JPS6114465B2 - - Google Patents
Info
- Publication number
- JPS6114465B2 JPS6114465B2 JP16551984A JP16551984A JPS6114465B2 JP S6114465 B2 JPS6114465 B2 JP S6114465B2 JP 16551984 A JP16551984 A JP 16551984A JP 16551984 A JP16551984 A JP 16551984A JP S6114465 B2 JPS6114465 B2 JP S6114465B2
- Authority
- JP
- Japan
- Prior art keywords
- glucagon
- peptide
- ser
- antigen
- pancreatic
- 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
Links
- 229960004666 glucagon Drugs 0.000 claims description 65
- 102000051325 Glucagon Human genes 0.000 claims description 59
- 108060003199 Glucagon Proteins 0.000 claims description 59
- MASNOZXLGMXCHN-ZLPAWPGGSA-N glucagon Chemical compound C([C@@H](C(=O)N[C@H](C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H]([C@@H](C)O)C(O)=O)C(C)C)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@H](C)NC(=O)[C@H](CCCNC(N)=N)NC(=O)[C@H](CCCNC(N)=N)NC(=O)[C@H](CO)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)[C@H](CCCCN)NC(=O)[C@H](CO)NC(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CO)NC(=O)[C@@H](NC(=O)[C@H](CC=1C=CC=CC=1)NC(=O)[C@@H](NC(=O)CNC(=O)[C@H](CCC(N)=O)NC(=O)[C@H](CO)NC(=O)[C@@H](N)CC=1NC=NC=1)[C@@H](C)O)[C@@H](C)O)C1=CC=CC=C1 MASNOZXLGMXCHN-ZLPAWPGGSA-N 0.000 claims description 59
- 239000000427 antigen Substances 0.000 claims description 25
- 108091007433 antigens Proteins 0.000 claims description 25
- 102000036639 antigens Human genes 0.000 claims description 25
- 108090000765 processed proteins & peptides Proteins 0.000 claims description 17
- 238000004519 manufacturing process Methods 0.000 claims description 8
- 102000004169 proteins and genes Human genes 0.000 claims description 6
- 108090000623 proteins and genes Proteins 0.000 claims description 6
- DLTISZBLYVHACM-RUCXOUQFSA-N OC(=O)[C@@H](N)CCO.OC(=O)[C@@H](N)CCO Chemical compound OC(=O)[C@@H](N)CCO.OC(=O)[C@@H](N)CCO DLTISZBLYVHACM-RUCXOUQFSA-N 0.000 claims 1
- 239000000243 solution Substances 0.000 description 11
- 238000000034 method Methods 0.000 description 10
- 238000006243 chemical reaction Methods 0.000 description 8
- BDAGIHXWWSANSR-UHFFFAOYSA-N methanoic acid Natural products OC=O BDAGIHXWWSANSR-UHFFFAOYSA-N 0.000 description 8
- 230000009260 cross reactivity Effects 0.000 description 7
- ZNZYKNKBJPZETN-WELNAUFTSA-N Dialdehyde 11678 Chemical compound N1C2=CC=CC=C2C2=C1[C@H](C[C@H](/C(=C/O)C(=O)OC)[C@@H](C=C)C=O)NCC2 ZNZYKNKBJPZETN-WELNAUFTSA-N 0.000 description 6
- 241000283973 Oryctolagus cuniculus Species 0.000 description 6
- 239000000872 buffer Substances 0.000 description 6
- 238000010790 dilution Methods 0.000 description 6
- 239000012895 dilution Substances 0.000 description 6
- 239000002504 physiological saline solution Substances 0.000 description 5
- 239000000725 suspension Substances 0.000 description 5
- 238000012360 testing method Methods 0.000 description 5
- OSWFIVFLDKOXQC-UHFFFAOYSA-N 4-(3-methoxyphenyl)aniline Chemical compound COC1=CC=CC(C=2C=CC(N)=CC=2)=C1 OSWFIVFLDKOXQC-UHFFFAOYSA-N 0.000 description 4
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 4
- WCUXLLCKKVVCTQ-UHFFFAOYSA-M Potassium chloride Chemical compound [Cl-].[K+] WCUXLLCKKVVCTQ-UHFFFAOYSA-M 0.000 description 4
- 102000005686 Serum Globulins Human genes 0.000 description 4
- 108010045362 Serum Globulins Proteins 0.000 description 4
- 235000019253 formic acid Nutrition 0.000 description 4
