (PBS, AZ-T2O), and the remaining PBS and AZ-T2O were removed using an aspirator.
（Ｂ）ブロッキング
１χのＢＳＡを含むＰＢＳ−Ａｚ （ＢＳＡ−ＰＢＳ＝
Ａｚ）を２５０μ＋−／ウェル注入し、１時間室温で放
置した。その後、アスピレータでＢＳＡ−ＰＢＳ−Ａｚ
を除去した。即日に以降の実験を行わないときは、この
状態で、水で湿したろ紙と共に４℃で保存した。(B) PBS-Az containing BSA with blocking 1χ (BSA-PBS=
Az) was injected at 250μ+/well and left at room temperature for 1 hour. Then, use an aspirator to aspirate BSA-PBS-Az.
was removed. If subsequent experiments were not to be performed on the same day, the sample was stored in this state at 4°C along with a filter paper moistened with water.
（Ｃ）抗体の反応
ＢＳＡ−Ｐ［３Ｓ−Ａ２で適時希釈した抗体（血清、培
養上清、精製抗体等）１００μＬ／ウエルを注入した。(C) Antibody reaction 100 μL/well of antibodies (serum, culture supernatant, purified antibodies, etc.) appropriately diluted with BSA-P[3S-A2 was injected.
インヒビジョンの実験を行うときはインヒビタ溶液５０
μｍ、／ウェルを注入し、振とうしながら抗体溶液５０
７１１−／ウェルをさらに加えた。常温で３時間保存し
た後、アスピレータで抗体溶液を除去し、ＰＢＡ、ＡＺ
−Ｔ２Ｏて３回洗浄し、アスピレータで残存するＰＢＳ
、ＡＺ−７２０を除去した。When conducting inhibition experiments, inhibitor solution 50
Inject 50 µm/well of antibody solution while shaking.
An additional 711−/well was added. After storing at room temperature for 3 hours, remove the antibody solution with an aspirator and add PBA, AZ.
- Wash 3 times with T2O and aspirate remaining PBS.
, AZ-720 was removed.
（Ｄ）第２抗体の反応
０．２μｇ／ｍＬのＡ７ギ由来ペルオキシダーゼ標識抗
マウス１ｇＧ抗体（ＫＰＬ、Ｃａｔ、１４］８０６ ｌ
ｏｔ、 ＨＬＩＯ−５）を１％ＢＳＡのＰＢＳ溶液に溶
解したもの（第２抗体溶液）５０７１１−／ウェル注入
し、常温で３０分放置した。アスピレータで第２抗体溶
液を除去し、０．０５％の界面活性剤（ポリオキシエチ
レングリコールソルビタンアルギルエステル類）を含む
、ＰＢＳ （ＰＢＳ−Ｔ２Ｏ）で３回洗浄し、さらにア
スピレータで残存するＰＢＳ−Ｔ２Ｏを除去した。(D) Reaction of second antibody 0.2 μg/mL A7-derived peroxidase-labeled anti-mouse 1gG antibody (KPL, Cat, 14) 806 l
ot, HLIO-5) dissolved in a 1% BSA PBS solution (second antibody solution) was injected into 50711-/well and left at room temperature for 30 minutes. Remove the second antibody solution with an aspirator, wash three times with PBS (PBS-T2O) containing 0.05% surfactant (polyoxyethylene glycol sorbitan argyl esters), and remove the remaining PBS with an aspirator. - T2O was removed.
（Ｅ）基質の反応と停止
０−フェニレンシアミン（セレン検出用）、２０Ｂを１
０ｍＬのクエン酸−リン酸バッファー（１）Ｈ５）に溶
解し、使用直前に３０％過酸化水素水４／ＩＬを加えた
溶液（基質溶液）を１００μＬ／ウエル注入し、室温放
置した。１０分後、４Ｎ硫酸を２５μＬ／ウエル注入し
て反応を停止した。(E) Substrate reaction and termination 0-phenylenecyamine (for selenium detection), 20B in 1
A solution (substrate solution) dissolved in 0 mL of citric acid-phosphate buffer (1) H5) and added with 30% hydrogen peroxide solution (4/IL) immediately before use was injected at 100 μL/well and left at room temperature. After 10 minutes, 25 μL/well of 4N sulfuric acid was injected to stop the reaction.
（Ｆ）測定
Ｔｉｔｅｒｔｅｋ Ｍｕｌｔｉｓｋａｎ ＭＣを用いて
４９２ｎｍ以上の吸光度を測定した。通常第１列は純水
を注入して参照値とし、適時（Ｃ）項のみを省いたブラ
ンク値を使用した。(F) Measurement Absorbance at 492 nm or higher was measured using Titertek Multiskan MC. Normally, pure water was injected into the first column to serve as a reference value, and a blank value in which only the term (C) was omitted was used at appropriate times.
