JPH0340832B2 - - Google Patents
Info
- Publication number
- JPH0340832B2 JPH0340832B2 JP59203453A JP20345384A JPH0340832B2 JP H0340832 B2 JPH0340832 B2 JP H0340832B2 JP 59203453 A JP59203453 A JP 59203453A JP 20345384 A JP20345384 A JP 20345384A JP H0340832 B2 JPH0340832 B2 JP H0340832B2
- Authority
- JP
- Japan
- Prior art keywords
- antibody
- enzyme
- antigenic determinant
- conjugate
- substance
- 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
- 238000000034 method Methods 0.000 claims description 51
- 102000004190 Enzymes Human genes 0.000 claims description 44
- 108090000790 Enzymes Proteins 0.000 claims description 44
- 230000000890 antigenic effect Effects 0.000 claims description 43
- 239000000126 substance Substances 0.000 claims description 41
- 230000000694 effects Effects 0.000 claims description 13
- 238000006911 enzymatic reaction Methods 0.000 claims description 9
- 229920003169 water-soluble polymer Polymers 0.000 claims description 3
- 229940088598 enzyme Drugs 0.000 description 42
- 239000003446 ligand Substances 0.000 description 22
- 239000000243 solution Substances 0.000 description 21
- 238000006243 chemical reaction Methods 0.000 description 13
- 239000002532 enzyme inhibitor Substances 0.000 description 11
- 229940125532 enzyme inhibitor Drugs 0.000 description 10
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 9
- 238000005259 measurement Methods 0.000 description 9
- 239000000203 mixture Substances 0.000 description 9
- 230000035945 sensitivity Effects 0.000 description 9
- 239000008363 phosphate buffer Substances 0.000 description 8
- 241000283707 Capra Species 0.000 description 7
- 102000013529 alpha-Fetoproteins Human genes 0.000 description 7
- 108010026331 alpha-Fetoproteins Proteins 0.000 description 7
- 239000004382 Amylase Substances 0.000 description 6
- 108010065511 Amylases Proteins 0.000 description 6
- 102000013142 Amylases Human genes 0.000 description 6
- 108010059892 Cellulase Proteins 0.000 description 6
- 235000019418 amylase Nutrition 0.000 description 6
- 239000000427 antigen Substances 0.000 description 6
- 102000036639 antigens Human genes 0.000 description 6
- 108091007433 antigens Proteins 0.000 description 6
- 229940106157 cellulase Drugs 0.000 description 6
- 238000002360 preparation method Methods 0.000 description 6
- 229920002307 Dextran Polymers 0.000 description 5
- 108010001682 Dextranase Proteins 0.000 description 5
- 238000002835 absorbance Methods 0.000 description 5
- 229920000642 polymer Polymers 0.000 description 5
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 5
- 101000827785 Homo sapiens Alpha-fetoprotein Proteins 0.000 description 4
- 101000848653 Homo sapiens Tripartite motif-containing protein 26 Proteins 0.000 description 4
- 241001465754 Metazoa Species 0.000 description 4
- 239000008351 acetate buffer Substances 0.000 description 4
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 4
- KZNICNPSHKQLFF-UHFFFAOYSA-N dihydromaleimide Natural products O=C1CCC(=O)N1 KZNICNPSHKQLFF-UHFFFAOYSA-N 0.000 description 4
- 102000046101 human AFP Human genes 0.000 description 4
- 210000002966 serum Anatomy 0.000 description 4
- 239000000758 substrate Substances 0.000 description 4
- 210000002700 urine Anatomy 0.000 description 4
- 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 3
- 229920005654 Sephadex Polymers 0.000 description 3
- 239000012507 Sephadex™ Substances 0.000 description 3
- 238000001042 affinity chromatography Methods 0.000 description 3
- 125000003277 amino group Chemical group 0.000 description 3
- 238000004458 analytical method Methods 0.000 description 3
- 239000007864 aqueous solution Substances 0.000 description 3
- 210000004369 blood Anatomy 0.000 description 3
- 239000008280 blood Substances 0.000 description 3
- 210000004027 cell Anatomy 0.000 description 3
- 239000003153 chemical reaction reagent Substances 0.000 description 3
- 239000012153 distilled water Substances 0.000 description 3
- 238000002523 gelfiltration Methods 0.000 description 3
- 238000003018 immunoassay Methods 0.000 description 3
- 229920001223 polyethylene glycol Polymers 0.000 description 3
- 238000006116 polymerization reaction Methods 0.000 description 3
- 239000000047 product Substances 0.000 description 3
- 235000018102 proteins Nutrition 0.000 description 3
- 102000004169 proteins and genes Human genes 0.000 description 3
- 108090000623 proteins and genes Proteins 0.000 description 3
- AHTFMWCHTGEJHA-UHFFFAOYSA-N s-(2,5-dioxooxolan-3-yl) ethanethioate Chemical compound CC(=O)SC1CC(=O)OC1=O AHTFMWCHTGEJHA-UHFFFAOYSA-N 0.000 description 3
- 238000003756 stirring Methods 0.000 description 3
- 229960002317 succinimide Drugs 0.000 description 3
- 238000012360 testing method Methods 0.000 description 3
- 125000003396 thiol group Chemical group [H]S* 0.000 description 3
- 102000009027 Albumins Human genes 0.000 description 2
- 108010088751 Albumins Proteins 0.000 description 2
- 102100032487 Beta-mannosidase Human genes 0.000 description 2
- 102000029816 Collagenase Human genes 0.000 description 2
- 108060005980 Collagenase Proteins 0.000 description 2
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 2
- 102000004882 Lipase Human genes 0.000 description 2
- 108090001060 Lipase Proteins 0.000 description 2
- 239000004367 Lipase Substances 0.000 description 2
- 241000283973 Oryctolagus cuniculus Species 0.000 description 2
- 102000016387 Pancreatic elastase Human genes 0.000 description 2
- 108010067372 Pancreatic elastase Proteins 0.000 description 2
- 108091005804 Peptidases Proteins 0.000 description 2
- 239000004365 Protease Substances 0.000 description 2
- 102100037486 Reverse transcriptase/ribonuclease H Human genes 0.000 description 2
- 229920002472 Starch Polymers 0.000 description 2
- 239000002671 adjuvant Substances 0.000 description 2
- 102000004139 alpha-Amylases Human genes 0.000 description 2
- 108090000637 alpha-Amylases Proteins 0.000 description 2
- 229940024171 alpha-amylase Drugs 0.000 description 2
- 108010055059 beta-Mannosidase Proteins 0.000 description 2
- 210000001124 body fluid Anatomy 0.000 description 2
- 239000010839 body fluid Substances 0.000 description 2
- 229920002678 cellulose Polymers 0.000 description 2
- 239000001913 cellulose Substances 0.000 description 2
- 229960002424 collagenase Drugs 0.000 description 2
- 150000001875 compounds Chemical class 0.000 description 2
- 238000000354 decomposition reaction Methods 0.000 description 2
- 201000010099 disease Diseases 0.000 description 2
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 2
- 239000012634 fragment Substances 0.000 description 2
- 125000000524 functional group Chemical group 0.000 description 2
- 239000003112 inhibitor Substances 0.000 description 2
- 230000005764 inhibitory process Effects 0.000 description 2
- 238000004255 ion exchange chromatography Methods 0.000 description 2
- 235000019421 lipase Nutrition 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
- 210000003200 peritoneal cavity Anatomy 0.000 description 2
- 125000001997 phenyl group Chemical group [H]C1=C([H])C([H])=C(*)C([H])=C1[H] 0.000 description 2
- 239000000376 reactant Substances 0.000 description 2
- 239000012086 standard solution Substances 0.000 description 2
- 239000008107 starch Substances 0.000 description 2
- 235000019698 starch Nutrition 0.000 description 2
- -1 succinimide ester Chemical class 0.000 description 2
- 229920003176 water-insoluble polymer Polymers 0.000 description 2
