JPH0441783B2 - - Google Patents
Info
- Publication number
- JPH0441783B2 JPH0441783B2 JP59153993A JP15399384A JPH0441783B2 JP H0441783 B2 JPH0441783 B2 JP H0441783B2 JP 59153993 A JP59153993 A JP 59153993A JP 15399384 A JP15399384 A JP 15399384A JP H0441783 B2 JPH0441783 B2 JP H0441783B2
- Authority
- JP
- Japan
- Prior art keywords
- antibody
- plasmin
- human
- plasmin inhibitor
- labeled
- 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.)
- Expired - Lifetime
Links
- 239000002806 plasmin inhibitor Substances 0.000 claims description 45
- 229940122791 Plasmin inhibitor Drugs 0.000 claims description 44
- 238000000034 method Methods 0.000 claims description 20
- 239000003153 chemical reaction reagent Substances 0.000 claims description 17
- 229940012957 plasmin Drugs 0.000 claims description 15
- 108010088842 Fibrinolysin Proteins 0.000 claims description 14
- 239000000126 substance Substances 0.000 claims description 9
- 108090000790 Enzymes Proteins 0.000 claims description 7
- 102000004190 Enzymes Human genes 0.000 claims description 7
- 101000605403 Homo sapiens Plasminogen Proteins 0.000 claims description 7
- 229940088598 enzyme Drugs 0.000 claims description 7
- 238000005259 measurement Methods 0.000 claims description 7
- 239000000427 antigen Substances 0.000 claims description 6
- 102000036639 antigens Human genes 0.000 claims description 6
- 108091007433 antigens Proteins 0.000 claims description 6
- 230000003480 fibrinolytic effect Effects 0.000 claims description 6
- 238000003018 immunoassay Methods 0.000 claims description 6
- 230000000694 effects Effects 0.000 claims description 5
- 239000003599 detergent Substances 0.000 claims description 3
- 239000004033 plastic Substances 0.000 claims description 3
- 229920003023 plastic Polymers 0.000 claims description 3
- 239000003795 chemical substances by application Substances 0.000 claims description 2
- 239000011521 glass Substances 0.000 claims description 2
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- 239000011324 bead Substances 0.000 claims 3
- 230000001900 immune effect Effects 0.000 claims 1
- 239000004816 latex Substances 0.000 claims 1
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- 230000002934 lysing effect Effects 0.000 claims 1
- 239000002923 metal particle Substances 0.000 claims 1
- 210000004027 cell Anatomy 0.000 description 18
- 239000000243 solution Substances 0.000 description 11
- 210000004408 hybridoma Anatomy 0.000 description 10
- 206010035226 Plasma cell myeloma Diseases 0.000 description 8
- 201000000050 myeloid neoplasm Diseases 0.000 description 8
- 238000005406 washing Methods 0.000 description 7
- 239000002609 medium Substances 0.000 description 6
- 210000004989 spleen cell Anatomy 0.000 description 6
- 241000699670 Mus sp. Species 0.000 description 5
- 230000004927 fusion Effects 0.000 description 5
- 238000002372 labelling Methods 0.000 description 5
- 108020004774 Alkaline Phosphatase Proteins 0.000 description 4
- 102000002260 Alkaline Phosphatase Human genes 0.000 description 4
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 4
- 230000003053 immunization Effects 0.000 description 4
- 238000004519 manufacturing process Methods 0.000 description 4
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 3
- 238000002965 ELISA Methods 0.000 description 3
- 102000018251 Hypoxanthine Phosphoribosyltransferase Human genes 0.000 description 3
- 108010091358 Hypoxanthine Phosphoribosyltransferase Proteins 0.000 description 3
- 238000002835 absorbance Methods 0.000 description 3
- 229940098773 bovine serum albumin Drugs 0.000 description 3
- 238000002649 immunization Methods 0.000 description 3
- 239000006152 selective media Substances 0.000 description 3
- LPXQRXLUHJKZIE-UHFFFAOYSA-N 8-azaguanine Chemical compound NC1=NC(O)=C2NN=NC2=N1 LPXQRXLUHJKZIE-UHFFFAOYSA-N 0.000 description 2
- 208000007536 Thrombosis Diseases 0.000 description 2
- IQFYYKKMVGJFEH-XLPZGREQSA-N Thymidine Chemical compound O=C1NC(=O)C(C)=CN1[C@@H]1O[C@H](CO)[C@@H](O)C1 IQFYYKKMVGJFEH-XLPZGREQSA-N 0.000 description 2
- 239000003146 anticoagulant agent Substances 0.000 description 2
- 239000012752 auxiliary agent Substances 0.000 description 2
- 230000027455 binding Effects 0.000 description 2
- 210000004369 blood Anatomy 0.000 description 2
- 239000008280 blood Substances 0.000 description 2
- 230000007910 cell fusion Effects 0.000 description 2
- 239000012228 culture supernatant Substances 0.000 description 2
- 208000009190 disseminated intravascular coagulation Diseases 0.000 description 2
- FDGQSTZJBFJUBT-UHFFFAOYSA-N hypoxanthine Chemical compound O=C1NC=NC2=C1NC=N2 FDGQSTZJBFJUBT-UHFFFAOYSA-N 0.000 description 2
- 239000008363 phosphate buffer Substances 0.000 description 2
- -1 polyethylene Polymers 0.000 description 2
- 239000000941 radioactive substance Substances 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 239000011780 sodium chloride Substances 0.000 description 2
- 238000002560 therapeutic procedure Methods 0.000 description 2
- 230000002537 thrombolytic effect Effects 0.000 description 2
- OBYNJKLOYWCXEP-UHFFFAOYSA-N 2-[3-(dimethylamino)-6-dimethylazaniumylidenexanthen-9-yl]-4-isothiocyanatobenzoate Chemical compound C=12C=CC(=[N+](C)C)C=C2OC2=CC(N(C)C)=CC=C2C=1C1=CC(N=C=S)=CC=C1C([O-])=O OBYNJKLOYWCXEP-UHFFFAOYSA-N 0.000 description 1
- TVZGACDUOSZQKY-LBPRGKRZSA-N 4-aminofolic acid Chemical compound C1=NC2=NC(N)=NC(N)=C2N=C1CNC1=CC=C(C(=O)N[C@@H](CCC(O)=O)C(O)=O)C=C1 TVZGACDUOSZQKY-LBPRGKRZSA-N 0.000 description 1
- 229960005508 8-azaguanine Drugs 0.000 description 1
- 229920000936 Agarose Polymers 0.000 description 1
- 206010003445 Ascites Diseases 0.000 description 1
- DWRXFEITVBNRMK-UHFFFAOYSA-N Beta-D-1-Arabinofuranosylthymine Natural products O=C1NC(=O)C(C)=CN1C1C(O)C(O)C(CO)O1 DWRXFEITVBNRMK-UHFFFAOYSA-N 0.000 description 1
- 210000003771 C cell Anatomy 0.000 description 1
- 229920002307 Dextran Polymers 0.000 description 1
- 108090000371 Esterases Proteins 0.000 description 1
- 102000009123 Fibrin Human genes 0.000 description 1