- 239000000203 mixture Chemical group 0.000 description 4
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 3
- 241000124008 Mammalia Species 0.000 description 3
- 241001465754 Metazoa Species 0.000 description 3
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 3
- 210000004369 blood Anatomy 0.000 description 3
- 239000008280 blood Substances 0.000 description 3
- KGBXLFKZBHKPEV-UHFFFAOYSA-N boric acid Chemical compound OB(O)O KGBXLFKZBHKPEV-UHFFFAOYSA-N 0.000 description 3
- 239000004327 boric acid Substances 0.000 description 3
- 229940098773 bovine serum albumin Drugs 0.000 description 3
- 238000002523 gelfiltration Methods 0.000 description 3
- 230000016784 immunoglobulin production Effects 0.000 description 3
- 238000005259 measurement Methods 0.000 description 3
- 230000004060 metabolic process Effects 0.000 description 3
- 238000011002 quantification Methods 0.000 description 3
- 230000005855 radiation Effects 0.000 description 3
- 238000011160 research Methods 0.000 description 3
- 230000035945 sensitivity Effects 0.000 description 3
- UKAUYVFTDYCKQA-UHFFFAOYSA-N -2-Amino-4-hydroxybutanoic acid Natural products OC(=O)C(N)CCO UKAUYVFTDYCKQA-UHFFFAOYSA-N 0.000 description 2
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 2
- 229920002307 Dextran Polymers 0.000 description 2
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 2
- QJPWUUJVYOJNMH-VKHMYHEASA-N L-homoserine lactone Chemical compound N[C@H]1CCOC1=O QJPWUUJVYOJNMH-VKHMYHEASA-N 0.000 description 2
- CDBYLPFSWZWCQE-UHFFFAOYSA-L Sodium Carbonate Chemical compound [Na+].[Na+].[O-]C([O-])=O CDBYLPFSWZWCQE-UHFFFAOYSA-L 0.000 description 2
- PXIPVTKHYLBLMZ-UHFFFAOYSA-N Sodium azide Chemical compound [Na+].[N-]=[N+]=[N-] PXIPVTKHYLBLMZ-UHFFFAOYSA-N 0.000 description 2
- 229910021538 borax Inorganic materials 0.000 description 2
- 238000007796 conventional method Methods 0.000 description 2
- ATDGTVJJHBUTRL-UHFFFAOYSA-N cyanogen bromide Chemical compound BrC#N ATDGTVJJHBUTRL-UHFFFAOYSA-N 0.000 description 2
- 238000003745 diagnosis Methods 0.000 description 2
- UQGFMSUEHSUPRD-UHFFFAOYSA-N disodium;3,7-dioxido-2,4,6,8,9-pentaoxa-1,3,5,7-tetraborabicyclo[3.3.1]nonane Chemical compound [Na+].[Na+].O1B([O-])OB2OB([O-])OB1O2 UQGFMSUEHSUPRD-UHFFFAOYSA-N 0.000 description 2
- 230000001575 pathological effect Effects 0.000 description 2
- 239000001103 potassium chloride Substances 0.000 description 2
- 235000011164 potassium chloride Nutrition 0.000 description 2
- 239000011541 reaction mixture Substances 0.000 description 2
- 239000004328 sodium tetraborate Substances 0.000 description 2
- 235000010339 sodium tetraborate Nutrition 0.000 description 2
- DDYAPMZTJAYBOF-ZMYDTDHYSA-N (3S)-4-[[(2S)-1-[[(2S)-1-[[(2S)-5-amino-1-[[(2S)-1-[[(2S)-1-[[(2S)-1-[[(2S)-4-amino-1-[[(2S,3R)-1-[[(2S)-6-amino-1-[[(2S)-1-[[(2S)-4-amino-1-[[(2S)-1-[[(2S)-4-amino-1-[[(2S)-4-amino-1-[[(2S,3S)-1-[[(1S)-1-carboxyethyl]amino]-3-methyl-1-oxopentan-2-yl]amino]-1,4-dioxobutan-2-yl]amino]-1,4-dioxobutan-2-yl]amino]-5-carbamimidamido-1-oxopentan-2-yl]amino]-1,4-dioxobutan-2-yl]amino]-5-carbamimidamido-1-oxopentan-2-yl]amino]-1-oxohexan-2-yl]amino]-3-hydroxy-1-oxobutan-2-yl]amino]-1,4-dioxobutan-2-yl]amino]-4-methylsulfanyl-1-oxobutan-2-yl]amino]-4-methyl-1-oxopentan-2-yl]amino]-3-(1H-indol-3-yl)-1-oxopropan-2-yl]amino]-1,5-dioxopentan-2-yl]amino]-3-methyl-1-oxobutan-2-yl]amino]-1-oxo-3-phenylpropan-2-yl]amino]-3-[[(2S)-5-amino-2-[[(2S)-2-[[(2S)-2-[[(2S)-2-[[(2S)-2-[[(2S)-2-[[(2S)-2-[[(2S)-2-[[(2S)-6-amino-2-[[(2S)-2-[[(2S)-2-[[(2S)-2-[[(2S)-2-[[(2S,3R)-2-[[(2S)-2-[[(2S,3R)-2-[[2-[[(2S)-5-amino-2-[[(2S)-2-[[(2S)-2-amino-3-(1H-imidazol-4-yl)propanoyl]amino]-3-hydroxypropanoyl]amino]-5-oxopentanoyl]amino]acetyl]amino]-3-hydroxybutanoyl]amino]-3-phenylpropanoyl]amino]-3-hydroxybutanoyl]amino]-3-hydroxypropanoyl]amino]-3-carboxypropanoyl]amino]-3-(4-hydroxyphenyl)propanoyl]amino]-3-hydroxypropanoyl]amino]hexanoyl]amino]-3-(4-hydroxyphenyl)propanoyl]amino]-4-methylpentanoyl]amino]-3-carboxypropanoyl]amino]-3-hydroxypropanoyl]amino]-5-carbamimidamidopentanoyl]amino]-5-carbamimidamidopentanoyl]amino]propanoyl]amino]-5-oxopentanoyl]amino]-4-oxobutanoic acid Chemical compound [H]N[C@@H](CC1=CNC=N1)C(=O)N[C@@H](CO)C(=O)N[C@@H](CCC(N)=O)C(=O)NCC(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CO)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](CO)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CO)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](C)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC1=CNC2=C1C=CC=C2)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](C)C(O)=O DDYAPMZTJAYBOF-ZMYDTDHYSA-N 0.000 description 1
- 108010039627 Aprotinin Proteins 0.000 description 1
- BTBUEUYNUDRHOZ-UHFFFAOYSA-N Borate Chemical compound [O-]B([O-])[O-] BTBUEUYNUDRHOZ-UHFFFAOYSA-N 0.000 description 1
- 241000700198 Cavia Species 0.000 description 1
- 108010088406 Glucagon-Like Peptides Proteins 0.000 description 1
- SXRSQZLOMIGNAQ-UHFFFAOYSA-N Glutaraldehyde Chemical compound O=CCCCC=O SXRSQZLOMIGNAQ-UHFFFAOYSA-N 0.000 description 1
- 108091006905 Human Serum Albumin Proteins 0.000 description 1
- 102000008100 Human Serum Albumin Human genes 0.000 description 1
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 description 1
- UKAUYVFTDYCKQA-VKHMYHEASA-N L-homoserine Chemical group OC(=O)[C@@H](N)CCO UKAUYVFTDYCKQA-VKHMYHEASA-N 0.000 description 1
- ZLMJMSJWJFRBEC-UHFFFAOYSA-N Potassium Chemical compound [K] ZLMJMSJWJFRBEC-UHFFFAOYSA-N 0.000 description 1
- 229920005654 Sephadex Polymers 0.000 description 1
- 239000012507 Sephadex™ Substances 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 239000002671 adjuvant Substances 0.000 description 1
- 150000001299 aldehydes Chemical class 0.000 description 1
- 125000000539 amino acid group Chemical group 0.000 description 1
- 150000001413 amino acids Chemical class 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- 235000015278 beef Nutrition 0.000 description 1
- 239000007853 buffer solution Substances 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 239000003610 charcoal Substances 0.000 description 1
- 230000007423 decrease Effects 0.000 description 1
- 206010012601 diabetes mellitus Diseases 0.000 description 1
- 238000000502 dialysis Methods 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 239000012530 fluid Substances 0.000 description 1
- 238000004108 freeze drying Methods 0.000 description 1
- 230000002496 gastric effect Effects 0.000 description 1
- ZJJXGWJIGJFDTL-UHFFFAOYSA-N glipizide Chemical compound C1=NC(C)=CN=C1C(=O)NCCC1=CC=C(S(=O)(=O)NC(=O)NC2CCCCC2)C=C1 ZJJXGWJIGJFDTL-UHFFFAOYSA-N 0.000 description 1
- 230000003053 immunization Effects 0.000 description 1
