JPH01269485A - Cell line capable of producing monoclonal antibody and monoclonal antibody - Google Patents
Cell line capable of producing monoclonal antibody and monoclonal antibodyInfo
- Publication number
- JPH01269485A JPH01269485A JP63097041A JP9704188A JPH01269485A JP H01269485 A JPH01269485 A JP H01269485A JP 63097041 A JP63097041 A JP 63097041A JP 9704188 A JP9704188 A JP 9704188A JP H01269485 A JPH01269485 A JP H01269485A
- Authority
- JP
- Japan
- Prior art keywords
- cell line
- trinitrobenzene
- monoclonal antibody
- derivative
- producing
- 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
- 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
- 150000005186 trinitrobenzenes Chemical class 0.000 claims abstract description 15
- 230000002163 immunogen Effects 0.000 claims abstract description 10
- 206010035226 Plasma cell myeloma Diseases 0.000 claims abstract description 8
- 201000000050 myeloid neoplasm Diseases 0.000 claims abstract description 8
- 239000000126 substance Substances 0.000 claims abstract description 7
- 239000012228 culture supernatant Substances 0.000 claims abstract description 5
- 108060003951 Immunoglobulin Proteins 0.000 claims abstract description 4
- 238000010367 cloning Methods 0.000 claims abstract description 4
- 102000018358 immunoglobulin Human genes 0.000 claims abstract description 4
- 210000004027 cell Anatomy 0.000 claims description 23
- 210000000628 antibody-producing cell Anatomy 0.000 claims description 9
- 239000002671 adjuvant Substances 0.000 claims description 5
- 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
- 241000271566 Aves Species 0.000 claims description 4
- 108010058846 Ovalbumin Proteins 0.000 claims description 4
- 229940092253 ovalbumin Drugs 0.000 claims description 4
- 239000000015 trinitrotoluene Substances 0.000 claims description 4
- 230000004927 fusion Effects 0.000 claims description 2
- 241000187479 Mycobacterium tuberculosis Species 0.000 claims 1
- 210000004989 spleen cell Anatomy 0.000 claims 1
- 238000002360 preparation method Methods 0.000 abstract description 7
- 125000000524 functional group Chemical group 0.000 abstract description 2
- 238000003018 immunoassay Methods 0.000 abstract description 2
- QCVGEOXPDFCNHA-UHFFFAOYSA-N 5,5-dimethyl-2,4-dioxo-1,3-oxazolidine-3-carboxamide Chemical compound CC1(C)OC(=O)N(C(N)=O)C1=O QCVGEOXPDFCNHA-UHFFFAOYSA-N 0.000 abstract 2
- 102000009027 Albumins Human genes 0.000 abstract 2
- 108010088751 Albumins Proteins 0.000 abstract 2
- 102000002322 Egg Proteins Human genes 0.000 abstract 2
- 108010000912 Egg Proteins Proteins 0.000 abstract 2
- 210000000991 chicken egg Anatomy 0.000 abstract 2
- 235000014103 egg white Nutrition 0.000 abstract 2
- 230000003393 splenic effect Effects 0.000 abstract 2
- 239000003153 chemical reaction reagent Substances 0.000 abstract 1
- 238000003113 dilution method Methods 0.000 abstract 1
- 239000000463 material Substances 0.000 abstract 1
- 239000000243 solution Substances 0.000 description 20
- 241000699670 Mus sp. Species 0.000 description 9
- 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
- 238000002474 experimental method Methods 0.000 description 5
- 238000002965 ELISA Methods 0.000 description 4
- 238000006243 chemical reaction Methods 0.000 description 4
- 210000004408 hybridoma Anatomy 0.000 description 4
- 238000000034 method Methods 0.000 description 4
- 238000003756 stirring Methods 0.000 description 4
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-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
- -1 polyoxyethylene Polymers 0.000 description 3
- 102000004169 proteins and genes Human genes 0.000 description 3
- 108090000623 proteins and genes Proteins 0.000 description 3
- 238000012216 screening Methods 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