以下、本発明のモノクローナル抗体産生細胞ラインを作
製する実験方法を記載する。Below, an experimental method for producing the monoclonal antibody-producing cell line of the present invention will be described.
１）免疫原の作製
Ｊｏｎ Ｗａｎｎｌｕｎｄらの報告（前出）に従ッテ、
免疫原の合成を行なった。以降の手順をステップ毎に記
す。1) Preparation of immunogen According to the report of Jon Wannlund et al. (cited above),
The immunogen was synthesized. The subsequent procedure will be described step by step.
（Ａ）ＫＬＩＩ溶液の作成
ＫＬＨ（Ｃａｌｂｉｏｃｈｅｍ、 ３７４８０５）
１５０ｍ８を、Ｏ，１Ｍ Ｎａ１ｌＣＯ３（ＦＩＨ８
，４） ２０ｍ１中に分散し、３０ｍ ｉ ｎ攪はんを
行った後、１９ 、０００ｒｐｍで３０ｍ団遠心分離を
行い、不溶成分を除去した。(A) Creation of KLII solution KLH (Calbiochem, 374805)
150m8 of O, 1M Na1lCO3 (FIH8
, 4) After dispersing in 20 ml and stirring for 30 min, centrifugation was performed for 30 m at 19,000 rpm to remove insoluble components.
（Ｂ）ＴＮＢＳ溶液の作成
トリニトロベンゼンスルホン酸ナトリウムニ水和物（以
下ＴＮＢＳ） １４１Ｔ１ｇを、Ｏ，］ＭＮａＨＣ０３
５ｍｌに溶解した。(B) Preparation of TNBS solution 141T1g of sodium trinitrobenzenesulfonate dihydrate (hereinafter referred to as TNBS), O, ]MNaHC03
It was dissolved in 5 ml.
（Ｃ） ＴＮＰ−Ｋｕｌの合成
ＴＮ［３Ｓ溶液とＫＬＨ溶液を混合【八 撹はんしなが
ら３７℃でインキュベートを行なった。２時間後ゲルク
ロマトグラフィーで低分子を除去し、３２．５ｍＬのＴ
ＮＰ−に１，１１溶液を得た。(C) Synthesis of TNP-Kul TN[3S solution and KLH solution were mixed [8] and incubated at 37°C with stirring. After 2 hours, remove low molecules by gel chromatography and add 32.5 mL of T.
A 1,11 solution was obtained in NP-.
（Ｄ）アジュバントコーマルジョンの調製合成によって
得たＴＮＰ−ｉＨをＰＢＳで希釈して、Ｉｍｇ／ｍｌ溶
液３■１．を得た。アジュバント（Ａｄｊｕｖａｎｔ、
Ｃｏｍｐｌｅｔ、ｅ Ｆｒｅｕｎｄヒト結核死菌含、
和光、Ｈ３７Ｒｖ）をよく攪はんしながら３ｍＬ取り、
ホモジナイザで攪はんしながら（１０，００叶ｐｍ）
ＴＮＴ−ＫＬｔｌ溶液３溶液３ｍ回に分けて加えた。十
分ｔこ乳化し、小量を水の上に落としても広がらなくな
ったのを確認した。(D) Preparation of adjuvant comulsion The TNP-iH obtained by synthesis was diluted with PBS to create an Img/ml solution 3.1. I got it. Adjuvant (Adjuvant)
Complete, including killed human tuberculosis bacterium e Freund,
Take 3 mL of Wako, H37Rv) while stirring well,
While stirring with a homogenizer (10,00 pm)
Three TNT-KLtl solutions were added in 3 m portions. It was confirmed that it was sufficiently emulsified and did not spread even when a small amount was dropped onto water.
２）ハイブリドーマ細胞の作製
（Ａ）マウスの免疫
生後約８週のマウス（Ｂａｌｂ／ｃ） ２０匹の腹腔に
アジュバントエマルジョンを２００μＬずつ注射した。2) Preparation of hybridoma cells (A) Immunization of mice 200 μL of the adjuvant emulsion was injected into the abdominal cavity of 20 mice (Balb/c) about 8 weeks old.
（１３）抗体産生のチエツク
免疫注射後、２８日を経過したマウスについて、眼静脈
より５０〜１００μＬの血液を遠心管に採取した。(13) Check for antibody production From mice 28 days after the immunization injection, 50 to 100 μL of blood was collected from the eye vein into a centrifuge tube.
血清を遠心分離し、ＥＬＩＳＡ法によるスクリーニング
を行ったところ、全てのマウスについて抗トリニトロヘ
ンセン抗体の産生が確認された。When the serum was centrifuged and screened by ELISA, production of anti-trinitrohensen antibodies was confirmed in all mice.