- 238000002649 immunization Methods 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 239000008363 phosphate buffer Substances 0.000 description 1
- 235000015277 pork Nutrition 0.000 description 1
- 229910052700 potassium Inorganic materials 0.000 description 1
- 239000011591 potassium Substances 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 238000001556 precipitation Methods 0.000 description 1
- 102000004196 processed proteins & peptides Human genes 0.000 description 1
- 210000002966 serum Anatomy 0.000 description 1
- 229910000029 sodium carbonate Inorganic materials 0.000 description 1
- 239000012475 sodium chloride buffer Substances 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 238000003756 stirring Methods 0.000 description 1
- 229940108519 trasylol 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
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/531—Production of immunochemical test materials
Landscapes
- Health & Medical Sciences (AREA)
- Immunology (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Molecular Biology (AREA)
- Biomedical Technology (AREA)
- Chemical & Material Sciences (AREA)
- Hematology (AREA)
- Urology & Nephrology (AREA)
- Biotechnology (AREA)
- Biochemistry (AREA)
- Cell Biology (AREA)
- Food Science & Technology (AREA)
- Medicinal Chemistry (AREA)
- Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- Microbiology (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Investigating Or Analysing Biological Materials (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
- Peptides Or Proteins (AREA)
Description
産業上の利用分野
本発明は新規なペプチド−蛋白複合体から成る
抗原を製造する方法、更に詳しくは膵グルカゴン
及びこれに類似するグルカゴン様物質（消化管グ
ルカゴン、GLI）の両者に対して強い交叉反応性
を示し、また得られる抗体の交叉反応率が一定で
あり、それ故血液内全グルカゴンの正確な定量、
惹いては上記GLIの正確な定量が可能な新しいグ
ルカゴン抗体を提供する抗原の製造法に関する。
従来の技術
従来より生理的膵臓ホモンの１種である膵グル
カゴン及びGLIが、殊に糖吸収代謝に関与し、従
つて血液内の之等グルカゴン濃度を定量すること
によつて、糖尿病等の各種病理状態の診断が可能
となることはよく知られている。しかして従来上
記膵グルカゴンに対する研究はさかんに行なわ
れ、その正確な定量を可能とする抗体即ちヒトの
膵グルカゴンに対して特異的に反応する抗体の開
発が種々行なわれ、本発明者らも先に該特異抗体
を膵グルカゴンの18−29もしくは19−29ペプチド
鎖をハプテンとする抗原より再現性よく製造する
に成功した（特開昭53−99320号）。しかしながら
GLIに対する研究はほとんど行なわれておらず、
その構造及び糖代謝との関連についても不明な点
が多く、勿論GLIに特異的に反応する抗体は未だ
全く知られていない。
発明が解決しようとする問題点
本発明者らはかねてよりグルカゴンの糖代謝に
対する役割、その関連を解明する過程において
種々のグルカゴン抗体（AGS）につき研究を重
ねてきたが、膵グルカゴン及びGLIに交叉反応す
る公知の各種抗体即ち非特異抗体は、上記膵グル
カゴン及びGLIに同程度に反応するものではな
く、その交叉性の程度が極めてばらついており
（両者に対する希釈スロープは平行でない）、従つ
て膵グルカゴンに相当量で表わされるGLI量は、
希釈倍率によつて実際のGLI量とはかけはなれた
ものとなり、しかも異なつた抗体によつて、同一
血漿を測定した場合に測定される全グルカゴン値
は抗体により大きく異なり全く測定間では比較で
きないものであつた。即ち膵グルカゴン特異抗体
により測定される膵グルカゴン値を差し引いて
も、全GLIの正確な定量は不可能であり、また各
非特異抗体のGLIに対する希釈スロープは該抗体
固有のものであり、他の非特異抗体については全
く利用できないものであつた。上記現状に鑑み本
発明者らは更に種々研究を重ねた結果、偶然にも
膵グルカゴンの１〜26ペプチド−ホモセリン及び
（又は）膵グルカゴンの１〜26ペプチド−ホモセ
リン・ラクトンからなるペプチドをハプテンとす
る特定の抗原の作成に成功すると共に、該抗原を
利用して得られる抗体は、膵グルカゴンとGLIと
の両者に完全な交叉反応性を有し、従つて該抗体
を利用して測定される全グルカゴン値は、膵グル
カゴンとGLIとの合計値に一致し、従つて該値よ
り膵グルカゴン特異抗体によつて測定した膵グル
カゴン値を差し引く時には、正確なGLI値が定量
でき、しかも上記抗原からは常に再現性よく、上
記完全な交叉反応性を有する抗体が収得できるこ
とを見い出した。
問題点を解決するための手段
本発明はこの新しい知見に基づいて完成された
ものである。即ち本発明は、平面構造式
Ｈ−His−Ser−Gln−Gly−Thr−Phe−Thr
−Ser−Asp−Tyr−Ser−Lys−Tyr−Leu
−Asp−Ser−Arg−Arg−Ala−Gln−Asp
−Phe−Val−Gln−Trp−Leu−ホモセリン
〔1a〕
で表わされる膵グルカゴン１〜26ペプチド−ホモ
セリン及び平面構造式
Ｈ−His−Ser−Gln−Gly−Thr−Phe−Thr
−Ser−Asp−Tyr−Ser−Lys−Tyr−Leu
−Asp−Ser−Arg−Arg−Ala−Gln−Asp
−Phe−Val−Gln−Trp−Leu−ホモセリン
・ラクトン 〔1b〕
で表わされる膵グルカゴン１〜26ペプチド−ホモ
セリン・ラクトンから選ばれた少なくとも１種の
ペブチドをハプテンとし、これを一般式
OHC−（CH2）o−CHO 〔〕
〔式中ｎは１〜５の整数を示す〕