- GOYDNIKZWGIXJT-UHFFFAOYSA-N 1,2-difluorobenzene Chemical compound FC1=CC=CC=C1F GOYDNIKZWGIXJT-UHFFFAOYSA-N 0.000 description 1
- RYHBNJHYFVUHQT-UHFFFAOYSA-N 1,4-Dioxane Chemical compound C1COCCO1 RYHBNJHYFVUHQT-UHFFFAOYSA-N 0.000 description 1
- AZQWKYJCGOJGHM-UHFFFAOYSA-N 1,4-benzoquinone Chemical compound O=C1C=CC(=O)C=C1 AZQWKYJCGOJGHM-UHFFFAOYSA-N 0.000 description 1
- OGMADIBCHLQMIP-UHFFFAOYSA-N 2-aminoethanethiol;hydron;chloride Chemical compound Cl.NCCS OGMADIBCHLQMIP-UHFFFAOYSA-N 0.000 description 1
- HRPVXLWXLXDGHG-UHFFFAOYSA-N Acrylamide Chemical compound NC(=O)C=C HRPVXLWXLXDGHG-UHFFFAOYSA-N 0.000 description 1
- 229940122816 Amylase inhibitor Drugs 0.000 description 1
- 244000186140 Asperula odorata Species 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- 102000004506 Blood Proteins Human genes 0.000 description 1
- 108010017384 Blood Proteins Proteins 0.000 description 1
- 108010022366 Carcinoembryonic Antigen Proteins 0.000 description 1
- 102100025475 Carcinoembryonic antigen-related cell adhesion molecule 5 Human genes 0.000 description 1
- 241000700198 Cavia Species 0.000 description 1
- 102000008186 Collagen Human genes 0.000 description 1
- 108010035532 Collagen Proteins 0.000 description 1
- 108010014258 Elastin Proteins 0.000 description 1
- 102000016942 Elastin Human genes 0.000 description 1
- 241000283086 Equidae Species 0.000 description 1
- 102000008857 Ferritin Human genes 0.000 description 1
- 108050000784 Ferritin Proteins 0.000 description 1
- 238000008416 Ferritin Methods 0.000 description 1
- 102000004641 Fetal Proteins Human genes 0.000 description 1
- 108010003471 Fetal Proteins Proteins 0.000 description 1
- 235000008526 Galium odoratum Nutrition 0.000 description 1
- 241000287828 Gallus gallus Species 0.000 description 1
- SXRSQZLOMIGNAQ-UHFFFAOYSA-N Glutaraldehyde Chemical compound O=CCCCC=O SXRSQZLOMIGNAQ-UHFFFAOYSA-N 0.000 description 1
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 description 1
- AVXURJPOCDRRFD-UHFFFAOYSA-N Hydroxylamine Chemical compound ON AVXURJPOCDRRFD-UHFFFAOYSA-N 0.000 description 1
- 108060003951 Immunoglobulin Proteins 0.000 description 1
- 229920000057 Mannan Polymers 0.000 description 1
- 229910019142 PO4 Inorganic materials 0.000 description 1
- 102000057297 Pepsin A Human genes 0.000 description 1
- 108090000284 Pepsin A Proteins 0.000 description 1
- 206010035226 Plasma cell myeloma Diseases 0.000 description 1
- 239000002202 Polyethylene glycol Substances 0.000 description 1
- 239000012506 Sephacryl® Substances 0.000 description 1
- 241000700605 Viruses Species 0.000 description 1
- 230000021736 acetylation Effects 0.000 description 1
- 238000006640 acetylation reaction Methods 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 230000029936 alkylation Effects 0.000 description 1
- 238000005804 alkylation reaction Methods 0.000 description 1
- 239000003392 amylase inhibitor Substances 0.000 description 1
- 230000006287 biotinylation Effects 0.000 description 1
- 238000007413 biotinylation Methods 0.000 description 1
- 230000037396 body weight Effects 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- 238000011088 calibration curve Methods 0.000 description 1
- 150000001718 carbodiimides Chemical class 0.000 description 1
- 238000003163 cell fusion method Methods 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 235000013330 chicken meat Nutrition 0.000 description 1
- 238000010367 cloning Methods 0.000 description 1
- 229920001436 collagen Polymers 0.000 description 1
- 230000002860 competitive effect Effects 0.000 description 1
- 230000008878 coupling Effects 0.000 description 1
- 238000010168 coupling process Methods 0.000 description 1
- 238000005859 coupling reaction Methods 0.000 description 1
- 239000003431 cross linking reagent Substances 0.000 description 1
- 238000005520 cutting process Methods 0.000 description 1
- XUJNEKJLAYXESH-UHFFFAOYSA-N cysteine Natural products SCC(N)C(O)=O XUJNEKJLAYXESH-UHFFFAOYSA-N 0.000 description 1
- 235000018417 cysteine Nutrition 0.000 description 1
- 238000006193 diazotization reaction Methods 0.000 description 1
- GYZLOYUZLJXAJU-UHFFFAOYSA-N diglycidyl ether Chemical compound C1OC1COCC1CO1 GYZLOYUZLJXAJU-UHFFFAOYSA-N 0.000 description 1
- 125000005442 diisocyanate group Chemical group 0.000 description 1
- LOKCTEFSRHRXRJ-UHFFFAOYSA-I dipotassium trisodium dihydrogen phosphate hydrogen phosphate dichloride Chemical compound P(=O)(O)(O)[O-].[K+].P(=O)(O)([O-])[O-].[Na+].[Na+].[Cl-].[K+].[Cl-].[Na+] LOKCTEFSRHRXRJ-UHFFFAOYSA-I 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 238000001035 drying Methods 0.000 description 1
- 229920002549 elastin Polymers 0.000 description 1
- 210000003372 endocrine gland Anatomy 0.000 description 1
- 230000002255 enzymatic effect Effects 0.000 description 1
- 238000004108 freeze drying Methods 0.000 description 1
- 150000004676 glycans Chemical class 0.000 description 1
- 108060003552 hemocyanin Proteins 0.000 description 1
- 229940088597 hormone Drugs 0.000 description 1
- 239000005556 hormone Substances 0.000 description 1
- 125000002887 hydroxy group Chemical group [H]O* 0.000 description 1
- 125000002883 imidazolyl group Chemical group 0.000 description 1
- 102000018358 immunoglobulin Human genes 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 238000011835 investigation Methods 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 210000003205 muscle Anatomy 0.000 description 1
- 201000000050 myeloid neoplasm Diseases 0.000 description 1
- 238000006396 nitration reaction Methods 0.000 description 1
- 235000014593 oils and fats Nutrition 0.000 description 1
- 210000000056 organ Anatomy 0.000 description 1
- 230000003647 oxidation Effects 0.000 description 1
- 238000007254 oxidation reaction Methods 0.000 description 1
- 229940111202 pepsin Drugs 0.000 description 1
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 1
- 239000010452 phosphate Substances 0.000 description 1
- 239000002953 phosphate buffered saline Substances 0.000 description 1
- 230000001766 physiological effect Effects 0.000 description 1
- 102000040430 polynucleotide Human genes 0.000 description 1
- 108091033319 polynucleotide Proteins 0.000 description 1
- 239000002157 polynucleotide Substances 0.000 description 1
- 229920001282 polysaccharide Polymers 0.000 description 1
- 239000005017 polysaccharide Substances 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 210000004989 spleen cell Anatomy 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 150000003573 thiols Chemical group 0.000 description 1
- 210000000689 upper leg Anatomy 0.000 description 1
- 238000005406 washing Methods 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
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
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)
- Microbiology (AREA)
- Cell Biology (AREA)
- Food Science & Technology (AREA)
- Medicinal Chemistry (AREA)
- Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Peptides Or Proteins (AREA)
Description
【発明の詳細な説明】
(産業上の利用分野)
血清、尿などの体液に含まれる薬物あるいは各
種疾患に由来する微量成分の分析は病気の診断あ
るいは治療経過の判定などに非常に有意義であ
り、日常の臨床検査に活用されている。本発明は
この微量成分を測定する方法に関するものであ
る。[Detailed Description of the Invention] (Field of Industrial Application) Analysis of trace components derived from drugs or various diseases contained in body fluids such as serum and urine is extremely meaningful for diagnosing diseases and determining the progress of treatment. , is used in daily clinical tests. The present invention relates to a method for measuring this trace component.