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 2
- 241000283707 Capra Species 0.000 description 2
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 description 2
- 229920003171 Poly (ethylene oxide) Polymers 0.000 description 2
- PXIPVTKHYLBLMZ-UHFFFAOYSA-N Sodium azide Chemical compound [Na+].[N-]=[N+]=[N-] PXIPVTKHYLBLMZ-UHFFFAOYSA-N 0.000 description 2
- UIIMBOGNXHQVGW-UHFFFAOYSA-M Sodium bicarbonate Chemical compound [Na+].OC([O-])=O UIIMBOGNXHQVGW-UHFFFAOYSA-M 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
- 238000001514 detection method Methods 0.000 description 2
- 239000000839 emulsion Substances 0.000 description 2
- LYCAIKOWRPUZTN-UHFFFAOYSA-N ethylene glycol Natural products OCCO LYCAIKOWRPUZTN-UHFFFAOYSA-N 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
- 230000028993 immune response Effects 0.000 description 2
- 229940072221 immunoglobulins Drugs 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
- 230000035755 proliferation Effects 0.000 description 2
- 210000002966 serum Anatomy 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
- ODIGIKRIUKFKHP-UHFFFAOYSA-N (n-propan-2-yloxycarbonylanilino) acetate Chemical compound CC(C)OC(=O)N(OC(C)=O)C1=CC=CC=C1 ODIGIKRIUKFKHP-UHFFFAOYSA-N 0.000 description 1
- GEYOCULIXLDCMW-UHFFFAOYSA-N 1,2-phenylenediamine Chemical compound NC1=CC=CC=C1N GEYOCULIXLDCMW-UHFFFAOYSA-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
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 102000003992 Peroxidases Human genes 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
- 239000002253 acid Substances 0.000 description 1
- 238000001042 affinity chromatography Methods 0.000 description 1
- BWZOPYPOZJBVLQ-UHFFFAOYSA-K aluminium glycinate Chemical compound O[Al+]O.NCC([O-])=O BWZOPYPOZJBVLQ-UHFFFAOYSA-K 0.000 description 1
- 239000012491 analyte Substances 0.000 description 1
- 230000000903 blocking effect Effects 0.000 description 1
- 239000012888 bovine serum Substances 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
- 238000007865 diluting Methods 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 230000008014 freezing Effects 0.000 description 1
- 238000007710 freezing Methods 0.000 description 1
- 238000005227 gel permeation chromatography Methods 0.000 description 1
- 239000003102 growth factor Substances 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
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 210000000496 pancreas Anatomy 0.000 description 1
- 108040007629 peroxidase activity proteins Proteins 0.000 description 1
- 239000008363 phosphate buffer Substances 0.000 description 1
- 239000008055 phosphate buffer solution Substances 0.000 description 1
- 238000002264 polyacrylamide gel electrophoresis Methods 0.000 description 1
- 230000002062 proliferating effect Effects 0.000 description 1
- 229910052711 selenium Inorganic materials 0.000 description 1
- 239000011669 selenium Substances 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- 229910052708 sodium Inorganic materials 0.000 description 1
- 239000011734 sodium Substances 0.000 description 1
- 229910000030 sodium bicarbonate Inorganic materials 0.000 description 1
- 235000017557 sodium bicarbonate Nutrition 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- 210000001541 thymus gland Anatomy 0.000 description 1
- 201000008827 tuberculosis Diseases 0.000 description 1
- 210000003462 vein 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関
するものである。この細胞ラインより産生きれるモノク
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ζこ有用である。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 detecting minute amounts of trinitrobenzene or trinitrobenzene derivatives in the air with high sensitivity 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、多くの場合爆発性の危険物である。Trinitrobenzene or derivatives of trinitrobenzene are often explosive hazards.