で表わされるジアルデヒドの存在下に、担体とす
る蛋白質と反応させることを特徴とするペプチド
−蛋白複合体から成る抗原の製造方法に係る。
本明細書においてハフテンとして用いる上記ペ
プチドの表示は、IUPACにより採択されている
アミノ酸命名法における略号によるアミノ酸残基
の表示法に従うものである。
本発明方法においては、ハプテンとして上記式
〔1a〕及び（又は）〔1b〕で表わされるペプチド
を用いることを必須とする。該ペプチドはヒトの
膵グルカゴンの１〜26ペプチド鎖にホモセリン又
はホモセリン・ラクトンが結合したもの及びそれ
らの混合物であり、例えばヒトの膵グルカゴンに
通常のペプタイドの化学反応を適用することによ
り容易に調製できる。より具体的には膵グルカゴ
ンのギ酸溶液にブロムシアンのギ酸溶液を加えて
反応させ、反応後ゲル過等を行なえばよい。
また上記一般式〔〕で表わされるジアルデヒ
ドは、上記ハプテンと、担体とする蛋白質とを結
合させる仲介物として働くものであり、具体的に
は、マロンアルデヒド、スクシンアルデヒド、グ
ルタルアルデヒド及びアジポアルデヒド等を使用
できる。
更に担体とする蛋白質は、従来よりこの種抗原
の製造に当り慣用される通常の蛋白質がいずれも
使用できる。代表的には例えば馬血清アルブミ
ン、牛血清アルブミン、兎血清アルブミン、ヒト
血清アルブミン、馬血清グロブリン、格子血清グ
ロブリン、兎血清グロブリン、ヒト血清グロブリ
ン等を例示できる。
本発明の抗原は上記ハプテンと蛋白質とをジア
ルデヒドの存在下に反応させることにより製造さ
れる。上記反応は、水溶液もしくはPH７〜10の通
常の緩衝液中好ましくはPH８〜９の緩衝液中で０
〜40℃好ましくは室温付近で行なわれ、約１〜24
時間で反応は完結する。上記において用いられる
代表的緩衝液としては、、次のようなものを例示
できる。
0.2N水酸化ナトリウム−0.2Mホウ酸−0.2M塩
化カリウム緩衝液、
0.2M炭酸ナトリウム−0.2Mホウ酸−0.2M塩化
カリウム緩衝液、
0.05M四ホウ酸ナトリウム−0.2Mホウ酸−
0.05M塩化ナトリウム緩衝液、
0.1Mリン酸二水素カリウム−0.05M四ホウ酸ナ
トリウム緩衝液、
上記においてハプテン、ジアルデヒド及び担体
の使用割合は適宜に決定できるが、通常担体に対
してハプテンを５〜20倍モル好ましくは10〜15倍
モル、及びジアルデヒド３〜20倍モル好ましくは
５〜10倍モルとするのがよい。上記反応によりジ
アルデヒドを仲介させて担体とハプテンとが結合
した本発明のペプチド−蛋白複合体から成る抗原
が収得される。反応終了後得られる抗原は常法に
従い、例えば透析法、ゲル過法、分別沈澱法等
により容易に単離精製できる。又該抗原は通常の
凍結乾燥法により保存できる。
上記で得られる抗原による抗体の作成に当つて
は、常法に従い抗原を哺乳動物に投与し、生体内
に産生される抗体を採取する方法を採用できる。
該抗体の製造に供せられる哺乳動物としては特に
制限はないが、通常兎やモルモツトを用いるのが
望ましい。抗体の産生に当つては、上記により得
られる抗原の所定量を生理食塩水で適当濃度に希
釈し、フロインドの補助液（Complete Freund′s
Adjuvant）と混合して懸濁液を調整し、これを
哺乳動物体に投与すればよい。例えば兎に上記懸
濁液を皮下注射（抗原の量として0.5〜２mg／
回）し、以後２週間毎に２〜10ケ月好ましくは４
〜６ケ月間投与し免疫化させればよい。抗体の採
取は、上記懸濁液の最終投与後抗体が多量産出さ
れる時期、通常上記最終投与１〜２週間経過後、
免疫化された動物から採血し、これを遠心分離後
血清を分離採取することにより行なわれる。殊に
上記方法によれば、用いる抗原の特殊性に基づい
て、常に安定して充分高力価、高感度のグルカゴ
ン抗体を再現性よく収得できる利点がある。
かくして得られる抗体は、上記の通り斯界で要
望されているRIA等法によるグルカゴンの定量を
可能とするものであり、糖尿病をはじめとして膵
グルカゴン及び消化管グルカゴンの関与する各種
病理状態の診断等に有用である。
以下本発明を更に詳しく説明するための参考例
及び実施例を挙げるが、本発明はこれらに限定さ
れるものではない。
参考例 １
ヒトの膵グルカゴン（シグマ社製、ビーフ・ポ
ーク・グルカゴン）60mgを70％ギ酸５mlに溶か
し、これに1.43M BrCNの70％ギ酸溶液１mlを加
え、室温下に24時間撹拌反応させ、反応終了後セ
フアデツクスＧ−25を用いてゲル過（展開溶媒
0.2M酢酸）して膵グルカゴンの１〜26ペプチド
鎖−ホモセリンおよび膵グルカゴンの１〜26ペプ
チド鎖−ホモセリン・ラクトンの混合物40mgを得
る。得られたペプチド混合物を次いで凍結乾燥す
る。
実施例 １
上記参考例１で得たペプチド混合物の30mg及び
牛血清アルブミン（BSA）40mgを0.1Mホウ酸緩
衝液15ml（PH＝8.5）に加えた液中に0.2Mグルタ
ルアルデヒド溶液５mlを滴下する。反応混合物を
室温下６時間撹拌して反応させる。
得られた反応混合物を４℃で24時間透析（透析
液生理食塩水）し、透析内液を凍結乾燥して白色
粉末状の本発明抗原（ペプチド−BSA複合体）
71mgを得る。
抗体の製造例１
無作為に選択した兎５羽（〜）に、実施例
１で得た本発明抗原（複合体）７mgを1.8mlの生
理食塩水に溶解後これにフロインドの補助液2.7
mlを加えて調製した懸濁液を、兎１羽につき１ml
づつ皮下投与し、２週間後更に同量を皮下投与す
る。以後２週間間隔で別途に調製した懸濁液（抗
原３mg、生理食塩水３ml及びフロインドの補助液
３ml）を同様にして3.5ケ月間投与していき、試
験動物を免疫化する。最終投与10日経過後試験動
物から採血し、遠心分離して抗血清を採取し、抗
体〜の夫々を得る。
〈力価の測定〉
得られた抗体〜の力価を次の通り測定す
る。即ち上記抗体を夫々生理食塩水で10、102、
103、104及び105倍に希釈し、之等の夫々100μ
に125I−グルカゴン100μ及び0.05Mリン酸緩衝
液（PH7.4）（0.25％BSA、0.1％NaN3及び0.01M
EDTAを含む）300μを加え４℃で48〜72時間
インキユベートし、生成した抗血清と、125I−グ
ルカゴンとの結合体を、デキストラン−活性炭法
及び遠心分離法（４℃、15分間、3000pm）によ
り未反応125I−グルカゴンから分離し、その放射
線をカウントし、各希釈濃度における抗血清の
125I−グルカゴンとの結合率（％）を測定する。
結合率（％）が50％となる抗血清の最終希釈倍率
即ち抗体の力価を下記第１表に示す。
INDUSTRIAL APPLICATION FIELD The present invention provides a method for producing an antigen consisting of a novel peptide-protein complex, and more specifically, a method for producing an antigen consisting of a novel peptide-protein complex, and more specifically, a method for producing an antigen consisting of a novel peptide-protein complex. The cross-reactivity of the antibodies obtained is constant and therefore accurate quantification of total glucagon in the blood.