- 108010073385 Fibrin Proteins 0.000 description 1
- BWGVNKXGVNDBDI-UHFFFAOYSA-N Fibrin monomer Chemical compound CNC(=O)CNC(=O)CN BWGVNKXGVNDBDI-UHFFFAOYSA-N 0.000 description 1
- 101710196208 Fibrinolytic enzyme Proteins 0.000 description 1
- 102000003886 Glycoproteins Human genes 0.000 description 1
- 108090000288 Glycoproteins Proteins 0.000 description 1
- UGQMRVRMYYASKQ-UHFFFAOYSA-N Hypoxanthine nucleoside Natural products OC1C(O)C(CO)OC1N1C(NC=NC2=O)=C2N=C1 UGQMRVRMYYASKQ-UHFFFAOYSA-N 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 241000699666 Mus <mouse, genus> Species 0.000 description 1
- 241000283973 Oryctolagus cuniculus Species 0.000 description 1
- 102000013566 Plasminogen Human genes 0.000 description 1
- 108010051456 Plasminogen Proteins 0.000 description 1
- 239000004698 Polyethylene Substances 0.000 description 1
- 239000002202 Polyethylene glycol Substances 0.000 description 1
- 239000004743 Polypropylene Substances 0.000 description 1
- 229920001213 Polysorbate 20 Polymers 0.000 description 1
- 239000004793 Polystyrene Substances 0.000 description 1
- 102000003990 Urokinase-type plasminogen activator Human genes 0.000 description 1
- 108090000435 Urokinase-type plasminogen activator Proteins 0.000 description 1
- 229960003896 aminopterin Drugs 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 108010005774 beta-Galactosidase Proteins 0.000 description 1
- 102000005936 beta-Galactosidase Human genes 0.000 description 1
- IQFYYKKMVGJFEH-UHFFFAOYSA-N beta-L-thymidine Natural products O=C1NC(=O)C(C)=CN1C1OC(CO)C(O)C1 IQFYYKKMVGJFEH-UHFFFAOYSA-N 0.000 description 1
- 230000000903 blocking effect Effects 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 1
- 239000000969 carrier Substances 0.000 description 1
- 239000006285 cell suspension Substances 0.000 description 1
- 238000010367 cloning Methods 0.000 description 1
- 238000012790 confirmation Methods 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- 238000003745 diagnosis Methods 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 238000003113 dilution method Methods 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- KAKKHKRHCKCAGH-UHFFFAOYSA-L disodium;(4-nitrophenyl) phosphate;hexahydrate Chemical compound O.O.O.O.O.O.[Na+].[Na+].[O-][N+](=O)C1=CC=C(OP([O-])([O-])=O)C=C1 KAKKHKRHCKCAGH-UHFFFAOYSA-L 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 229950003499 fibrin Drugs 0.000 description 1
- MHMNJMPURVTYEJ-UHFFFAOYSA-N fluorescein-5-isothiocyanate Chemical compound O1C(=O)C2=CC(N=C=S)=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 MHMNJMPURVTYEJ-UHFFFAOYSA-N 0.000 description 1
- 150000004676 glycans Chemical class 0.000 description 1
- 230000023597 hemostasis Effects 0.000 description 1
- 230000003100 immobilizing effect Effects 0.000 description 1
- 238000000760 immunoelectrophoresis Methods 0.000 description 1
- 238000010324 immunological assay Methods 0.000 description 1
- 239000012535 impurity Substances 0.000 description 1
- 239000003112 inhibitor Substances 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 210000004698 lymphocyte Anatomy 0.000 description 1
- 239000002184 metal Substances 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 238000012544 monitoring process Methods 0.000 description 1
- 210000005170 neoplastic cell Anatomy 0.000 description 1
- 230000001613 neoplastic effect Effects 0.000 description 1
- 230000009871 nonspecific binding Effects 0.000 description 1
- 239000000123 paper Substances 0.000 description 1
- 210000003200 peritoneal cavity Anatomy 0.000 description 1
- 108040007629 peroxidase activity proteins Proteins 0.000 description 1
- 102000013415 peroxidase activity proteins Human genes 0.000 description 1
- 229920002239 polyacrylonitrile Polymers 0.000 description 1
- 229920000728 polyester Polymers 0.000 description 1
- 229920000573 polyethylene Polymers 0.000 description 1
- 229920001223 polyethylene glycol Polymers 0.000 description 1
- 229920000642 polymer Polymers 0.000 description 1
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 description 1
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 description 1
- 229920001155 polypropylene Polymers 0.000 description 1
- 229920001282 polysaccharide Polymers 0.000 description 1
- 239000005017 polysaccharide Substances 0.000 description 1
- 229920002223 polystyrene Polymers 0.000 description 1
- 230000002265 prevention Effects 0.000 description 1
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 1
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- 230000003248 secreting effect Effects 0.000 description 1
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- 239000002594 sorbent Substances 0.000 description 1
- 210000000952 spleen Anatomy 0.000 description 1
- 210000004988 splenocyte Anatomy 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 229940104230 thymidine Drugs 0.000 description 1
- 229960005356 urokinase Drugs 0.000 description 1
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/86—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving blood coagulating time or factors, or their receptors
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/40—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against enzymes
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/56—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving blood clotting factors, e.g. involving thrombin, thromboplastin, fibrinogen
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/81—Protease inhibitors
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/90—Enzymes; Proenzymes
- G01N2333/914—Hydrolases (3)
- G01N2333/948—Hydrolases (3) acting on peptide bonds (3.4)
- G01N2333/968—Plasmin, i.e. fibrinolysin
Description
【発明の詳細な説明】
ａ 産業上の利用分野
本発明は、溶液状態にあるプラスミン−ヒトα2
−プラスミンインヒビター複合体（plasmin−α2
−plasmin inhibitor complex、plasmin−α2−
antiplasmin complex）を免疫学的に測定する試
薬及びそのキツトに関する。DETAILED DESCRIPTION OF THE INVENTION a. Field of Industrial Application The present invention provides plasmin-human α 2 in a solution state.