従来の技術
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
ド マーレン・チル力、アナリティカル バイオケミス
トリー、１２２．３８５〜３９３（１９８２）、 Ｊ
ｏｎ Ｗａｎｎｌｕｎｄ ａｎｄ Ｍａｒｌｅｎｅ Ｄ
ｅｌｕｃａ、Ａｎａｌｙｔ、１ｃａｌ Ｂｉｏｃｈｅｍ
ｉｓｔｒｙ、 １２２．３８５〜３９３（１９８２））
。Prior Art Antibodies against trinitrobenzene or derivatives of trinitrobenzene have been reported by John Vanland et al. (See, for example, John Vanlandt and Maren Chill, Analytical Biochemistry, 122.385-393 (1982), J
on Wannlund and Marlene D
eluca, Analyt, 1cal Biochem
istry, 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. In addition, polyclonal antibodies are a mixture of antibodies with various affinities for antigens, so their average affinity is low;
It could not be used for high-sensitivity measurements.
課題を解決するだめの手段
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、トリ卵白アルブミンとの結合物質からなる免疫原で
感作されたマウスの膵臓細胞と、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モ
ノクローナル抗体産生細胞ラインを構成する。Another way to solve the problem: Cloning after fusion of mouse pancreatic cells sensitized with an immunogen consisting of trinitrobenzene or a derivative of trinitrobenzene and a substance that binds to avian ovalbumin and a myeloma-derived cell line. The immunoglobulins obtained and produced in the culture supernatant constitute a monoclonal antibody-producing cell line that specifically binds to trinitrobenzene or a derivative 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.
またハイブリド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, monoclonal antibodies with certain characteristics can be provided as long as hybridoma cell lines are cultured.
実施例 免疫グロブリンを産生ずる細胞は膵臓内に蓄積される。Example Cells that produce immunoglobulins accumulate within the pancreas.
膵臓細胞はそれ自体増殖能力を持たないが骨髄腫細胞ラ
インと融合することによって、増殖しながら抗体を産生
ずるハイブリドーマ細胞ラインを作製することができる
。もつ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胞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。 −本発
明において、目的抗原はトリニトロベンゼンまたはトリ
ニトロベンゼンの誘導体である。Pancreatic cells themselves do not have the ability to proliferate, but by fusing them with myeloma cell lines, it is possible to create hybridoma cell lines that proliferate and produce antibodies. When one hybridoma cell that produces an antibody with excellent affinity and has a high proliferative ability is selected (cloned) and cultured, a monoclonal antibody with high affinity is produced. - In the present invention, the target antigen is trinitrobenzene or a derivative of trinitrobenzene.
ここで、トリニトロベンゼンの誘導体とは、例えは以下
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 derivative of trinitrobenzene refers to a substance having the structure shown below, and in many cases, it is a dangerous explosive substance like trinitrobenzene.
Ｘは、ＣＨ３、Ｓ０３、Ｃｏｏ、 ＳＨ，ＮＨ２，０
Ｈを含む官能基を示す。X is CH3, S03, Coo, SH, NH2,0
Indicates a functional group containing H.
これらの物質は何れも低分子であるため、蛋白質等の高
分子に結合させることにより、初めて免疫原として作用
する。トリ卵白アルブミンは比較的大量に精製タンパク
質が得られ、かつ高い免疫応答が期待できる。Since these substances are all low molecules, they act as immunogens only by binding to macromolecules such as proteins. Avian ovalbumin can be purified in relatively large quantities and is expected to produce a high immune response.
また、Ｂａ ｌ ｂ／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, Bal b/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膵臓細
胞とマウス骨髄腫由来細胞ラインを融合し、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ーナル抗体を産生ず
る細胞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 created a cell line that produces monoclonal antibodies with high affinity for trinitrobenzene or trinitrobenzene derivatives by fusing pancreatic cells from immunized mice with a mouse myeloma-derived cell line. It was extremely successful.
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ＥＬ Ｉ ＳＡ法を用いて行った。In carrying out the present invention, evaluation of fused cells, evaluation of monoclonal antibodies, detection experiments of trinitrobenzene or trinitrobenzene derivatives, etc. were all performed using ELISA method 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
の文中で、リン酸緩衝液すリン（円１ｏｓｐｈａｔ、ｅ
−Ｂｃ＋４ｆｅｒｃｄ ５ａｌｉｎｅ）をＰＢＳと略す
。First, the experimental procedure of the ELISA method will be explained. In addition, in the following text, phosphate buffer solution (1 osphat, e
-Bc+4fercd5aline) is abbreviated as PBS.