(従来の技術)
血液等の体液には多種多様の成分が含まれてお
り、そのなかには、分子量の近似した物質、生理
活性の似た物質あるいは構造の近似した物質など
も含まれていることも多い。そこで、この分析法
は特異性が高く、かつ微少量まで定量しうること
が要求される。さらに、日常検査として利用され
るために、簡単かつルーチン化しうることが望ま
しい。(Prior art) Body fluids such as blood contain a wide variety of components, some of which may include substances with similar molecular weights, substances with similar physiological activities, or substances with similar structures. many. Therefore, this analytical method is required to have high specificity and to be able to quantify down to minute amounts. Furthermore, since it is used as a daily test, it is desirable that it be simple and routine.
血液のこれらの微量成分を検出する方法が種々
開発されているが、感度、特異性、大量検体の短
時間処理などの点にすぐれる酵素免疫測定法が賞
用されている。しかしながら、従来の酵素免疫測
定法の場合には、未だ感度が充分とはいえず、ま
た洗浄操作が繁雑であつたり、チユーブの移しか
えが必要であつたりして正確な濃度を求めること
が容易でなかつた。 Various methods have been developed for detecting these trace components of blood, but enzyme immunoassay is the preferred method due to its excellent sensitivity, specificity, and ability to process large amounts of samples in a short time. However, in the case of conventional enzyme immunoassay methods, the sensitivity is still not sufficient, and the washing operation is complicated and the tube needs to be changed, making it difficult to determine accurate concentrations. It wasn't.
そこで、本発明者らはさらに感度を高めかつ繁
雑な操作の少ない分析方法を開発するべく検討を
行ない、測定対象の抗原決定基具有物質に対する
抗体と酵素に対する抗体との結合物を利用する方
法を開発した。この方法は、この結合物に対して
測定対象の抗原決定基具有物質と酵素を競争反応
させその後この酵素の活性を測定することによつ
て抗原決定基具有物質を定量する方法(特願昭58
−38975号、特開昭59−164960号公報)である。
その際、この抗原決定基具有物質に対する抗体と
反応する抗体あるいは酵素に対する抗体と反応す
る抗体をさらに競争反応させる方法(特願昭58−
51494号、特開昭59−178360号)、あるいは抗原決
定基具有物質と高分子化合物との結合物又は抗原
決定基具有物質の重合物を競争反応させる方法
(特願昭58−51495号、特開昭59−178361号)も併
せて開発した。そして、その後さらに検討を進
め、この技術に近縁の種々の抗原決定基具有物質
測定法を次々と開発して特許出願を行なつた。そ
のなかに、測定対象の抗原決定基具有物質にその
抗体と水に不溶性の高分子物質に作用しうる酵素
との結合物を作用させてその後結合物の酵素活性
を測定する方法(特願昭58−231241号、特開昭60
−123767号)があつた。また、測定対象の抗原決
定基具有物質にこの抗原決定基具有物質の一の抗
原決定基に対する抗体とアミラーゼとの結合物及
びこの抗原決定基具有物質の他の抗原決定基に対
する抗体を接触させてその後結合物のアミラーゼ
活性等を測定する方法(特願昭59−143801号、特
開昭61−23970号)もあつた。 Therefore, the present inventors conducted studies to develop an analysis method with higher sensitivity and fewer complicated operations, and developed a method that utilizes a combination of an antibody against the antigenic determinant-containing substance to be measured and an antibody against an enzyme. developed. This method is a method for quantifying antigenic determinant-containing substances by competitively reacting an antigenic determinant-containing substance to be measured with an enzyme against this bound substance, and then measuring the activity of this enzyme.
-38975, Japanese Unexamined Patent Publication No. 164960/1983).
At that time, there is a method in which an antibody that reacts with the antibody against the antigenic determinant-containing substance or an antibody that reacts with the antibody against the enzyme is further subjected to a competitive reaction (Japanese Patent Application No. 1983-
51494, Japanese Patent Application Laid-open No. 59-178360), or a method of competitively reacting a combination of an antigenic determinant-containing substance with a polymer compound or a polymer of an antigenic determinant-containing substance (Japanese Patent Application No. 58-51495, 178361) was also developed. After further investigation, they developed a series of methods for measuring substances containing antigenic determinants that are closely related to this technique, and filed patent applications. Among them, there is a method in which a conjugate of the antibody and an enzyme capable of acting on a water-insoluble polymer substance is applied to a substance containing an antigenic determinant to be measured, and then the enzymatic activity of the conjugate is measured (patent application No. 58-231241, Japanese Patent Publication No. 1983
-123767) was received. Further, the antigenic determinant-containing substance to be measured is brought into contact with a combination of an antibody and amylase against one antigenic determinant of the antigenic determinant-containing substance and an antibody against another antigenic determinant of this antigenic determinant-containing substance. Subsequently, a method for measuring the amylase activity, etc. of the conjugate was developed (Japanese Patent Application No. 143801/1983, Japanese Patent Application Laid-open No. 23970/1983).
(発明が解決しようとする問題点)
前者の方法に比し後者の方法は測定感度がさら
に1桁ないし2桁高くなる点ですぐれていたが、
後者の方法は血清などの検体にはアミラーゼが含
まれているところから検体中のアミラーゼに特異
的に作用するアミラーゼインヒビターを添加する
必要があつた。(Problems to be Solved by the Invention) Compared to the former method, the latter method was superior in that the measurement sensitivity was one to two orders of magnitude higher;
The latter method requires the addition of an amylase inhibitor that specifically acts on amylase in the sample, since the sample such as serum contains amylase.