- Plasmin inhibitor complex (plasmin-α 2
−plasmin inhibitor complex, plasmin−α 2 −
The present invention relates to a reagent and kit for immunologically measuring antiplasmin complex.
更に詳しくは、ヒトα2−プラスミンインヒビタ
ーを特異的に認識して結合するモノクローナル抗
体とプラスミン（plasmin）を認識して結合し得
る抗体を用いるサンドイツチ法によるプラスミン
−ヒトα2−プラスミンインヒビター複合体の免疫
学的測定試薬及びそのキツトに関する。 More specifically, a plasmin-human α 2 -plasmin inhibitor complex was prepared by the Sandersch method using a monoclonal antibody that specifically recognizes and binds to human α 2 -plasmin inhibitor and an antibody that can recognize and bind to plasmin. This invention relates to an immunological assay reagent and a kit thereof.
ｂ 従来技術
ヒトのα2−プラスミンインヒビターは、青木と
諸井によつて最初に単離・精製され、線維素溶解
酵素のプラスミン（plasmin）のエステラーゼ活
性を瞬間的に阻害する強力なプラスミンインヒビ
ターであり、11.7％の糖を含む分子量約67000の
１本鎖の糖蛋白質であることが知られている
〔Moroi ＆ Aoki；The Journal of
Biological Chemistry，251，5956−5965（1976）
参照〕。b. Prior Art Human α 2 -plasmin inhibitor was first isolated and purified by Aoki and Moroi, and is a strong plasmin inhibitor that instantly inhibits the esterase activity of the fibrinolytic enzyme plasmin. , is known to be a single-chain glycoprotein with a molecular weight of approximately 67,000 containing 11.7% sugar [Moroi &Aoki; The Journal of
Biological Chemistry, 251, 5956-5965 (1976)
reference〕.
一方ヒトのα2−プラスミンインヒビターには３
種類の活性部位があることが知られている。第１
はプラスミンの線維素溶解作用を阻止する部位
（以下これを“リアクテイブサイド”ということ
がある）〔B.Wiman ＆ D.Collen；The
Journal of Biological Chemistry，254，9291〜
9297（1979）参照〕であり、第２はカルボキシル
基末端側のプラスミン結合部位〔B.Wiman ＆
D.Collen；European Journal of
Biochemistry，84，573−578（1978）参照〕であ
り、第３はアミノ基末端のフイブリン結合部位で
ある〔Y.Sakata，et al.，Thrombosis
Research，16 279〜282（1979）参照〕。 On the other hand, human α 2 -plasmin inhibitor has 3
It is known that there are different types of active sites. 1st
is the site that blocks the fibrinolytic action of plasmin (hereinafter referred to as the “reactive side”) [B. Wiman & D. Collen;
Journal of Biological Chemistry, 254 , 9291~
9297 (1979)], and the second is the plasmin binding site at the carboxyl terminal side [B. Wiman &
D.Collen；European Journal of
Biochemistry, 84, 573-578 (1978)], and the third is the fibrin binding site at the amino terminal [Y. Sakata, et al., Thrombosis
Research, 16 279-282 (1979)].
ヒトα2−プラスミンインヒビターは、プラスミ
ン活性をほとんど瞬間的に阻害し、α2−プラスミ
ンインヒビターは、プラスミンと１：１の割合で
結合し複合体を形成する。（B.Wiman ＆ D.
Collen；The Journal of Biological
Chemistry、254、9291〜9297（1979）、D.
Collen；Thrombosis and Haemostasis、43、
77−89（1980）参照）
汎発性血管内凝固（DIC）やウロキナーゼによ
る血栓溶解療法など、プラスミノーゲンの活性化
のみられる状態では、生成したプラスミンとα2−
プラスミンインヒビターは反応し、複合体を形成
する。 Human α 2 -plasmin inhibitor inhibits plasmin activity almost instantaneously, and α 2 -plasmin inhibitor binds to plasmin at a ratio of 1:1 to form a complex. (B. Wiman & D.
Collen; The Journal of Biology
Chemistry, 254 , 9291-9297 (1979), D.
Collen; Thrombosis and Haemostasis, 43 ,
77-89 (1980)) In conditions where plasminogen is activated, such as in disseminated intravascular coagulation (DIC) or thrombolytic therapy using urokinase, the generated plasmin and α 2 −
Plasmin inhibitors react and form complexes.
最近、血漿中のプラスミン−α2−プラスミンイ
ンヒビター複合体の定量は、血栓溶解療法のモニ
ターやDICの診断等に有効であると考えられてい
る（例えばN.A.Booth ＆ B.Bennett：British
Journal of Haematology、50、537〜541（1982）
参照）。 Recently, quantifying the plasmin-α 2 -plasmin inhibitor complex in plasma is considered to be effective for monitoring thrombolytic therapy and diagnosing DIC (for example, NABooth & B.Bennett: British
Journal of Haematology, 50 , 537–541 (1982)
reference).
従つて、血液中のプラスミン−α2−プラスミン
インヒビター複合体の量を正確且つ簡便に測定す
ることができれば、種々の病気に対してその予
防、診断に極めて役立つことである。 Therefore, if the amount of plasmin-α 2 -plasmin inhibitor complex in blood could be measured accurately and easily, it would be extremely useful for the prevention and diagnosis of various diseases.
従来知られたプラスミン−α2−プラスミンイン
ヒビター複合体の測定方法として、第１の方法は
二次元交叉免疫電気泳動を用いる方法であり、第
２の方法は抗血清より得た抗体を固定化し、酵素
抗体法を応用したいわゆるサンドイツチ法による
ものである。 As conventionally known methods for measuring the plasmin-α 2 -plasmin inhibitor complex, the first method uses two-dimensional cross-immunoelectrophoresis, and the second method involves immobilizing antibodies obtained from antiserum. This is based on the so-called Sandermansch method, which is an application of the enzyme-antibody method.