（Ａ）抗原のコーチインク
ウシ血清アルアミン（ＢＳＡ） １分子あたりトリニト
ロベンゼンが０．５分子結合したコンジュゲート（ＴＮ
Ｐ［１、５−ＢＳＡ）を０．０４γのアシ化ナトリウム
を含むＰＢＳ （ＰＢＳ−Ａｚ）て希釈して、ＢＳＡの
濃度として０．１ｍｇ／ｍＬの抗原溶液を調製した。(A) Antigen coachin bovine serum alamine (BSA) Conjugate (TN) with 0.5 molecules of trinitrobenzene bound per molecule
P[1,5-BSA) was diluted with PBS containing 0.04γ sodium acylate (PBS-Az) 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、ＤＹＮＡＴＥＣｌ
ｌｌ、ΔＢＯＲＡＴＯＲＩ ＥＳ社製）に抗原溶液を１
００ツノ１．／ウェル注入し、２０℃で１夜保存した。Microplate (96-well plate made of vinyl chloride,
Manufactured by Dynatic Laboratories, DYNATECl
11 of the antigen solution (manufactured by ΔBORATORI ES)
00 horn 1. / well and stored at 20°C overnight.
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クリコールソルビタンア
ルキルエステル類）および０．０４′Ｉｌ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−Ｔ２
Ｏを除去した。After removing the antigen solution with an aspirator, it was washed three times with PBS (PBS, AZ-T2O) containing 0.05χ of surfactant (polyoxyethylene glycol sorbitan alkyl esters) and 0.04'Il of sodium azide. Wash and aspirate remaining PBS, AZ-T2
O was removed.
（Ｂ）ブロッキング
］％ＢＳＡを含むＰＢＳ−Ａｚ （ＢＳＡ−ＰＢＳ−Ａ
ｚ）を２５０７１１−戸ンエル注入し、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) Blocking] PBS-Az containing % BSA (BSA-PBS-A
z) was injected into 250,711 units 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−ＰＢＳ−Ａｚで適時希釈した抗体（血清、培養
」−清、精製抗体等）１００μｍ、／ウェルを注入した
。インヒビジョンの実験を行うときはインヒビタ溶液５
０〃Ｌ／ウエルを注入し、振とうしながら抗体溶液５０
μＬ／ウエルをさらに加えた。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−Ｔ
２Ｏで３回洗浄し、アスピレータで残存するＰＢＳ、Ａ
Ｚ−Ｔ２Ｏを除去した。(C) Antibody reaction Antibodies (serum, culture supernatant, purified antibodies, etc.) appropriately diluted with BSA-PBS-Az were injected at 100 μm/well. When conducting inhibition experiments, inhibitor solution 5
Inject 0〃L/well and add 50mL of antibody solution while shaking.
Additional μL/well was added. After storing at room temperature for 3 hours, remove the antibody solution with an aspirator and add PBA, AZ-T.
Wash 3 times with 2O and aspirate remaining PBS, A
Z-T2O was removed.
（Ｄ）第２抗体の反応
０．２μｇ／ｍＬのヤギ由来ペルオキシダーゼ標識抗マ
ウスＩｇＧ抗体（ＫＰＩ、Ｃａｔ、Ｉ４１８０６ ｌｏ
ｔ、 Ｉ−ＩＬＩＯ−５）を１χＢＳＡのＰＢＳ溶液に
溶解した。もの（第２抗体溶液）５０７ｚＬ／ウエル注
入し、常温で３０分放置した。アスピレ−タで第２抗体
溶液を除去し、０ 、０５Ｘの界面活性剤（ポリオキシ
エチレンクリコールソルビタンアルキルエステル類）を
含む、ＰＢＳ （ＰＢＳ−Ｔ２Ｏ） テ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 goat-derived peroxidase-labeled anti-mouse IgG antibody (KPI, Cat, I41806 lo
t, I-ILIO-5) was dissolved in a PBS solution of 1χBSA. 507 zL/well of the second antibody solution was injected and left at room temperature for 30 minutes. Remove the second antibody solution with an aspirator, wash three times with PBS (PBS-T2O) containing 0.05X surfactant (polyoxyethylene glycol sorbitan alkyl esters), and remove the remaining antibody with an aspirator. PBS-T2O was removed.