The present invention also relates to a method for producing an antigen that provides a new glucagon antibody capable of accurately quantifying GLI. Conventional technology Pancreatic glucagon and GLI, which are types of physiological pancreatic homomones, have been particularly involved in sugar absorption and metabolism. It is well known that it enables the diagnosis of pathological conditions. However, research on the pancreatic glucagon has been actively conducted in the past, and various antibodies that enable accurate quantification, that is, antibodies that specifically react with human pancreatic glucagon, have been developed. They succeeded in producing the specific antibody with high reproducibility using an antigen containing the 18-29 or 19-29 peptide chain of pancreatic glucagon as a hapten (Japanese Patent Application Laid-open No. 99320/1983). however
There has been little research on GLI,
There are many unknowns about its structure and its relationship to sugar metabolism, and of course, there are still no known antibodies that specifically react with GLI. Problems to be Solved by the Invention The present inventors have been conducting research on various glucagon antibodies (AGS) in the process of elucidating the role and relationship of glucagon in sugar metabolism, The various antibodies known to react, that is, non-specific antibodies, do not react to the same degree with pancreatic glucagon and GLI, and their degree of cross-reactivity varies greatly (the dilution slopes for both are not parallel). The amount of GLI expressed as the equivalent amount of glucagon is
Depending on the dilution ratio, the actual GLI amount may be far from the actual amount, and the total glucagon value measured when the same plasma is measured with different antibodies varies greatly depending on the antibody and cannot be compared between measurements at all. It was hot. That is, even if the pancreatic glucagon value measured by a pancreatic glucagon-specific antibody is subtracted, accurate quantification of total GLI is not possible, and the dilution slope of each non-specific antibody against GLI is unique to that antibody, and other Non-specific antibodies could not be used at all. In view of the above-mentioned current situation, the present inventors further conducted various studies, and as a result, coincidentally, a peptide consisting of pancreatic glucagon 1-26 peptide - homoserine and/or pancreatic glucagon 1-26 peptide - homoserine lactone was used as a hapten. In addition to successfully creating a specific antigen, the antibody obtained using the antigen has complete cross-reactivity with both pancreatic glucagon and GLI, and therefore can be measured using the antibody. The total glucagon value corresponds to the sum of pancreatic glucagon and GLI, and therefore, when subtracting the pancreatic glucagon value measured by a pancreatic glucagon-specific antibody from this value, an accurate GLI value can be quantified. found that antibodies having the above-mentioned perfect cross-reactivity could be obtained with good reproducibility. Means for Solving the Problems The present invention has been completed based on this new knowledge. That is, the present invention has a planar structural formula H-His-Ser-Gln-Gly-Thr-Phe-Thr -Ser-Asp-Tyr-Ser-Lys-Tyr-Leu -Asp-Ser-Arg-Arg-Ala-Gln- Pancreatic glucagon 1-26 peptide-homoserine represented by Asp -Phe-Val-Gln-Trp-Leu-homoserine [1a] and the planar structural formula H-His-Ser-Gln-Gly-Thr-Phe-Thr -Ser-Asp -Tyr-Ser-Lys-Tyr-Leu -Asp-Ser-Arg-Arg-Ala-Gln-Asp -Phe-Val-Gln-Trp-Leu-Homoserine lactone [1b] Pancreatic glucagon 1-26 peptide - At least one peptide selected from homoserine lactone is used as a hapten, and this is a dialdehyde represented by the general formula OHC-(CH 2 ) o -CHO [] [wherein n represents an integer from 1 to 5] The present invention relates to a method for producing an antigen comprising a peptide-protein complex, which is characterized by reacting the antigen with a protein used as a carrier in the presence of a peptide-protein complex. The representation of the above-mentioned peptides used as haftenes herein is in accordance with the method of representing amino acid residues by abbreviations in the amino acid nomenclature adopted by IUPAC. In the method of the present invention, it is essential to use a peptide represented by the above formula [1a] and/or [1b] as the hapten. The peptide is a peptide chain 1 to 26 of human pancreatic glucagon bound to homoserine or homoserine lactone, or a mixture thereof, and can be easily prepared, for example, by applying a common peptide chemical reaction to human pancreatic glucagon. can. More specifically, a formic acid solution of bromocyanide may be added to a formic acid solution of pancreatic glucagon to cause a reaction, and after the reaction, gel filtration or the like may be performed. Further, the dialdehyde represented by the above general formula [] acts as an intermediary that binds the above hapten to the protein used as a carrier. Aldehydes etc. can be used. Further, as the protein used as a carrier, any conventional protein conventionally used in the production of this type of antigen can be used. Representative examples include horse serum albumin, bovine serum albumin, rabbit serum albumin, human serum albumin, horse serum globulin, lattice serum globulin, rabbit serum globulin, and human serum globulin. The antigen of the present invention is produced by reacting the above hapten and protein in the presence of dialdehyde. The above reaction can be carried out in an aqueous solution or in a conventional buffer of pH 7-10, preferably in a buffer of pH 8-9.
~40℃, preferably around room temperature, about 1~24℃
The reaction completes in time. Typical buffer solutions used in the above may include the following. 0.2N sodium hydroxide - 0.2M boric acid - 0.2M potassium chloride buffer, 0.2M sodium carbonate - 0.2M boric acid - 0.2M potassium chloride buffer, 0.05M sodium tetraborate - 0.2M boric acid -
0.05M sodium chloride buffer, 0.1M potassium dihydrogen phosphate-0.05M sodium tetraborate buffer. In the above, the ratio of hapten, dialdehyde and carrier can be determined as appropriate, but usually 50% of hapten to carrier is used. -20 times the mole, preferably 10 to 15 times the mole, and dialdehyde 3 to 20 times the mole, preferably 5 to 10 times the mole. Through the above reaction, an antigen consisting of the peptide-protein complex of the present invention, in which a carrier and a hapten are bonded via dialdehyde, is obtained. The antigen obtained after completion of the reaction can be easily isolated and purified by conventional methods such as dialysis, gel filtration, and fractional precipitation. The antigen can also be preserved by conventional freeze-drying methods. In producing antibodies using the antigen obtained above, a conventional method can be employed in which the antigen is administered to a mammal and the antibodies produced in the body are collected.
There are no particular restrictions on the mammal used for the production of the antibody, but it is usually desirable to use rabbits or guinea pigs. For antibody production, dilute the prescribed amount of the antigen obtained above with physiological saline to an appropriate concentration and add Freund's auxiliary solution (Complete Freund's
Adjuvant) to prepare a suspension, which may be administered to a mammal. For example, subcutaneously inject the above suspension into rabbits (the amount of antigen is 0.5-2 mg/
2 times) and then every 2 weeks for 2 to 10 months, preferably 4 times.
It is sufficient to administer the drug for ~6 months to achieve immunization. Antibodies are collected at a time when antibodies are produced in large quantities after the final administration of the suspension, usually 1 to 2 weeks after the final administration.