しかし、前者の方法は感度と定量性が低いとい
う欠点があつた。また、後者の方法はきわめて感
度が高く有効な方法であるが、ヒトα2−プラスミ
ンインヒビターに対する一定の活性を有する抗血
清を安定して得ることが極めて困難という欠点が
あつた。 However, the former method had the drawback of low sensitivity and quantitative performance. Furthermore, although the latter method is extremely sensitive and effective, it has the drawback that it is extremely difficult to stably obtain an antiserum having a certain level of activity against human α 2 -plasmin inhibitor.
ｃ 発明の構成
そこで本発明者らは、ヒトα2−プラスミンイン
ヒビターに対するモノクローナル抗体について研
究を重ねたところ、ヒトα2−プラスミンインヒビ
ターにおけるプラスミンの線維素溶解作用を阻止
する部位以外の場所を特異的に認識するモノクロ
ーナル抗体を見出し、またこのモノクローナル抗
体を産生するハイブリドーマ細胞を創作し得、既
に提案した。c. Structure of the Invention The present inventors have conducted repeated research on monoclonal antibodies against human α 2 -plasmin inhibitor, and have found that they have been found to specifically target a site other than the site that blocks the fibrinolytic action of plasmin in human α 2 -plasmin inhibitor. We have discovered a monoclonal antibody that recognizes this monoclonal antibody, and have created hybridoma cells that produce this monoclonal antibody, which we have already proposed.
本発明者らは、かゝる新しく見出した前記モノ
クローナル抗体の特異的な作用を利用すれば、溶
液状態にあるヒトプラスミン−α2−プラスミンイ
ンヒビター複合体の量を直接に且つ正確に測定し
得ることを見出し本発明に到達した。 By utilizing the newly discovered specific action of the monoclonal antibody, the present inventors can directly and accurately measure the amount of human plasmin-α 2 -plasmin inhibitor complex in solution. This discovery led to the present invention.
すなわち、本発明の、サンドイツチ法による免
疫学的測定試薬において、不溶性担体に結合した
抗体と標識抗体とはヒトα2−プラスミンインヒビ
ターを認識し結合するモノクローナル抗体及び、
ヒトプラスミンを認識し、結合する抗プラスミノ
ーゲル抗血清の抗体成分である。 That is, in the immunoassay reagent according to the Sand-Deutsch method of the present invention, the antibody bound to the insoluble carrier and the labeled antibody are a monoclonal antibody that recognizes and binds to human α 2 -plasmin inhibitor;
It is an antibody component of anti-plasminogel antiserum that recognizes and binds to human plasmin.
一般に抗原の２つの異なつた位置に結合した抗
体を作つて抗原の有無又はその量を測定する方法
は、サンドイツチ法と呼ばれ、例えばワイド
（Wide）の「放射線免疫検定法
（Radiomunoassay Methods）」199−206（1970）
に記載されている。 In general, the method of measuring the presence or absence of an antigen or its amount by making antibodies that bind to two different positions on an antigen is called the Sanderutsch method, for example Wide's "Radiomunoassay Methods" 199 −206 (1970)
It is described in.
かくして本発明によれば、試薬の品質差がな
く、恒常的に精度よく溶液状態の（例えば血漿中
の）プラスミン−α2−プラスミンインヒビター複
合体を測定することが可能となる。また直接プラ
スミン−α2−プラスミンインヒビター複合体を測
定するので他の挾雑物の影響は全く受けず正確に
且つ短時間に測定することができる。従つて、本
発明によれば、プラスミン−α2−プラスミンイン
ヒビター複合体を正確且つ迅速に測定し得る試薬
及びそのキツトが提供される。 Thus, according to the present invention, it is possible to consistently and accurately measure the plasmin-α 2 -plasmin inhibitor complex in a solution state (for example, in plasma) without any quality differences in reagents. Furthermore, since the plasmin-α 2 -plasmin inhibitor complex is directly measured, it is not affected by other impurities and can be measured accurately and in a short time. Therefore, according to the present invention, there are provided a reagent and a kit thereof that can accurately and rapidly measure the plasmin-α 2 -plasmin inhibitor complex.
次に本発明による測定試薬及びプラスミン−α2
−プラスミンインヒビター複合体の含有量の測定
方法を具体的に説明する。 Next, the measurement reagent according to the present invention and plasmin-α 2
- A method for measuring the content of plasmin inhibitor complex will be specifically explained.
ヒトα2−プラスミンインヒビターに対するモノ
クローナル抗体（第１抗体）を適当な不溶性担体
（例えばプラスチツク容器）に固定化する（以下
これを“固定化抗体”という）。ついで不溶性担
体と測定しようとする試薬又は検体試料との非特
異的結合を避けるために適当な物質（例えば牛血
清アルブミン）で不溶性担体の表面を被覆する。 A monoclonal antibody (first antibody) against human α 2 -plasmin inhibitor is immobilized on a suitable insoluble carrier (for example, a plastic container) (hereinafter referred to as "immobilized antibody"). Next, the surface of the insoluble carrier is coated with a suitable substance (for example, bovine serum albumin) to avoid non-specific binding between the insoluble carrier and the reagent or specimen sample to be measured.
このようにして得られた第１抗体が固定化され
た不溶性担体を検体試料と一定時間及び温度で接
触させ反応させる。この間に固定化抗体（第１抗
体）と検体試料中のプラスミン−α2−プラスミン
インヒビター複合体が結合する。ついで適当な洗
浄液で洗つた後、適当な標識物質で標識したヒト
プラスミノーゲンに対する抗体（第２抗体）の溶
液（例えば水溶液）を、不溶性担体における固定
化抗体に結合したプラスミン−α2−プラスミンイ
ンヒビター複合体と一定時間及び温度で接触させ
第２抗体と反応させる。これを適当な洗浄液で洗
い、次いで不溶性担体上に存在する第２抗体に標
識された標識物質の量を測定する。かくしてその
値から検体試料中のプラスミン−α2−プラスミン
インヒビター複合体の量を算出することができ
る。 The insoluble carrier on which the first antibody thus obtained is immobilized is brought into contact with the specimen sample for a certain period of time and at a certain temperature to cause a reaction. During this time, the immobilized antibody (first antibody) and the plasmin-α 2 -plasmin inhibitor complex in the specimen sample bind to each other. After washing with an appropriate washing solution, a solution (e.g., aqueous solution) of an antibody (second antibody) against human plasminogen labeled with an appropriate labeling substance is added to the plasmin-α 2 -plasmin bound to the immobilized antibody in the insoluble carrier. It is brought into contact with the inhibitor complex for a certain period of time and at a certain temperature to react with the second antibody. This is washed with an appropriate washing solution, and then the amount of the labeling substance labeled with the second antibody present on the insoluble carrier is measured. Thus, the amount of plasmin-α 2 -plasmin inhibitor complex in the specimen sample can be calculated from this value.