（Ｃ）マウスのブースト
（Ｂ）項のスクリーニングで特にタイターの高かった２
匹のマウスについてマウスの脾臓を肥大させるためにブ
ースト（弱い免疫原の注射）を行った。免疫原としてＴ
ＮＰ−ＫＩＨをＰＢＳで希釈して得た０、５ｍ３／ｍＬ
溶液を、アジュバントを加えずにそのまま用いた。免疫
後６週を経過した時点でこの免疫原１００７ノＬをマウ
スの腹腔に注射した。(C) Mouse boost (B) 2 with particularly high titer in screening
A boost (injection of a weak immunogen) was given to two mice to enlarge their spleens. T as an immunogen
0.5 m3/mL obtained by diluting NP-KIH with PBS
The solution was used as is without addition of adjuvant. Six weeks after immunization, this immunogen 1007noL was injected into the abdominal cavity of the mouse.
（’Ｄ ）細胞融合
ブースト後３日を経過したマウスの脾臓細胞を摘出し、
平均分子ｆｆｉ＋、５００のポリエチレングリコールを
用いた常法ζこより、マウス骨髄腫由来細胞ライン（Ｐ
３Ｘ６３−Ａｇ８．６５３） ？＝融合シタ。フィータ
ー（成長因子を供給する細胞）として同しマウスの脾臓
細胞を用い、９６ウ工ルプレート５枚の上で１０％のウ
シ胎児血清を含むＨＡＴ培地で培養した。１週間後、１
０χのウシ胎児血清を含むＨＴ培地と交換した。('D) Spleen cells of mice 3 days after cell fusion boost were removed,
A mouse myeloma-derived cell line (P
3X63-Ag8.653)? = fusion shita. Spleen cells from the same mouse were used as feeders (cells that supply growth factors) and cultured in HAT medium containing 10% fetal bovine serum on five 96-well plates. 1 week later, 1
The medium was replaced with HT medium containing 0x fetal bovine serum.
３）モノクローナル抗体の作製
（Ａ）クローニング
ＥＬＩＳＡ法によるスクリーニングを行へ タイターの
高いものから上位５ウエルを選択した。ウェルあたり１
ケの細胞が含まれる濃度に希釈（限界希釈）し、９６ウ
エルのマイクロプレート１０枚に分注した。3) Preparation of monoclonal antibodies (A) Cloning Proceed to screening by ELISA The top 5 wells with the highest titers were selected. 1 per well
The mixture was diluted (limiting dilution) to a concentration containing 50 cells, and dispensed into 10 96-well microplates.
フィーダーとして生後４週のマウス（Ｂａｌｂ／ｃ）の
胸腺細胞を用いて初期増殖を促した。プレートのサイズ
を上げながら培養を進め、適時上清ｔこつぃてＥＬＩＳ
Ａによるスクリーニングを繰り返し、ＴＮＴに対して高
いタイターを示し、かつ良好な増殖を示している細胞ラ
インを最終的に選別し、２０’Ｏｍｌ中で５Ｘ １０５
ケ／ｍｌの濃度に至るまで培養を進めた。Initial proliferation was promoted using 4-week-old mouse (Balb/c) thymocytes as feeders. Proceed with the culture while increasing the plate size, and remove the supernatant from ELIS at appropriate times.
The screening by A was repeated and the cell lines showing high titer for TNT and good proliferation were finally selected and incubated at 5X 105 in 20'Oml.
The culture was continued until the concentration reached 100 mg/ml.
（Ｂ）最終的に選別された細胞ラインは上清を遠心分離
し、５Ｘ １０６／ｍＬ（７）濃度テＦＣ５： ＤＭＳ
Ｏ＝９ ： １（７） 溶液１ｍ１．に浮遊させ、−８
０℃で凍結した後、液体窒素内に移して長期保存状態に
した。(B) The final sorted cell line was centrifuged with supernatant and diluted to 5X 106/mL (7) concentration TeFC5: DMS.
O=9:1(7) solution 1ml1. -8
After freezing at 0°C, it was transferred to liquid nitrogen for long-term storage.
（Ｃ）ファルマシア製Ｐｒｏｔｅｉｎ Ａ−５ｅｐｈａ
ｒｏｓｅ ＣＬ−４８を用いたアフィニティークロマト
グラフィにより細胞培養上清からモノクローナル抗体を
精製した。このモノクローナル抗体はＳＤＳポリアクリ
ルアミドゲル電気泳動により、゛標ｉ蛋白との比較から
、精製抗探は′分子量約５０’、０００のＨ鎖と約２０
、０００のＬ鎖からなるＩｇＧであることを確認した
。(C) Protein A-5epha manufactured by Pharmacia
Monoclonal antibodies were purified from cell culture supernatants by affinity chromatography using rose CL-48. This monoclonal antibody was analyzed by SDS polyacrylamide gel electrophoresis and was found to have a molecular weight of approximately 50,000 H chains and a molecular weight of approximately 20,000.