(問題点を解決するための手段)
本発明は、この後者の発明を拡張するものであ
り、アミラーゼのかわりに他の酵素のうち水に不
溶性の高分子物質に作用しうる酵素を用いること
によつても抗原決定基具有物質を高感度で測定で
きるという知見に基いている。この酵素のうち検
体に含まれていない酵素を用いれば特に酵素阻害
物質などを用いなくとも測定できるという利点が
ある。(Means for Solving the Problems) The present invention extends this latter invention by using, in place of amylase, an enzyme that can act on water-insoluble polymeric substances. This method is based on the knowledge that substances containing antigenic determinants can be measured with high sensitivity. Among these enzymes, if an enzyme not included in the sample is used, there is an advantage that the measurement can be performed without using any particular enzyme inhibitor.
すなわち、本発明は、検体に含まれる2以上の
抗原決定基を具有する物質を測定する方法におい
て、該抗原決定基具有物質に、この抗原決定基具
有物質の一の抗原決定基に対する抗体と水に不溶
性の高分子物質に作用しうる酵素との結合物及び
他の抗原決定基に対する抗体を接触せしめて反応
させ、さらに前記の高分子物質に前記の結合物を
接触せしめて酵素反応させ、酵素活性を測定する
ことを特徴とする抗原決定基具有物質の測定方法
に関するものである。 That is, the present invention provides a method for measuring a substance containing two or more antigenic determinants contained in a specimen, in which the antigenic determinant-containing substance is coated with an antibody against one antigenic determinant of the antigenic determinant-containing substance and water. A conjugate with an enzyme capable of acting on an insoluble polymeric substance and an antibody against other antigenic determinants are brought into contact with each other to react, and the conjugate is further brought into contact with the polymeric substance to cause an enzymatic reaction. The present invention relates to a method for measuring a substance containing an antigenic determinant, which is characterized by measuring activity.
本発明における測定対象は検体に含まれる抗原
決定基具有物質である。検体の種類は限定されな
いが、例えば血清、尿などである。血清、尿など
の場合には、通常は特別な前処理を必要とせず、
そのまま測定を行うことができる。 The object to be measured in the present invention is a substance containing an antigenic determinant contained in a specimen. The type of specimen is not limited, but includes, for example, serum and urine. For serum, urine, etc., no special pretreatment is usually required;
Measurements can be made as is.
抗原決定基具有物質(以下リガンドという。)
は抗原決定基を二以上有しているものであり、例
えば、各種内分泌腺に由来するホルモン類、免疫
グロブリン、アルブミン、フエリチン等の血漿蛋
白質、HB抗原等のウイルス、バクテリア類、α
−フエトプロテイン、癌胎児性抗原等の各種臓器
あるいは血中、尿中に存在する抗原などである。 Substances containing antigenic determinants (hereinafter referred to as ligands)
has two or more antigenic determinants, such as hormones derived from various endocrine glands, plasma proteins such as immunoglobulin, albumin, and ferritin, viruses such as HB antigen, bacteria, α
- Antigens present in various organs, blood, and urine, such as fetoprotein and carcinoembryonic antigen.
結合物を構成している抗体はリガンドと反応す
るものでなければならない。この抗体にはF
(ab′)2、Fab′、Fabなどのフラグメントも含まれ
る。 The antibodies making up the conjugate must be reactive with the ligand. This antibody has F
Also included are fragments such as (ab′) 2 , Fab′, and Fab.
抗体の製造方法としては、リガンド又はリガン
ドと蛋白との結合物を兎、山羊、馬、モルモツ
ト、ニワトリなどの温血動物に体重1Kgあたり
0.3〜2mgを1〜数回背中皮下、フツトパツド、
大腿筋等にアジユバントとともに注射して当該動
物の体内に形成させる。この抗体は各種の抗原決
定基を認識する抗体の混合物であるからこれを分
離して用いる。分離方法にはアフイニテイークロ
マトグラフイーを用いるのがよく、例えば、リガ
ンドを酵素あるいは化学試薬により分解してゲル
過、イオン交換クロマトグラフイーなどで分離
し、この各抗原フラクシヨンを不溶化したアフイ
ニテイーカラムを作製し、このカラムを用いて前
記の抗体混合物を分離することができる。また、
この抗体は市販品も存在する。本発明の方法にお
いては、抗体は単一抗体に分離しなくともよく、
少なくとも2群に分割すれば足りる。 As a method for producing antibodies, a ligand or a conjugate of a ligand and a protein is administered per kilogram of body weight to warm-blooded animals such as rabbits, goats, horses, guinea pigs, and chickens.
0.3 to 2 mg once to several times subcutaneously on the back, foot pads,
It is injected into the thigh muscle etc. together with an adjuvant to form within the animal's body. Since this antibody is a mixture of antibodies that recognize various antigenic determinants, it is used after being separated. Affinity chromatography is preferably used as a separation method. For example, affinity chromatography is performed in which the ligand is decomposed with enzymes or chemical reagents, separated by gel filtration, ion exchange chromatography, etc., and each antigen fraction is insolubilized. A column can be constructed and used to separate the antibody mixture described above. Also,
This antibody is also commercially available. In the method of the present invention, antibodies do not need to be separated into single antibodies,
It is sufficient to divide it into at least two groups.
一方、この抗体はモノクローナル抗体として取
得することもできる。その場合には、マウスに前
記のいずれかの抗原をアジユバントとともに数回
腹腔等に注射し、脾臓細胞を取り出してポリエチ
レングリコール等を用いてマウスミエローマ細胞
と融合させる。そして、この融合細胞のなかから
当該抗体を産生するものをクローニングによつて
モノクローン細胞として増殖させ、マウス腹腔中
で増殖させることによつて単一抗体、すなわちモ
ノクローナル抗体を大量に製造することができ
る。 On the other hand, this antibody can also be obtained as a monoclonal antibody. In that case, one of the antigens mentioned above is injected into the peritoneal cavity of a mouse several times together with an adjuvant, and spleen cells are taken out and fused with mouse myeloma cells using polyethylene glycol or the like. Then, by cloning those fused cells that produce the antibody, they are grown as monoclonal cells, and by growing them in the peritoneal cavity of a mouse, a single antibody, that is, a monoclonal antibody, can be produced in large quantities. can.
結合物を構成している酵素は水に不溶性の高分
子化合物に作用しうるものであるが、そのなかで
は活性の測定方法が容易なものがよい。このよう
な酵素は、例えばアミラーゼ、デキストラナー
ゼ、セルラーゼ、コラーゲナーゼ、マンナーゼ、
プロテアーゼ、エラスターゼ、リパーゼ、などで
ある。 The enzymes constituting the conjugate can act on water-insoluble polymer compounds, and among them, enzymes whose activity can be easily measured are preferred. Such enzymes include, for example, amylase, dextranase, cellulase, collagenase, mannase,
protease, elastase, lipase, etc.