かくして本発明の測定試薬は、第１抗体が不溶
性担体に結合した固定化抗体と標識化された第２
抗体とより主として構成される。この試薬を能率
よく且つ簡便に利用するために、これら抗体以外
に種々の補助剤を含めてキツトを形成することが
できる。かゝる補助剤としては、例えば固体状の
試薬を溶解させるための溶解剤、不溶化担体を洗
浄するために使用される洗浄剤、抗体の標識物質
として酵素を使用した場合、酵素活性を測定する
ための基質、その反応停止剤などの免疫学的測定
試薬のキツトとして通常使用されるものが挙げら
れる。 Thus, the measurement reagent of the present invention comprises a first antibody bound to an insoluble carrier, an immobilized antibody, and a labeled second antibody.
Mainly composed of antibodies. In order to utilize this reagent efficiently and conveniently, a kit can be formed containing various auxiliary agents in addition to these antibodies. Such auxiliary agents include, for example, a dissolving agent for dissolving solid reagents, a detergent used for washing an insolubilized carrier, and when an enzyme is used as a labeling substance for an antibody, a detergent for measuring enzyme activity. Examples include those commonly used as kits for immunoassay reagents, such as substrates and reaction terminators for immunoassays.
本発明の測定試薬に使用される不溶性担体とし
ては、例えばポリスチレン、ポリエチレン、ポリ
プロピレン、ポリエステル、ポリアクリルニトリ
ル、弗素樹脂、架橋デキストラン、ポリサツカラ
イドなどの高分子、その他紙、ガラス、金属、ア
ガロース及びこれらの組合せなどを例示すること
ができる。 Examples of insoluble carriers used in the measurement reagent of the present invention include polymers such as polystyrene, polyethylene, polypropylene, polyester, polyacrylonitrile, fluororesin, crosslinked dextran, polysaccharide, and other paper, glass, metal, agarose, and Combinations of these can be exemplified.
また不溶性担体の形状としては、例えばトレイ
状、球状、繊維状、棒状、盤状、容器状、セル、
試験管などの種々の形状であることができる。 In addition, the shape of the insoluble carrier is, for example, tray-shaped, spherical, fibrous, rod-shaped, disc-shaped, container-shaped, cell,
It can be of various shapes, such as a test tube.
また標識物質としては放射性物質、酵素又は螢
光物質を使用するのが有利である。放射性物質と
しては 125I 131I、 14C、 3Hなどを、酵素として
はアルカリ性フオスフアターゼ、パーオキシダー
ゼ、β−Ｄ−ガラクトシダーゼなど、また螢光物
質としてはフルオレツセインイソチオシアネー
ト、テトラメチルローダミンイソチオシアネート
などを使用することができるが、これらは例示し
たものに限らず、免疫学的測定方法に使用し得る
ものであれば、他のものでも使用できる。 It is also advantageous to use radioactive substances, enzymes or fluorescent substances as labeling substances. Radioactive substances include 125 I 131 I, 14 C, and 3 H, enzymes include alkaline phosphatase, peroxidase, and β-D-galactosidase, and fluorescent substances include fluorescein isothiocyanate and tetramethylrhodamine isothiocyanate. However, these are not limited to the exemplified ones, and other types can also be used as long as they can be used in the immunoassay method.
本発明の前記モノクローナル抗体及びその製造
方法については、先に特許出願された（昭和59年
４月17日出願：発明の名称“モノクローナル抗
体、ハイブリドーマ細胞及びモノクローナル抗体
の製造方法”特願昭59−75778（特開60−
222426））。 Regarding the monoclonal antibody of the present invention and the method for producing the same, a patent application was previously filed (Application filed on April 17, 1982: Title of the invention: "Monoclonal antibody, hybridoma cells, and method for producing monoclonal antibody" Patent application filed in 1982) 75778 (JP 60-
222426)).
本発明の前記モノクローナル抗体及びその製造
方法については前記特許出願明細書に詳細に説明
されている通り、ヒトα2−プラスミンインヒビタ
ーにおけるプラスミンの線維素溶解作用の阻止部
位以外の場所を特異的に認識して結合し得るモノ
クローナル抗体である。以下にその内容を簡単に
説明する。 As explained in detail in the patent application, the monoclonal antibody of the present invention and its production method specifically recognize a site other than the site of inhibiting the fibrinolytic action of plasmin in human α 2 -plasmin inhibitor. It is a monoclonal antibody that can bind to The contents will be briefly explained below.
≪モノクローナル抗体及びその製造方法≫
Ａ 抗原の単離、精製；
抗原に用いるヒトα2−プラスミンインヒビタ
ーは、前記青木と諸井の方法によりヒト血漿中
より単離精製された。<<Monoclonal antibody and method for producing the same>> A. Isolation and purification of antigen; Human α 2 -plasmin inhibitor used as an antigen was isolated and purified from human plasma by the method of Aoki and Moroi.
Ｂ ヒトα2−プラスミンインヒビターによるマウ
スの免疫；
雄Balb／ｃマウスを用いるが、他の系
（strains）のマウスを使用することもできる。
その際、免疫計画及びヒトα2−プラスミンイン
ヒビターの濃度は十分な量の抗原刺激を受けた
リンパ球が形成されるよう選ばれるべきであ
る。例えばマウスに少量のヒトα2−プラスミン
インヒビターで或る間隔で腹腔に数回免疫の
後、さらに数回静脈に投与した。最終免疫の数
日後に融合の為に脾臓細胞を取り出す。B. Immunization of mice with human α 2 -plasmin inhibitor; male Balb/c mice are used, but mice of other strains can also be used.
The immunization regimen and the concentration of human α 2 -plasmin inhibitor should then be chosen so that a sufficient number of antigen-stimulated lymphocytes are formed. For example, mice were immunized intraperitoneally several times at certain intervals with a small amount of human α 2 -plasmin inhibitor, and then intravenously administered several times. Spleen cells are removed for fusion several days after the final immunization.