, 000 L chain was confirmed to be IgG.
発明の効果
本発明により、トリニトロベンゼンまたはトリニトロベ
ンゼンの誘導体に対して高いアフィニティーを有するモ
ノクロごナル抗体を提供することが可能になった。Effects of the Invention The present invention has made it possible to provide a monoclonal antibody having high affinity for trinitrobenzene or a derivative of trinitrobenzene.
Claims (6)
誘導体と、スカシ貝由来のヘモシアニンの結合物質から
なる免疫原で感作されたマウスの脾臓細胞と、骨髄腫由
来の細胞ラインとを融合後、クローニングして得られ、
培養上清中に産生されるイムノグロブリンが、トリニト
ロベンゼンまたはトリニトロベンゼンの誘導体に特異的
に結合することを特徴とするモノクローナル抗体産生細
胞ライン。(1) Obtained by fusion of mouse spleen cells sensitized with an immunogen consisting of trinitrobenzene or a derivative of trinitrobenzene and a binding substance of hemocyanin derived from keyhole limpet and a myeloma-derived cell line, followed by cloning. is,
A monoclonal antibody-producing cell line characterized in that immunoglobulin produced in the culture supernatant specifically binds to trinitrobenzene or a derivative of trinitrobenzene.
またはＡ／Ｊ系統であることを特徴とする特許請求の範
囲第１項記載のモノクローナル抗体産生細胞ライン。(2) The monoclonal antibody-producing cell line according to claim 1, wherein the mouse sensitized with the immunogen is of the Balb/c strain or the A/J strain.
６５３であることを特徴とする特許請求の範囲第１項記
載のモノクローナル抗体産生細胞ライン。(3) Myeloma-derived cell line is P3X63-Ag8.
653, the monoclonal antibody-producing cell line according to claim 1.
アジユバントからなることを特徴とする特許請求の範囲
第１項記載のモノクローナル抗体産生細胞ライン。(4) The monoclonal antibody-producing cell line according to claim 1, wherein the immunogen consists of Freund's complete adjuvant containing killed Mycobacterium tuberculosis.
産生細胞ラインより産生される、トリニトロベンゼンま
たはトリニトロベンゼンの誘導体に特異的に結合するモ
ノクローナル抗体。(5) A monoclonal antibody that specifically binds to trinitrobenzene or a derivative of trinitrobenzene, which is produced by the monoclonal antibody-producing cell line according to claim 1.
産生細胞ラインより産生される、トリニトロトルエンに
特異的に結合するモノクローナル抗体。(6) A monoclonal antibody that specifically binds to trinitrotoluene and is produced by the monoclonal antibody-producing cell line according to claim 1.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP63097044A JPH01269488A (en) | 1988-04-20 | 1988-04-20 | Monoclonal antibody-producing cell line and monoclonal antibody therefrom |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP63097044A JPH01269488A (en) | 1988-04-20 | 1988-04-20 | Monoclonal antibody-producing cell line and monoclonal antibody therefrom |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| JPH01269488A true JPH01269488A (en) | 1989-10-26 |
Family
ID=14181609
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP63097044A Pending JPH01269488A (en) | 1988-04-20 | 1988-04-20 | Monoclonal antibody-producing cell line and monoclonal antibody therefrom |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPH01269488A (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5484709A (en) * | 1993-09-10 | 1996-01-16 | Ensys, Inc. | Immunoassay method for detecting an immunologically non-remarkable compound, its components and a kit for use in performing the same |
| WO1996013521A1 (en) * | 1993-09-10 | 1996-05-09 | Ensys, Inc. | An immunoassay for detecting immunologically non-remarkable compounds, its components and an immunoassay kit |
| WO2000043774A3 (en) * | 1999-01-25 | 2001-03-01 | Yissum Res Dev Co | Detection of small molecules by use of a piezoelectric sensor |
-
1988
- 1988-04-20 JP JP63097044A patent/JPH01269488A/en active Pending
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5484709A (en) * | 1993-09-10 | 1996-01-16 | Ensys, Inc. | Immunoassay method for detecting an immunologically non-remarkable compound, its components and a kit for use in performing the same |
| WO1996013521A1 (en) * | 1993-09-10 | 1996-05-09 | Ensys, Inc. | An immunoassay for detecting immunologically non-remarkable compounds, its components and an immunoassay kit |
| WO2000043774A3 (en) * | 1999-01-25 | 2001-03-01 | Yissum Res Dev Co | Detection of small molecules by use of a piezoelectric sensor |
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