酵素と抗体との結合方法は双方の官能基を考慮
して決定すればよい。官能基は、アミノ基、カル
ボキシル基、水酸基、チオール基、イミダゾール
基、フエニル基などを利用することができ、例え
ばアミノ基相互間を結合させる場合には、ジイソ
シアネート法、グルタルアルデヒド法、ジフルオ
ロベンゼン法、ベンゾキノン法等数多く知られて
いる。また、アミノ基とカルボキシル基との間を
結合させる方法としては、カルボキシル基をサク
シンイミドエステル化する方法のほかカルボジイ
ミド法、ウツドワード試薬法等が知らており、ア
ミノ基と糖鎖を架橋する過ヨウ素酸酸化法
(Nakane法)もある。チオール基を利用する場
合には、例えばもう一方の側のカルボキシル基を
サクシンイミドエステル化してこれにシステイン
を反応させてチオール基を導入し、チオール基反
応性二価架橋試薬を用いて双方を結合することが
できる。フエニル基を利用する方法としてはジア
ゾ化法、アルキル化法などがある。結合方法はこ
れらの例示に限られるものではなく、このほか例
えば(Method in Immunology and
Immunochemistry」あるいは「酵素免疫測定法」
等の成書に記載されている方法のなかから適宜選
択して利用することができる。結合比は1:1に
限らず、目的に応じて任意の比率をとることがで
きるということはいうまでもない。反応後は、ゲ
ル過法、イオン交換クロマトグラフイー、アフ
イニテイークロマトグラフイーなどを適宜組み合
わせて精製を行い、必要により凍結乾燥法等で乾
燥する。 The method of binding the enzyme and antibody may be determined by taking into consideration the functional groups of both. As the functional group, an amino group, a carboxyl group, a hydroxyl group, a thiol group, an imidazole group, a phenyl group, etc. can be used. For example, when bonding between amino groups, a diisocyanate method, a glutaraldehyde method, a difluorobenzene method can be used. , benzoquinone method, etc. are known. In addition, as a method for bonding between an amino group and a carboxyl group, in addition to the method of converting the carboxyl group into a succinimide ester, the carbodiimide method and the Woodward reagent method are known. There is also an acid oxidation method (Nakane method). When using a thiol group, for example, the carboxyl group on the other side is esterified with succinimide, this is reacted with cysteine to introduce a thiol group, and the two are bonded using a thiol group-reactive divalent cross-linking reagent. can do. Methods that utilize phenyl groups include diazotization and alkylation. The binding method is not limited to these examples, and in addition, for example (Method in Immunology and
"Immunochemistry" or "Enzyme immunoassay"
You can select and use the methods as appropriate from among the methods described in books such as . It goes without saying that the coupling ratio is not limited to 1:1 and can be any ratio depending on the purpose. After the reaction, purification is performed using an appropriate combination of gel filtration, ion exchange chromatography, affinity chromatography, etc., and if necessary, drying is performed using freeze-drying or the like.
この結合物の抗体とともにリガンドに作用させ
る抗体は結合物の抗体が反応する抗原決定基と異
なる抗原決定基に対して反応するものである。こ
の抗体はIgG、IgMあるいはIgAであり、F
(ab′)2、Fabなどのフラグメントであつてもよ
く、また、例えばDNP化、アセチル化、ビオチ
ニル化、ニトロ化などの化学修飾が施されたもの
であつてもよい。この抗体は1種類に限られるも
のではなく、2種類以上あつてもよい。 The antibody that is allowed to act on the ligand together with the conjugate antibody reacts with a different antigenic determinant from the antigenic determinant that the conjugate antibody reacts with. This antibody is IgG, IgM or IgA, and F
It may be a fragment such as (ab') 2 or Fab, or it may be one that has been chemically modified, such as DNP, acetylation, biotinylation, or nitration. This antibody is not limited to one type, and may include two or more types.
この異なる抗原決定基を認識する抗体は前述の
細胞融合法によるモノクローナル抗体を製造する
方法により容易に取得することができる。また、
前述の温血動物を利用して抗体群を産生させ、こ
れを分離してもよい。その場合には単一抗体まで
分離しなくともよく、例えば2群に分割してその
一方を前述の酵素と結合させ、もう一方をこの抗
体に利用してもよい。また、この抗体は完全に分
離しなくともよく、測定を阻害しない程度に他方
の抗体が混入していてもよい。 Antibodies that recognize these different antigenic determinants can be easily obtained by the method for producing monoclonal antibodies using the cell fusion method described above. Also,
The above-mentioned warm-blooded animals may be used to produce antibodies, which may then be isolated. In that case, it is not necessary to separate the antibodies into a single antibody; for example, the antibodies may be divided into two groups, one of which may be bound to the above-mentioned enzyme, and the other may be used for this antibody. Furthermore, this antibody does not need to be completely separated, and the other antibody may be mixed to the extent that the measurement is not inhibited.
この抗体には水溶性高分子を結合させたほうが
感度を高める点で好ましい場合がある。水溶性高
分子は分子量が1000以上のものであり、例えばア
ルブミン、ヘモシアニン等の蛋白質、ポリサツカ
ライド、ポリエチレングリコール、ポリヌクレオ
チド等である。結合方法は前述の酵素に抗体を結
合させる方法のなかから適宜選択すればよい。 It may be preferable to bind a water-soluble polymer to this antibody in order to increase sensitivity. Water-soluble polymers have a molecular weight of 1000 or more, and include proteins such as albumin and hemocyanin, polysaccharides, polyethylene glycols, and polynucleotides. The binding method may be appropriately selected from among the methods described above for binding antibodies to enzymes.
同様に、この抗体にさらにこの抗体に対する抗
体を反応させて高分子化してもよい。この第2抗
体は例えばヤギIgGに対するウサギIgGなどであ
り、第1抗体あるいは第1抗体とリガンドの結合
物を抗原として前述の抗体の取得方法に準じて取
得することができる。この第2抗体を接触させる
時期は第1抗体をリガンドに接触させる前であつ
ても後であつてもよいが、同時に加えることが操
作上簡便である。 Similarly, this antibody may be further reacted with an antibody against this antibody to form a polymer. The second antibody is, for example, rabbit IgG against goat IgG, and can be obtained using the first antibody or a combination of the first antibody and a ligand as an antigen according to the method for obtaining antibodies described above. The second antibody may be brought into contact with the first antibody before or after the first antibody is brought into contact with the ligand, but it is operationally convenient to add it at the same time.
測定対象のリガンドに、前記一つの抗原決定基
に対する抗体と酵素との結合物及び他の抗原決定
基に対する抗体を溶液中で接触させる。その際、
溶液の温度は20〜45℃程度、そしてPHは通常4〜
8.5程度が適当である。PHを一定に保つために、
必要により、リン酸緩衝液、酢酸緩衝液などの緩
衝液を用いてもよい。その際、結合物及び抗体の
適当な量は、その種類、リガンドの種類、あるい
は接触時の条件などによつて異なるので予め試験
をして定めるのがよい。リガンドへの結合物及び
抗体の接触順序は問うところではなく、いずれが
先であつてもまた両方同時であつてもよい。 The ligand to be measured is brought into contact with a conjugate of an antibody to the one antigenic determinant and an enzyme and an antibody to another antigenic determinant in a solution. that time,
The temperature of the solution is about 20-45℃, and the pH is usually 4-45℃.
Approximately 8.5 is appropriate. To keep the pH constant,
If necessary, a buffer such as a phosphate buffer or an acetate buffer may be used. In this case, the appropriate amounts of the conjugate and antibody vary depending on the type thereof, the type of ligand, the contact conditions, etc., and are therefore preferably determined by testing in advance. The order in which the ligand is contacted with the conjugate and the antibody is not critical; either may come first or both may be brought into contact simultaneously.