Ｃ 細胞融合
脾臓を無菌的に取り出し、それから単細胞懸
濁液を調製する。それらの脾臓細胞を適当なラ
インからのマウス骨髄腫細胞と適当な融合促進
剤の使用により細胞融合させる。脾臓細胞対骨
髄腫細胞の好ましい比率は約20：１〜約２：１
の範囲である。約108個の脾臓細胞について0.5
〜1.5mlの融合媒体の使用が適当である。C Cell Fusion The spleen is aseptically removed and a single cell suspension prepared from it. The spleen cells are fused with mouse myeloma cells from an appropriate line by use of an appropriate fusion promoter. The preferred ratio of spleen cells to myeloma cells is about 20:1 to about 2:1.
is within the range of 0.5 for approximately 10 8 splenocytes
The use of ~1.5 ml of fusion medium is appropriate.
細胞融合に用いる骨髄腫細胞は多く知られて
いるが、本発明では、P3−X63−Ag8−U1細
胞（以下P3−U1と略記する）〔Yelton，D.Eet
al.，Current Topics in Microbiolgy and
Immunology 81、１（1978）参照〕を用いた。
これは８−アザグアニン耐性の細胞ラインであ
り、酵素ヒポキサンチン−グアニンホスホリボ
シルトランスフエラーゼ（hypoxanthine−
guanine phosphoribosyl transferase）が欠失
しており、それゆえにHAT（ヒポキサンチン、
アミノプテリン、チミジン）培地中では生存し
ない。また、この細胞ラインはそれ自体抗体を
分泌しない、いわゆる非分泌型である。 Many myeloma cells are known for use in cell fusion, but in the present invention, P3-X63-Ag8-U1 cells (hereinafter abbreviated as P3-U1) [Yelton, D.Eet
al.，Current Topics in Microbiology and
Immunology 81, 1 (1978)] was used.
This is a cell line resistant to 8-azaguanine, and the enzyme hypoxanthine-guanine phosphoribosyltransferase (hypoxanthine-guanine phosphoribosyltransferase)
guanine phosphoribosyl transferase) and therefore HAT (hypoxanthine,
(aminopterin, thymidine) does not survive in medium. Furthermore, this cell line itself does not secrete antibodies, and is a so-called non-secreting type.
好ましい融合促進剤としては、例えば平均分
子量が1000〜4000のポリエチレングリコールを
有利に使用できるが、この分野で知られている
他の融合促進剤を使用することもできる。 As a preferred fusion promoter, for example polyethylene glycol having an average molecular weight of 1000 to 4000 can be advantageously used, but other fusion promoters known in the art can also be used.
Ｄ 融合した細胞の選択；
別の容器内（例えばマイクロタイタープレー
ト）で未融合の脾臓細胞、未融合の骨髄腫細胞
および融合した細胞の混合物を、未融合の骨髄
腫細胞を支持しない選択培地で希釈し、未融合
の細胞を死滅させるのに十分な時間（約１週
間）培養する。培地は薬物抵抗性（例えば８−
アザグアニン抵抗性）で未融合の骨髄腫細胞を
支持しないもの（例えば前記HAT培地）が使
用される。この選択培地中では未融合の骨髄腫
細胞は死滅する。また未融合の脾臓細胞は非腫
瘍性細胞なのである一定期間後（約１週間後）
死滅する。これらに対して融合した細胞は骨髄
腫の親細胞の腫瘍性と親脾臓細胞の性質をあわ
せ持つために選択培地中で生存できる。D. Selection of fused cells; in a separate container (e.g., a microtiter plate), a mixture of unfused spleen cells, unfused myeloma cells, and fused cells is grown in a selective medium that does not support unfused myeloma cells. Dilute and culture for sufficient time (about 1 week) to kill unfused cells. The medium should be drug resistant (e.g. 8-
A medium (such as the HAT medium described above) that is resistant to azaguanine and does not support unfused myeloma cells is used. Unfused myeloma cells die in this selective medium. Also, unfused spleen cells are non-neoplastic cells, so after a certain period of time (about 1 week)
die out. Cells fused to these cells can survive in selective media because they have both the neoplastic properties of myeloma parent cells and the properties of parent spleen cells.
Ｅ 各容器中のα2−プラスミンインヒビターに対
する抗体の確認；
かくしてハイブリドーマが細胞が検出された
後、その培養上清を採取し、ヒトα2−プラスミ
ンインヒビターに対する抗体について酵素免疫
定量法（Enzyme Linked Immuno Sorbent
Assay）によりスクリーニングする。E. Confirmation of antibodies against α 2 -plasmin inhibitor in each container; thus, after hybridoma cells have been detected, the culture supernatant is collected and tested for antibodies against human α 2 -plasmin inhibitor by enzyme linked immunoassay (Enzyme Linked Immunoassay). Sorbent
Assay).
Ｆ 目的の抗体を産生するハイブリドーマ細胞の
クロー化；
目的の抗体を産生するハイブリドーマ細胞を
適当な方法（例えば限定希釈法）でクローン化
すると、抗体は２つの異なつた方法で産生され
る。その第１の方法によればハイブリドーマ細
胞を一定時間適当な培地で培養することにより
その培養上清から、そのハイブリドーマ細胞の
産生するモノクローナル抗体を得ることができ
る。第２の方法によればハイブリドーマ細胞は
同質遺伝子又は半同質遺伝子を持つマウスの腹
腔に注射することができる。一定時間後の宿主
動物の血液中及び腹水中より、そのハイブリド
ーマ細胞の産出するモノクローナル抗体を得る
ことができる。F. Cloning of hybridoma cells producing the antibody of interest; When hybridoma cells producing the antibody of interest are cloned by an appropriate method (e.g. limited dilution method), antibodies can be produced in two different ways. According to the first method, monoclonal antibodies produced by hybridoma cells can be obtained from the culture supernatant by culturing hybridoma cells in an appropriate medium for a certain period of time. According to a second method, hybridoma cells can be injected into the peritoneal cavity of isogenic or semi-isogenic mice. Monoclonal antibodies produced by the hybridoma cells can be obtained from the blood and ascites of the host animal after a certain period of time.