（Ｅ）基質の反応と停止
０−フェニレンジアミン（セレン検出用） ２０ｍｇを
］ＯｍＬのクエン酸−リン酸バッファー（ｐＨ５）に溶
解し、使用直前に３０χ過酸化水素水４μＬを加えた溶
液（基質溶液）を１００μｍ、／ウェル注入し、室温放
置した。１０分後、４Ｎｆｆｆｉ酸を２５μＬ／ウエル
注入して反応を停止した。(E) Substrate reaction and termination 20 mg of 0-phenylenediamine (for selenium detection) was dissolved in] OmL of citric acid-phosphate buffer (pH 5), and immediately before use, 4 μL of 30× hydrogen peroxide was added to the solution (substrate Solution) was injected at 100 μm/well and left at room temperature. After 10 minutes, 25 μL/well of 4Nfffi acid was injected to stop the reaction.
（Ｆ）測定
Ｔｉｔ、ｅｒｔｅｋ 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 Tit, absorbance at 492 nm or higher was measured using ertek 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）に従フて、
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 by Jon Wannlund et al. (cited above),
The immunogen was synthesized. The subsequent procedure will be described step by step.
（Ａ）トリ卵白アルブミン（ＣＥＡ）溶液の作成
ＣＥＡ（ＳＩＧＭＡ！Ｊ Ａ３５０３） ５０ｍｇヲ、
Ｏ，１Ｍ ＮａＨＣＯ３（ｐＨ８，４） ２０ｍ１中に
分散し、３０ｍ１ｎ攪はんを行った後、１９．０００ｒ
ｐｍで３０ｍ１ｎ遠心分離を行い、不溶成分を除去した
。(A) Preparation of avian ovalbumin (CEA) solution CEA (SIGMA!J A3503) 50mg,
After dispersing in 20ml of O, 1M NaHCO3 (pH 8,4) and stirring for 30ml, 19.000r
Centrifugation was performed at 30 ml at pm 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） １４ｍｇを、Ｏ，１ＭＮａＨＣ０３５ｍ
ｌニ溶解した。(B) Preparation of TNBS solution 14 mg of sodium trinitrobenzenesulfonate dihydrate (hereinafter referred to as TNBS) was mixed with O, 1M NaHC035m
1 was dissolved.
（Ｃ） ＴＮＰ−ＣＥＡの合成
ＴＮ ＢＳ溶液とＣＥＡ溶液を混合し、攪はんしながら
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−ＣＥＡ溶液を得た。(C) Synthesis of TNP-CEA TN BS solution and CEA solution were mixed and incubated at 37° C. with stirring. After 2 hours, low molecules were removed by gel chromatography and 32.5 ml of T
A NP-CEA solution was obtained.
（Ｄ）アジュバントエマルジョンの調製合成ここよって
得たＴＮＰ−ＣＥＡをＰＢＳて希釈して、１ｍｇ／畦温
溶液３畦得た。アジュバント（Ａｄｊｕｖａｎｔ、 Ｃ
ｏｍｐｌｅｔｅ Ｆｒｅｕｎｄヒト結核死菌含、和光、
）１３７Ｒｖ）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（１０，００Ｏｒｐｍ） Ｔ
ＮＴ−ＣＥＡ溶液３ｍｌ、を３回に分けて加えた。十分
に乳化し、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 and Synthesis of Adjuvant Emulsion The TNP-CEA thus obtained was diluted with PBS to obtain 1 mg/3 wells of a warm solution. Adjuvant (C
complete Freund, including killed human tuberculosis bacteria, Wako,
) 137Rv) while stirring well, and stir with a homogenizer (10,00 Orpm) T
3 ml of NT-CEA solution was added in three portions. It was confirmed that it was sufficiently emulsified and did not spread even when a small amount was dropped on 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 each of 20 mice (Balb/c) about 8 weeks old.