This is carried out by collecting blood from an immunized animal, centrifuging it, and then separating and collecting the serum. In particular, the above method has the advantage that glucagon antibodies with a sufficiently high titer and high sensitivity can always be obtained stably and reproducibly based on the specificity of the antigen used. As mentioned above, the antibodies obtained in this way enable the quantitative determination of glucagon using methods such as RIA, which are in demand in the field, and are useful for the diagnosis of various pathological conditions involving pancreatic glucagon and gastrointestinal glucagon, including diabetes. Useful. Reference examples and examples are given below to explain the present invention in more detail, but the present invention is not limited thereto. Reference Example 1 Dissolve 60 mg of human pancreatic glucagon (Beef Pork Glucagon, manufactured by Sigma) in 5 ml of 70% formic acid, add 1 ml of 70% formic acid solution of 1.43 M BrCN, and stir and react at room temperature for 24 hours. After the reaction is complete, gel filtration (developing solvent) is carried out using Sephadex G-25.
0.2M acetic acid) to obtain 40 mg of a mixture of 1-26 peptide chains of pancreatic glucagon-homoserine and 1-26 peptide chains of pancreatic glucagon-homoserine lactone. The resulting peptide mixture is then lyophilized. Example 1 5 ml of 0.2 M glutaraldehyde solution is dropped into a solution in which 30 mg of the peptide mixture obtained in Reference Example 1 and 40 mg of bovine serum albumin (BSA) are added to 15 ml of 0.1 M borate buffer (PH = 8.5). . The reaction mixture was stirred at room temperature for 6 hours to react. The resulting reaction mixture was dialyzed at 4°C for 24 hours (dialysate physiological saline), and the dialyzed fluid was lyophilized to give the antigen of the present invention (peptide-BSA complex) in the form of a white powder.
Get 71 mg. Antibody Production Example 1 Five randomly selected rabbits (~) were given 7 mg of the antigen (complex) of the present invention obtained in Example 1, dissolved in 1.8 ml of physiological saline, and then 2.7 mL of Freund's auxiliary solution was added to the solution.
1 ml of the suspension prepared by adding 1 ml to each rabbit.
Two weeks later, the same amount is administered subcutaneously. Thereafter, a separately prepared suspension (3 mg of antigen, 3 ml of physiological saline, and 3 ml of Freund's auxiliary solution) was administered in the same manner at 2-week intervals for 3.5 months to immunize the test animals. 10 days after the final administration, blood is collected from the test animal and centrifuged to collect antiserum to obtain each of the antibodies. <Measurement of titer> The titer of the obtained antibody ~ is determined as follows. That is, the above antibodies were dissolved in physiological saline at 10, 10 2 ,
Diluted 10 3 , 10 4 and 10 5 times, and 100μ of each of these.
125I -glucagon 100μ and 0.05M phosphate buffer (PH7.4) (0.25% BSA, 0.1% NaN3 and 0.01M
EDTA (containing EDTA) was added and incubated for 48 to 72 hours at 4°C. The resulting antiserum and 125 I-glucagon conjugate were separated using the dextran-activated charcoal method and centrifugation (4°C, 15 minutes, 3000 pm). 125 I-glucagon is separated from unreacted 125 I-glucagon, its radiation is counted, and the antiserum at each dilution concentration is
Measure the binding rate (%) with 125 I-glucagon.
The final dilution ratio of the antiserum at which the binding rate (%) is 50%, that is, the antibody titer, is shown in Table 1 below.
【表】
〈感度及び交叉性の測定〉
この試験は、一定量のグルカゴン抗体に結合す
る標識グルカゴンと非標識グルカゴンの比は、溶
液中の之等各グルカゴン濃度比に一致し、標識グ
ルカゴン濃度を一定にした時非標識グルカゴン
（測定されるべきグルカゴン）の濃度が増加する
に従い、グルカゴン抗体と結合する結合型標識グ
ルカゴン（Ｂ）の量は減少し、溶液中に遊離して
存在する遊離型標識グルカゴン（Ｆ）の量は増加
するという原理に基づき行なわれたものである。
供試試料として膵グルカゴン（標識グルカゴン、
濃度10pｇ/ml〜100000nｇ/ml、）並びにgut GLI
（濃度0.203〜400μｇ凍結乾燥物／ml、ケニーの
方法〔Kenny.A.J.、J.Clin.Endocrinol.Matab.、
15、1089〜1105（1955）〕により得られたピーク
）を使用する。また標識グルカゴンとして125I
−グルカゴン（10000cpm）を用いる。
上記供試グルカゴン試料又はgut GLI試料の
200μ、125I−グルカゴン200μ、抗体の製造
例２で得た適当な力価の抗体〜の夫々一者づ
つ200μ及びトラジロール（バイエル社製、
1000KIU）100μを混合し、４℃で48〜72時間
インキユベート後、デキストラン炭末法により結
合型標識グルカゴン（Ｂ）と遊離型標識グルカゴ
ン（Ｆ）との夫々の放射線をカウントし、用いた
各抗体の力価に相当する結合率（Bo）を100％と
して、各供試試料の濃度における結合型標識グル
カゴン（Ｂ）の百分率を求める。供試膵グルカゴ
ンにおける希釈スロープ及び該膵グルカゴンの等
価としての供試gut GLIにおける希釈スロープを
第１図乃至第５図に示す。各図は夫々抗体〜
を用いて得られた上記各スロープを示すものであ
り、各図中縦軸は結合％（Ｂ／Bo×100）を、横
軸は、膵グルカゴン濃度（pｇ/ml）とgut GLI濃
度（μｇ/ml）とを示す。又各図において曲線イ
は膵グルカゴンを、曲線ロはgut GLIを夫々示
す。
また上記第１図〜第５図より膵グルカゴンと
gut GLIとの交叉性及び結合％が５０％の時の抗
体〜の感度を、膵グルカゴン濃度（nｇ/
ml）として求めると、下記第２表の通りとなる。[Table] <Measurement of sensitivity and cross-reactivity> In this test, the ratio of labeled glucagon to unlabeled glucagon that binds to a fixed amount of glucagon antibody corresponds to the ratio of the respective glucagon concentrations in the solution, and the labeled glucagon concentration is As the concentration of unlabeled glucagon (glucagon to be measured) increases, the amount of bound labeled glucagon (B) that binds to the glucagon antibody decreases, reducing the amount of free labeled glucagon (B) present in the solution. This was done based on the principle that the amount of glucagon (F) increases.