一方、結合物の酵素と同種の酵素が検体に含ま
れている場合には、この検体中の酵素を阻害する
程度が前記の結合物に結合されている酵素の活性
を阻害する程度より大きい酵素阻害物質を接触さ
せるのがよい。 On the other hand, if the sample contains an enzyme of the same type as the enzyme of the conjugate, the degree of inhibition of the enzyme in this sample is greater than the degree of inhibition of the activity of the enzyme bound to the conjugate. It is preferable to bring the inhibitor into contact with the inhibitor.
この酵素阻害物質は検体に含まれている酵素を
完全に失活させかつ結合物に結合されている酵素
を全く阻害しないものが最も望ましいことはいう
までもないが、実用上は要は測定時においてブラ
ンク値を上昇させなければよく、測定後に酵素阻
害物質が失活するなどしてこの酵素活性が回復し
てもよい。この酵素阻害物質の作用が問題になる
もう一方の、酵素は抗体に結合されている状態の
ものであり、遊離状態では酵素阻害物質によつて
失活するものであつてもよい。この酵素阻害物質
にはこのような特異性を有する公知の酵素阻害物
質を利用すればよいが、そのほか、検体に含まれ
ている酵素を温血動物に投与してその抗体を取得
し、これを酵素阻害物質として用いることもでき
る。抗体の取得方法は前述のリガンドに対する抗
体の取得方法と同様でよい。酵素阻害物質の添加
時期は、検体中の酵素による後述する水に不溶性
の高分子物質の分解を実質的に防止できればよ
く、通常はこの高分子物質の添加前に添加する。
しかしながら、一般に酵素阻害物質による阻害作
用は酵素による基質の分解速度よりもはるかには
やいので酵素阻害物質を高分子物質と同時あるい
は多少遅れて添加してもよい。 It goes without saying that it is most desirable for this enzyme inhibitor to be one that completely deactivates the enzyme contained in the sample and does not inhibit the enzyme bound to the conjugate at all. The blank value may not be increased in the measurement, and the enzyme activity may be recovered by deactivating the enzyme inhibitor after the measurement. On the other hand, the effect of the enzyme inhibitor is a problem, and the enzyme is in a state bound to an antibody, and in a free state, it may be inactivated by the enzyme inhibitor. Known enzyme inhibitors with such specificity may be used as this enzyme inhibitor, but it is also possible to administer the enzyme contained in the sample to a warm-blooded animal to obtain its antibodies. It can also be used as an enzyme inhibitor. The method for obtaining antibodies may be the same as the method for obtaining antibodies against the aforementioned ligands. The enzyme inhibitor may be added at any time as long as it can substantially prevent the decomposition of a water-insoluble polymeric substance, which will be described later, by the enzyme in the sample, and is usually added before the addition of this polymeric substance.
However, since the inhibitory effect of an enzyme inhibitor is generally much faster than the rate of decomposition of a substrate by the enzyme, the enzyme inhibitor may be added at the same time as the polymeric substance or after some delay.
リガンドと反応させた結合物は高分子物質に接
触させて反応させる。 The conjugate reacted with the ligand is brought into contact with a polymeric substance and reacted.
高分子物質と接融させる結合物は反応物から分
離したものでもよいが、通常は反応物に含まれて
いる状態のままでよい。 The bond to be fused with the polymeric substance may be separated from the reactant, but usually it may remain contained in the reactant.
この高分子物質は結合物中の酵素が酵素反応し
うるものであり、通常は基質であるが、水に不溶
性のものである。高分子物質の例としてはα−ア
ミラーゼの場合には不溶性デンプン、セルラーゼ
の場合にはセルロース、コラーゲナーゼの場合に
はコラーゲン、マンナーゼの場合にはマンナン、
プロテアーゼの場合には不溶性蛋白質、エラスタ
ーゼの場合にはエラスチン、そしてリパーゼの場
合には各種油脂類を挙げることができる。この高
分子物質はそれ自身が可溶性であつても、不溶性
の担体に結合させるとか、重合させるなどして不
溶化して用いることもできる。 This polymeric substance is one in which the enzyme in the conjugate can undergo an enzymatic reaction, and is usually a substrate, but it is insoluble in water. Examples of polymeric substances include insoluble starch for α-amylase, cellulose for cellulase, collagen for collagenase, and mannan for mannase.
Examples include insoluble proteins in the case of protease, elastin in the case of elastase, and various oils and fats in the case of lipase. Even if this polymer substance itself is soluble, it can also be used after being bound to an insoluble carrier or made insoluble by polymerization.
酵素反応条件は用いる酵素に応じて適当になる
ように定めればよい。 Enzyme reaction conditions may be determined appropriately depending on the enzyme used.
酵素反応後は酵素活性を求める。酵素活性は、
この酵素反応による分解物の増加、原料である高
分子物質の減少、その他、酵素反応による系の変
化を追跡すればよい。 After the enzymatic reaction, determine the enzyme activity. Enzyme activity is
What is necessary is to track the increase in decomposed products due to this enzymatic reaction, the decrease in the polymeric material that is the raw material, and other changes in the system due to the enzymatic reaction.
(作用)
本発明の方法においては、リガンドが結合物の
抗体部分に結合することによつてその後の酵素反
応に立体障害を生じさせることを利用している。
酵素反応させる高分子物質が不溶性であるために
結合物の酵素部分との接触の大部分が固−液間に
なり、その結果、酵素の高分子化による立体障害
が大きく現われる。本発明者らはこのことを確認
するためにα−アミラーゼの系を用いて検討した
ところ、ペンタオースの場合には酵素の高分子化
による酵素活性の低下がほとんど認められず、一
方、不溶化デンプンの場合には酵素活性が著しく
低下した。(Function) The method of the present invention utilizes the fact that the ligand binds to the antibody portion of the conjugate, thereby causing steric hindrance to the subsequent enzymatic reaction.
Since the polymer substance subjected to the enzymatic reaction is insoluble, most of the contact between the conjugate and the enzyme moiety occurs between solid and liquid, resulting in significant steric hindrance due to the polymerization of the enzyme. The present inventors investigated this using an α-amylase system to confirm this, and found that in the case of pentaose, there was almost no decrease in enzyme activity due to polymerization of the enzyme, whereas in the case of insoluble starch, In some cases, enzyme activity was significantly reduced.
このような系に結合物の抗体とは異なる抗原決
定基を認識する新たな抗体を導入したところに本
発明の特徴がある。すなわち、結合物の抗体部分
に一の抗原決定基部分が結合したリガンドの他の
抗原決定基部分にこの抗体が結合することによつ
て結合物を巨大化してその立体障害をさらに大き
くしている。それによつて、リガンドの測定感度
を大きく高めているのである。 A feature of the present invention is that a new antibody that recognizes an antigenic determinant different from that of the antibody of the conjugate is introduced into such a system. In other words, one antigenic determinant portion is bound to the antibody portion of the conjugate, and this antibody binds to the other antigenic determinant portion of the ligand, thereby making the conjugate larger and further increasing its steric hindrance. . This greatly increases the sensitivity of ligand measurement.