本発明の測定試薬においては、かゝるモノクロ
ーナル抗体を第１抗体或いは第２抗体のいずれか
に使用する。すなわち、前記モノクローナル抗体
は、不溶性担体に結合させて固定化抗体として使
用することもできるし、また標識物質を付けて標
識抗体としても使用することもできる。 In the measurement reagent of the present invention, such a monoclonal antibody is used as either the first antibody or the second antibody. That is, the monoclonal antibody can be bound to an insoluble carrier and used as an immobilized antibody, or can be attached with a labeling substance and used as a labeled antibody.
前記モノクローナル抗体と共に使用される他の
抗体としては、ヒトプラスミンを認識し、結合し
得るものであればよい。 Other antibodies used in conjunction with the monoclonal antibody may be any antibody that can recognize and bind to human plasmin.
以上本発明によれば、プラスミン−α2−プラス
ミンインヒビター複合体を含む検体（例えばヒト
血漿）中のプラスミン−α2−プラスミンインヒビ
ター複合体の量を正確に且つ容易に測定すること
が可能である。 As described above, according to the present invention, it is possible to accurately and easily measure the amount of plasmin-α 2 -plasmin inhibitor complex in a sample containing the plasmin-α 2 -plasmin inhibitor complex (for example, human plasma). .
以下実施例を掲げて本発明を詳述する。 The present invention will be described in detail below with reference to Examples.
実施例 １
本実施例で使用した第１及び第２抗体は、本発
明者らが先に出願した明細書（昭和59年４月17日
付出願；発明の名称“モノクローナル抗体、ハイ
ブリドーマ細胞及びモノクローナル抗体の製造方
法”特願昭59−75778（特開昭60−222426））に記
載された方法によつて得られた下記のものを使用
した。Example 1 The first and second antibodies used in this example were described in the specification previously filed by the present inventors (filed on April 17, 1982; title of the invention: "Monoclonal antibodies, hybridoma cells and monoclonal antibodies"). The following product obtained by the method described in Japanese Patent Application No. 59-75778 (Japanese Unexamined Patent Publication No. 60-222426) was used.
第１抗体
前記出願明細書の実施例３において得られた
抗体名“1B10G11”微工研条寄第1782号
（FERMBP−1782）を使用し、これを下記の
如く不溶性担体（マイクロタイタープレート）
に固定化させて用いた。この抗体はα2−プラス
ミンインヒビターにおけるプラスミンの線維素
溶解作用の阻止部位（リアクテイブサイト）以
外の部位を特異的に認識し得るモノクローナル
抗体である。First antibody: The antibody named "1B10G11" obtained in Example 3 of the above application specification (FERMBP-1782) was used, and it was transferred to an insoluble carrier (microtiter plate) as shown below.
It was immobilized and used. This antibody is a monoclonal antibody that can specifically recognize a site other than the reactive site for blocking the fibrinolytic action of plasmin in α 2 -plasmin inhibitor.
第２抗体
ウサギをヒトプラスミノーゲンで免疫して得
たヒトプラスミノーゲンに対する抗血清より抗
体成分を精製し、アルカリ性フオスフアターゼ
で標識化して使用した。Second Antibody An antibody component was purified from an antiserum against human plasminogen obtained by immunizing a rabbit with human plasminogen, labeled with alkaline phosphatase, and used.
濃度20μg／mlのヒトα2−プラスミンインヒビ
ターを特異的に認識するモノクローナル抗体
（1B10G11）をマイクロタイタープレート上に４
℃で一晩放置し固定化した。これに１％牛血清ア
ルブミンを含む緩衝液（15mM Na2CO3、35mM
NaHCO3、3mM NaN3）を加え室温で４時間放
置した後、１％牛血清アルブミンを含む洗浄液
（20mMリン酸緩衝液、0.135M NaCl、2mM
NaN3、0.05％Tween20）で５回洗浄した。次に
希釈用溶液（20mMリン酸緩衝液、0.135M
NaCl）で種々の濃度となるように希釈したプラ
スミン−α2−プラスミンインヒビター複合体を加
え、室温で４時間放置した。 A monoclonal antibody (1B10G11) that specifically recognizes human α 2 -plasmin inhibitor at a concentration of 20 μg/ml was plated on a microtiter plate.
It was left to stand overnight at °C for immobilization. A buffer containing 1% bovine serum albumin (15mM Na 2 CO 3 , 35mM
After adding NaHCO 3 , 3mM NaN 3 ) and leaving it at room temperature for 4 hours, wash solution containing 1% bovine serum albumin (20mM phosphate buffer, 0.135M NaCl, 2mM
Washed 5 times with NaN 3 , 0.05% Tween 20). Next, dilution solution (20mM phosphate buffer, 0.135M
Plasmin-α 2 -plasmin inhibitor complexes diluted with NaCl) to various concentrations were added and left at room temperature for 4 hours.
その後前記洗浄液で５回洗浄し、さらにアルカ
リ性フオスフアターゼで標識したヒトプラスミノ
ーゲンに対する抗体を350ng／mlの濃度で加え４
℃で一晩放置した。前記洗浄液で洗浄後アルカリ
性フオスフアターゼ基質溶液を１mg／mlの濃度で
加え、ELISA ANALYZER〔東洋測器(株)製ETY
−96〕で405nmの波長における１分間当りの吸光
度変化を測定した。その結果を添付図面第１図に
示した。この図面からプラスミン−α2−プラスミ
ンインヒビター複合体の濃度と吸光度変化との関
係は直線関係になることが理解できる。従つてα2
−プラスミンインヒビターにおけるリアクテイブ
サイト以外の場所を特異的に認識するモノクロー
ナル抗体をサンドイツチ法における一つの抗体と
して使用することによつて、プラスミン−α2−プ
ラスミンインヒビター複合体の量を容易に測定す
ることができる。 After that, the cells were washed 5 times with the above washing solution, and an antibody against human plasminogen labeled with alkaline phosphatase was added at a concentration of 350 ng/ml.
It was left overnight at °C. After washing with the above washing solution, alkaline phosphatase substrate solution was added at a concentration of 1 mg/ml, and an ELISA ANALYZER [ETY manufactured by Toyo Sokki Co., Ltd.] was added.
-96] and the change in absorbance per minute at a wavelength of 405 nm was measured. The results are shown in Figure 1 of the attached drawing. From this figure, it can be understood that the relationship between the concentration of the plasmin-α 2 -plasmin inhibitor complex and the change in absorbance is a linear relationship. Therefore α 2
- Easily measure the amount of plasmin-α 2 -plasmin inhibitor complex by using a monoclonal antibody that specifically recognizes a site other than the reactive site in plasmin inhibitor as one antibody in the Sand-Deutsch method. I can do it.