Pancreatic glucagon (labeled glucagon,
Concentration 10pg/ml ~ 100000ng/ml, ) and gut GLI
(Concentration 0.203-400μg lyophilizate/ml, Kenny's method [Kenny.AJ, J.Clin.Endocrinol.Matab.,
15, 1089-1105 (1955)] is used. Also labeled glucagon as 125I
- Using glucagon (10000cpm). of the above test glucagon sample or gut GLI sample.
200μ, 200μ of 125 I-glucagon, 200μ of each of the antibodies of appropriate titer obtained in Antibody Production Example 2, and trasylol (manufactured by Bayer).
After mixing 100μ of 1000KIU) and incubating at 4°C for 48 to 72 hours, the radiation of bound labeled glucagon (B) and free labeled glucagon (F) was counted by the dextran charcoal method, and the radiation of each antibody used was counted. Assuming that the binding rate (Bo) corresponding to the titer is 100%, determine the percentage of bound labeled glucagon (B) at the concentration of each test sample. The dilution slope of the pancreatic glucagon sample and the gut GLI sample as an equivalent of the pancreatic glucagon are shown in FIGS. 1 to 5. Each figure is an antibody~
In each figure, the vertical axis represents binding % (B/Bo×100), and the horizontal axis represents pancreatic glucagon concentration (pg/ml) and gut GLI concentration (μg /ml). In each figure, curve A indicates pancreatic glucagon, and curve B indicates gut GLI. Also, from Figures 1 to 5 above, pancreatic glucagon and
gut Glucagon concentration (ng/
ml), it is as shown in Table 2 below.
【表】
上記第１図〜第５図及び第２表より明らかな通
り、本発明抗原の利用によれば、無作為に選択し
た兎５羽中４羽において、膵グルカゴン及びgut
GLIに対し完全に交叉反応性を示す抗体を収得で
き、その再現性は極めて高いことが判る。また之
等の抗体は感度も良好であり、しかも上記の通り
完全な交叉性を有する所から、その利用によつて
gut GLIの正確な定量を可能とすることが明らか
である。[Table] As is clear from Figures 1 to 5 and Table 2 above, by using the antigen of the present invention, pancreatic glucagon and gut
It can be seen that an antibody showing complete cross-reactivity to GLI was obtained, and its reproducibility was extremely high. In addition, these antibodies have good sensitivity and, as mentioned above, have complete cross-reactivity, so their use
It is clear that gut GLI can be accurately quantified.
第１図乃至第５図は本発明方法によつて得られ
る抗原から得た抗体の膵グルカゴン及びgut GLI
に対する交叉性を示すグラフである。
Figures 1 to 5 show pancreatic glucagon and gut GLI of antibodies obtained from antigens obtained by the method of the present invention.
FIG.
Claims (1)
セリン及び平面構造式 Ｈ−His−Ser−Gln−Gly−Thr−Phe−Thr −Ser−Asp−Tyr−Ser−Lys−Tyr−Leu −Asp−Ser−Arg−Arg−Ala−Gln−Asp −Phe−Val−Gln−Trp−Leu−ホモセリン ・ラクトン で表わされる膵グルカゴン１〜26ペプチド−ホモ
セリン・ラクトンから選ばれた少なくとも１種の
ペプチドをハプテンとし、これを一般式 OHC−（CH2）o−CHO 〔式中ｎは１〜５の整数を示す〕 で表わされるジアルデヒドの存在下に、担体とす
る蛋白質と反応させることを特徴とするペプチド
−蛋白複合体から成る抗原の製造方法。[Claims] 1 Planar structural formula H-His-Ser-Gln-Gly-Thr-Phe-Thr -Ser-Asp-Tyr-Ser-Lys-Tyr-Leu -Asp-Ser-Arg-Arg-Ala- Gln-Asp -Phe-Val-Gln-Trp-Leu-Peptide of pancreatic glucagon 1-26 expressed by homoserine-homoserine and the planar structural formula H-His-Ser-Gln-Gly-Thr-Phe-Thr -Ser-Asp- Pancreatic glucagon 1-26 peptide expressed by Tyr-Ser-Lys-Tyr-Leu -Asp-Ser-Arg-Arg-Ala-Gln-Asp -Phe-Val-Gln-Trp-Leu-homoserine lactone-homoserine lactone A hapten is at least one peptide selected from 1. A method for producing an antigen comprising a peptide-protein complex, which comprises reacting with a protein.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP16551984A JPS60149971A (en) | 1984-08-06 | 1984-08-06 | Preparation of antigen |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP16551984A JPS60149971A (en) | 1984-08-06 | 1984-08-06 | Preparation of antigen |
Related Parent Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP54098840A Division JPS6037428B2 (en) | 1979-08-01 | 1979-08-01 | Antibodies and their production methods |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPS60149971A JPS60149971A (en) | 1985-08-07 |
| JPS6114465B2 true JPS6114465B2 (en) | 1986-04-18 |
Family
ID=15813931
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP16551984A Granted JPS60149971A (en) | 1984-08-06 | 1984-08-06 | Preparation of antigen |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPS60149971A (en) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5198367A (en) * | 1989-06-09 | 1993-03-30 | Masuo Aizawa | Homogeneous amperometric immunoassay |
-
1984
- 1984-08-06 JP JP16551984A patent/JPS60149971A/en active Granted
Also Published As
| Publication number | Publication date |
|---|---|
| JPS60149971A (en) | 1985-08-07 |
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