(実施例)
実施例 1
セルラーゼ基質の調製
紙を20cm×20cmの大きさに切断し、あらか
じめ用意しておいたリアクテイブブルー溶液
(5gリアクテイブブルー、5gNa2CO3蒸留
水200ml)中に浸した。60℃に加温し、時々撹
拌しながら、3日間加熱を続けた。この紙を
蒸留水で十分に洗浄し、過剰の染料を除去し
た。続いて、恒温乾燥器で乾燥させ、1cm×5
cmの大きさに切断して、目的の基質を得た。(Example) Example 1 Preparation of cellulase substrate Paper was cut into pieces of 20 cm x 20 cm and immersed in a previously prepared reactive blue solution (5 g reactive blue, 5 g Na 2 CO 3 distilled water 200 ml). did. The mixture was heated to 60°C and continued to be heated for 3 days with occasional stirring. The paper was thoroughly washed with distilled water to remove excess dye. Next, dry it in a constant temperature dryer and cut it into 1cm x 5
The desired substrate was obtained by cutting into cm-sized pieces.
セルラーゼ−抗ヒトIgGマウスIgG結合物の
調製
セルラーゼ10mgをPH6.0の0.1Mリン酸緩衝液
2mlに溶かし4−(マレイミドメチルシクロヘ
キサン−1−カルボン酸)サクシンイミドエス
テル(CHMS)のジメチルスルホキシド溶液
(2mg/ml)200μを加え、室温で1時間、放
置した。この反応液をセフアデツクスG−25を
用いてゲル過し、未反応のCHMSを除去し
た。このCHMS化セルラーゼを1mlまで濃縮
した。 Preparation of cellulase-anti-human IgG mouse IgG conjugate Dissolve 10 mg of cellulase in 2 ml of 0.1 M phosphate buffer at pH 6.0 and prepare a dimethyl sulfoxide solution of 4-(maleimidomethylcyclohexane-1-carboxylic acid) succinimide ester (CHMS) ( 2mg/ml) was added thereto, and the mixture was left at room temperature for 1 hour. This reaction solution was gel-filtered using Sephadex G-25 to remove unreacted CHMS. This CHMS-modified cellulase was concentrated to 1 ml.
一方、抗ヒトIgGマウスIgG10mlを5mA
EDTAを含むPH7.5の0.1Mリン酸緩衝液2mlに
とかし9mg/mlのS−アセチルメルカプトコハ
ク酸無水物(SAMS)のジオキサン溶液200μ
加えた。それから37℃で一時間放置後、1N
ヒドロキシルアミン水溶液(PH7.5)200μ加
えた。30分後反応液をセフアデツクスG−25で
ゲル過し未反応のSAMSを除いた。このHS
−抗ヒトIgGマウスIgG溶液を前述のCHM化セ
ルテーゼ1mlに加え、37℃で2時間放置した。
この反応液をセフアクリルS−300でゲル過
し目的のセルラーゼ−抗ヒトIgGマウスIgG結
合物を得た。 Meanwhile, 10 ml of anti-human IgG mouse IgG was added at 5 mA.
200 μl of a dioxane solution of 9 mg/ml S-acetylmercaptosuccinic anhydride (SAMS) dissolved in 2 ml of 0.1 M phosphate buffer, pH 7.5 containing EDTA.
added. Then, after leaving it at 37℃ for 1 hour, 1N
200μ of hydroxylamine aqueous solution (PH7.5) was added. After 30 minutes, the reaction solution was gel-filtered through Sephadex G-25 to remove unreacted SAMS. This HS
-Anti-human IgG Mouse IgG solution was added to 1 ml of the above CHM-modified celltase and left at 37°C for 2 hours.
This reaction solution was gel-filtered with Sephacryl S-300 to obtain the desired cellulase-anti-human IgG mouse IgG conjugate.
ヒトIgGの測定
セルラーゼ−抗ヒトIgGマウスIgG結合物を
含む溶液50μにヒトIgGを含む標準溶液50μ
及び結合物の抗体と別の抗原決定基を認識する
抗ヒトIgGマウスIgG50μ(0.1mg/ml)を加
え、37℃で30分間放置した。これにPH5.0の
0.1M酢酸緩衝液を1mlを加え、次にで調製
したブルーセルロース紙を1枚加えた。1時
間後反応液の吸光度を波長650nmで測定した。
第1図は標準溶液中のヒトIgG量と吸光度を示
したものである。尚、図中白丸は抗ヒトIgGマ
ウスIgGを加えた場合をそして黒丸は加えなか
つた場合をそれぞれ示している。 Measurement of human IgG Add 50μ of a standard solution containing human IgG to 50μ of a solution containing a cellulase-anti-human IgG mouse IgG conjugate.
Then, 50μ (0.1 mg/ml) of anti-human IgG mouse IgG that recognizes an antigenic determinant other than the antibody of the conjugate was added, and the mixture was left at 37°C for 30 minutes. This has a PH5.0
1 ml of 0.1M acetate buffer was added, and then one sheet of the blue cellulose paper prepared above was added. After 1 hour, the absorbance of the reaction solution was measured at a wavelength of 650 nm.
Figure 1 shows the amount of human IgG in the standard solution and the absorbance. In the figure, white circles indicate the case where anti-human IgG mouse IgG was added, and black circles indicate the case where it was not added.
実施例 2
不溶化ブルーデキストランの作製
ブルーデキストラン2000(フアルマシア社製
品)2gを0.6N NaOH水溶液25mlに溶かし、
これにセルロースパウダー1g(フアルマシア
社製品)及びNaBH430mgを加えた。撹拌しつ
つジグリシジルエーテル8mlを加え、室温にて
一夜撹拌した。反応後、生じた塊をスパーテル
で破砕し、蒸留水で十分洗浄した。遠心して不
溶化ブルーデキストランを分取した。この不溶
化ブルーデキストラン1gを0.1M酢酸緩衝液
50mlに懸濁した。Example 2 Preparation of insolubilized blue dextran 2 g of Blue Dextran 2000 (product of Pharmacia) was dissolved in 25 ml of 0.6N NaOH aqueous solution.
To this were added 1 g of cellulose powder (product of Pharmacia) and 30 mg of NaBH 4 . 8 ml of diglycidyl ether was added with stirring, and the mixture was stirred overnight at room temperature. After the reaction, the resulting lumps were crushed with a spatula and thoroughly washed with distilled water. The insolubilized blue dextran was fractionated by centrifugation. Add 1g of this insolubilized blue dextran to 0.1M acetate buffer.
Suspended in 50ml.
CHM化デキストラナーゼの作製
デキストラナーゼ1mgをPH7.0の0.1Mリン酸
緩衝液1mlに溶かし、CHMS1mg/mlのDMF
溶液100μを加えて室温で1時間放置して反
応させた。この反応液をBio Gel P−2のカ
ラムに入れ、PH7.0の0.1Mリン酸緩衝液を流し
てゲル過を行ない、素通り分画を分取した。 Preparation of CHM-modified dextranase Dissolve 1 mg of dextranase in 1 ml of 0.1M phosphate buffer (PH7.0), and dissolve 1 mg of CHMS/ml in DMF.
100μ of the solution was added and left to react at room temperature for 1 hour. This reaction solution was put into a column of Bio Gel P-2, and gel filtration was performed by flowing 0.1M phosphate buffer at pH 7.0, and the flow-through fraction was collected.