（’Ｂ）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。('B) 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ベ
ンゼン抗体の産生が確認された。When the serum was centrifuged and screened by ELISA, production of anti-trinitrobenzene 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ａｍ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−ＣＥＡをＰＢＳで希釈して得た０、５ｍｇ／畦溶
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１００μｍ、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 performed on two mice to enlarge the mouse am. T as an immunogen
A 0.5 mg/row solution obtained by diluting NP-CEA with PBS was used as it was without adding any adjuvant. Six weeks after immunization, 100 μm of this immunogen was injected into the abdominal cavity of the mouse.
（Ｄ） ５ｓ胞融合
ブースト後３日を経過したマウスの膵臓細胞を摘出し、
平均分子量１，５００のポリエチレングリコールを用い
た常法により、マウス骨髄腫由来細胞ライン（Ｐ３Ｘ６
３−Ａｇ８．６５３）と融合した。フィーダー（成長因
子を供給する細胞）として同じマウスの膵臓細胞を用い
、９６ウ工ルプレート５枚の上で１０％のウシ胎児血清
を含むＩＩＡＴ培地で培養した。１週問後、１０χのウ
シ胎児血清を含むＨＴ培地と交換した。(D) Pancreatic cells from mice were removed 3 days after the 5s cell fusion boost,
A mouse myeloma-derived cell line (P3X6
3-Ag8.653). Pancreatic cells from the same mouse were used as feeders (cells that supply growth factors) and cultured in IIAT medium containing 10% fetal bovine serum on five 96-well plates. After one week, the medium was replaced with HT medium containing 10x 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い、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 Screening was performed by ELISA method, and 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
胸線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、適時上清についてＥｌ、Ｉｓ
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、２００ｍ１中で５×１０５ケ／
ｍｌの濃度に至るまで培養を進めた。Initial proliferation was promoted using thymus cells from 4-week-old mice (Balb/c) as feeders. Proceed with the culture while increasing the plate size, and remove El and Is of the supernatant from time to time.
The screening by A was repeated, and a cell line showing a high titer for TNT and good proliferation was finally selected, and 5 x 10 cells/cell line was selected in 200 ml.
The culture was continued until the concentration reached 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、５×１０６１畦の濃度でＦＣ５：ＤＭＳＯ＝９：１
の溶液１ｍＬに浮遊させ、−８０℃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 obtained by centrifuging the supernatant and using FC5:DMSO = 9:1 at a concentration of 5 x 1061 cells.
After floating in 1 mL of solution and freezing at -80°C, it was transferred to liquid nitrogen for long-term storage.
（Ｃ）ファルマシア製Ｐｒｏｔｅｉｎ Ａ−５ｅｐｈａ
ｒｏｓｅ ＣＬ−４Ｂ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ＳＯＳポリアクリ
ルアミドゲル電気泳動により、標準蛋白との比較から、
精製抗体は分子量的５０，０００のＨ鎖と約２０ 、０
００のＬ鎖からなる１８Ｇ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-4B. This monoclonal antibody was analyzed by SOS polyacrylamide gel electrophoresis and compared with standard proteins.
The purified antibody has a heavy chain with a molecular weight of 50,000 and a molecular weight of approximately 20,000.
It was confirmed that it was 18G consisting of a 00 L chain.
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
ノクロー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 that has high affinity for trinitrobenzene or a derivative of trinitrobenzene.
Claims (6)
誘導体と、トリ卵白アルブミンと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 substance binding to avian ovalbumin and a myeloma-derived cell line, followed by cloning. , 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.
アジュ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 |
|---|---|---|---|
| JP63097041A JPH01269485A (en) | 1988-04-20 | 1988-04-20 | Cell line capable of producing monoclonal antibody and monoclonal antibody |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP63097041A JPH01269485A (en) | 1988-04-20 | 1988-04-20 | Cell line capable of producing monoclonal antibody and monoclonal antibody |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| JPH01269485A true JPH01269485A (en) | 1989-10-26 |
Family
ID=14181491
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP63097041A Pending JPH01269485A (en) | 1988-04-20 | 1988-04-20 | Cell line capable of producing monoclonal antibody and monoclonal antibody |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPH01269485A (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 JP63097041A patent/JPH01269485A/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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