添付図面は、本発明の実施例におけるプラスミ
ン−α2−プラスミンインヒビター複合体の濃度と
吸光度変化との関係を示すものである。
The accompanying drawings show the relationship between the concentration of plasmin-α 2 -plasmin inhibitor complex and the change in absorbance in Examples of the present invention.
Claims (1)
いて、不溶性担体に結合した抗体と標識抗体との
いずれか一方が、ヒトα2−プラスミンインヒビタ
ーにおけるプラスミンの線維素溶解作用を阻止す
る部位以外の場所を特異的に認識するモノクロー
ナル抗体であり、他の一方がヒトプラスミノーゲ
ンに対する抗体であることを特徴とする、ヒトα2
−プラスミンインヒビターに対するモノクローナ
ル抗体を用いたプラスミン−α2−プラスミンイン
ヒビター複合体の免疫学的測定試薬。 ２ 該不溶性担体が、プラスチツク容器、プラス
チツクビーズ、ラテツクスビーズ、ガラスビーズ
又は金属粒子である第１項記載の測定試薬。 ３ 該標識抗体が、酵素、放射性同位元素又は螢
光物質で標識化された抗体である第１項記載の測
定試薬。 ４ 不溶性担体に結合した抗体と標識抗体を含
み、これら抗体のいずれか一方はα2−プラスミン
インヒビターにおけるプラスミンの線維素溶解作
用を阻止する部位以外の場所を特異的に認識する
モノクローナル抗体であり、他の一方はヒトプラ
スミノーゲンを抗原として認識して結合する抗体
であり、これに(a)溶解剤、(b)洗浄剤及び酵素で標
識化した抗体を用いる場合には、(c)酵素活性を測
定するための基質及びその反応停止剤を組合せて
なる免疫学的測定のためのキツト。[Scope of Claims] 1. In an immunoassay reagent based on the Sand-Deutsch method, either the antibody bound to the insoluble carrier or the labeled antibody inhibits the fibrinolytic action of plasmin in human α 2 -plasmin inhibitor. Human α2 is a monoclonal antibody that specifically recognizes a site other than human plasminogen, and the other one is an antibody against human plasminogen.
- An immunological measurement reagent for plasmin-α 2 -plasmin inhibitor complex using a monoclonal antibody against plasmin inhibitor. 2. The measurement reagent according to item 1, wherein the insoluble carrier is a plastic container, plastic beads, latex beads, glass beads, or metal particles. 3. The measurement reagent according to item 1, wherein the labeled antibody is an antibody labeled with an enzyme, a radioisotope, or a fluorescent substance. 4 comprises an antibody bound to an insoluble carrier and a labeled antibody, one of these antibodies being a monoclonal antibody that specifically recognizes a site other than the site that inhibits the fibrinolytic action of plasmin in the α 2 -plasmin inhibitor; The other is an antibody that recognizes and binds to human plasminogen as an antigen, and when using an antibody labeled with (a) a lysing agent, (b) a detergent, and an enzyme, (c) an enzyme. A kit for immunoassay comprising a combination of a substrate for measuring activity and a reaction terminator.
Priority Applications (4)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP15399384A JPS6134465A (en) | 1984-07-26 | 1984-07-26 | Immunoassay reagent and kit using monoclonal antibody against human alpha2-plasmin inhibitor |
| EP19850109227 EP0169549B1 (en) | 1984-07-26 | 1985-07-23 | Immunological determination of human plasmin/alpha2-plasmin inhibitor |
| DE19853586577 DE3586577T2 (en) | 1984-07-26 | 1985-07-23 | IMMUNOLOGICAL DETERMINATION OF HUMAN PLASMIN / ALPHA-2-PLASMIN INHIBITOR. |
| DK339985A DK339985A (en) | 1984-07-26 | 1985-07-25 | HUMAN PLASMIN / ALFA2 PLASMIN INHIBITOR IMMUNOLOGICAL DETERMINATION AND REAGENT SYSTEM FOR USE |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP15399384A JPS6134465A (en) | 1984-07-26 | 1984-07-26 | Immunoassay reagent and kit using monoclonal antibody against human alpha2-plasmin inhibitor |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPS6134465A JPS6134465A (en) | 1986-02-18 |
| JPH0441783B2 true JPH0441783B2 (en) | 1992-07-09 |
Family
ID=15574568
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP15399384A Granted JPS6134465A (en) | 1984-07-26 | 1984-07-26 | Immunoassay reagent and kit using monoclonal antibody against human alpha2-plasmin inhibitor |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPS6134465A (en) |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS57551A (en) * | 1980-04-30 | 1982-01-05 | Merck Patent Gmbh | Immunologic measuring method and means of enzyme |
| JPS5716355A (en) * | 1980-04-25 | 1982-01-27 | Hoffmann La Roche | Immunological method |
| JPS5794660A (en) * | 1980-09-30 | 1982-06-12 | Behringwerke Ag | Measurement of inhibitor-enzyme compound |
| JPS57136165A (en) * | 1981-02-18 | 1982-08-23 | Mochida Pharmaceut Co Ltd | Immunological measuring reagent |
| JPS58148963A (en) * | 1982-03-02 | 1983-09-05 | Maruko Seiyaku Kk | Reagent for measuring bound material of alpha-1- antitrypsin-trypsin in human body fluid by sandwich method |
-
1984
- 1984-07-26 JP JP15399384A patent/JPS6134465A/en active Granted
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS5716355A (en) * | 1980-04-25 | 1982-01-27 | Hoffmann La Roche | Immunological method |
| JPS57551A (en) * | 1980-04-30 | 1982-01-05 | Merck Patent Gmbh | Immunologic measuring method and means of enzyme |
| JPS5794660A (en) * | 1980-09-30 | 1982-06-12 | Behringwerke Ag | Measurement of inhibitor-enzyme compound |
| JPS57136165A (en) * | 1981-02-18 | 1982-08-23 | Mochida Pharmaceut Co Ltd | Immunological measuring reagent |
| JPS58148963A (en) * | 1982-03-02 | 1983-09-05 | Maruko Seiyaku Kk | Reagent for measuring bound material of alpha-1- antitrypsin-trypsin in human body fluid by sandwich method |
Also Published As
| Publication number | Publication date |
|---|---|
| JPS6134465A (en) | 1986-02-18 |
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