抗ヒトα−フエトプロテインヤギIgG F
(ab′)2の作製
抗ヒトα−フエトプロテインヤギIgG10mgを
0.1M酢酸緩衝液(PH4.0)2mlにペプシン300μ
gを加え、37℃で18時間撹拌した。0.1N
NaOHを加えてPHを6.0に調節しこの反応液を
予め0.1Mリン酸緩衝1mM EDTA溶液(PH
6.3)で緩衝化したセフアクリルS−300ゲルカ
ラムに入れ、上記のリン酸緩衝液で溶出した。
分子量約10万付近に溶出されたピーク部分を集
めて1mlに濃縮し、目的の抗ヒトα−フエトプ
ロテインヤギIgG F(ab′)2を得た。 Anti-human α-fetoprotein goat IgG F
Preparation of (ab′) 2 10mg of anti-human α-fetoprotein goat IgG
300μ of pepsin in 2ml of 0.1M acetate buffer (PH4.0)
g was added thereto, and the mixture was stirred at 37°C for 18 hours. 0.1N
The pH was adjusted to 6.0 by adding NaOH, and the reaction solution was preliminarily mixed with 0.1M phosphate buffered 1mM EDTA solution (PH
6.3) and eluted with the above phosphate buffer.
The peak portion eluted at a molecular weight of approximately 100,000 was collected and concentrated to 1 ml to obtain the target anti-human α-fetoprotein goat IgG F(ab') 2 .
デキストラナーゼ−抗ヒトα−フエトプロテ
インヤギIgG Fab′結合物の作製
で調製した抗原ヒトα−フエトプロテイン
ヤギIgG F(ab′)23mgを含む0.1Mリン酸緩衝1
mM EDTA溶液(PH6.0)1mlに10mg/mlの
2−メルカプトエチルアミン塩酸塩水溶液
100μを加え、37℃で90分間撹拌した。この
反応液を予め0.1Mリン酸緩衝液(PH7.0)で緩
衝化したセフアデツクスG−25カラムでゲル
過して未反応の2−メルカプトメチルアミンを
除去し、HS−Fab′を得た。これにで調製し
たCHM化デキストラナーゼ1mgを加え、37℃
で90分間反応後4℃で一夜放置した。次にこの
反応液を20mMリン酸緩衝化生理食塩水(PH
7.0)で緩衝化したグリセルCPGカラムでゲル
過して分子量17万以上の分画を集め、これを
濃縮して目的の結合物を得た。 Preparation of dextranase-anti-human α-fetoprotein goat IgG Fab′ conjugate Antigen human α-fetoprotein goat IgG F(ab′) 2 containing 3 mg in 0.1 M phosphate buffer 1
10 mg/ml 2-mercaptoethylamine hydrochloride aqueous solution in 1 ml of mM EDTA solution (PH6.0)
100μ was added and stirred at 37°C for 90 minutes. This reaction solution was gel-filtered through a Sephadex G-25 column buffered in advance with 0.1M phosphate buffer (PH7.0) to remove unreacted 2-mercaptomethylamine, yielding HS-Fab'. Add 1 mg of CHM-modified dextranase prepared above, and cool at 37°C.
After reacting for 90 minutes, the mixture was left at 4°C overnight. Next, this reaction solution was mixed with 20mM phosphate buffered saline (PH
7.0) and collected fractions with a molecular weight of 170,000 or more, which were concentrated to obtain the target conjugate.
ヒトα−フエトプロテインの測定
濃度0〜1mgのα−フエトプロテイン溶液
(8%PEG含有)50μに、で調製した結合
物溶液50μ及び抗ヒトα−フエトプロテイン
マウスIgG(モノクローナル抗体)50μ(500μ
g/ml)を加えて20分間反応させた。反応液に
不溶化ブルーデキストラン懸濁液1.0mlを加え
て37℃で30分間さらに反応させ、0.5N
NaOH1mlを加えて反応を停止させた。これを
撹拌後、3500rpmで2分間遠心し、得られた上
清の620nmにおける吸光度を測定した。 Measurement of human α-fetoprotein Add 50μ of an α-fetoprotein solution (containing 8% PEG) with a concentration of 0 to 1 mg, and 50μ of the conjugate solution prepared in and 50μ of anti-human α-fetoprotein mouse IgG (monoclonal antibody). (500μ
g/ml) and allowed to react for 20 minutes. Add 1.0 ml of insolubilized blue dextran suspension to the reaction solution and further react at 37°C for 30 minutes.
The reaction was stopped by adding 1 ml of NaOH. After stirring, the mixture was centrifuged at 3500 rpm for 2 minutes, and the absorbance of the resulting supernatant at 620 nm was measured.
得られた吸光度とヒトα−フエトプロテイン
の濃度との関係を示す検量線を第2図に示す。
図中白丸は抗ヒトα−フエトプロテインマウス
IgGを加えた場合を示し、一方、黒丸は加えな
かつた場合を示している。 FIG. 2 shows a calibration curve showing the relationship between the obtained absorbance and the concentration of human α-fetoprotein.
The white circle in the figure is anti-human α-fetoprotein mouse.
The case where IgG was added is shown, while the black circle shows the case where it was not added.
(発明の効果)
本発明の方法は、検体中のリガンドを特異性高
くかつ極めて高感度で測定できる。この本発明の
方法は先願(特願昭59−27710号、特開昭60−
171461)の方法に比し、感度をさらに1桁ないし
2桁向上させることができる。また、操作が簡単
であり、安価かつ容易にリガンドを定量すること
が可能である。本発明の方法はリガンドの種類を
問わず測定できる。本発明の方法に用いる試薬に
はリガンドを直接使用せず、リガンドは抗体の製
造に用いられるだけであるから微量で足りるとい
う利点も有する。従つて、本発明の方法は測定対
象と同じリガンドが入手しにくい場合とか、高価
な場合に特に有効である。(Effects of the Invention) The method of the present invention can measure a ligand in a specimen with high specificity and extremely high sensitivity. The method of the present invention has been disclosed in earlier patent applications (Japanese Patent Application No. 59-27710, Japanese Patent Application Laid-open No. 60-1989).
171461), the sensitivity can be further improved by one to two orders of magnitude. In addition, the operation is simple, and the ligand can be quantified easily and inexpensively. The method of the present invention can be used to measure any type of ligand. The reagent used in the method of the present invention has the advantage that a trace amount is sufficient because the ligand is not used directly and the ligand is only used for producing the antibody. Therefore, the method of the present invention is particularly effective when the same ligand as the target to be measured is difficult to obtain or expensive.
第1図は酵素にセルラーゼを用い、ヒトIgGの
濃度と吸光度の関係を他の抗原決定基に対する抗
体を加えた場合(白丸)と加えなかつた場合(黒
丸)を比較した結果を示すものであり、第2図は
酵素にデキストラナーゼを用い、ヒトα−フエト
プロテインについて同様に比較した結果を示すも
のである。
Figure 1 shows the results of comparing the relationship between human IgG concentration and absorbance when cellulase was used as the enzyme and when antibodies against other antigenic determinants were added (white circles) and when antibodies against other antigenic determinants were not added (black circles). FIG. 2 shows the results of a similar comparison for human α-fetoprotein using dextranase as the enzyme.
Claims (1)
る物質を測定する方法において、 該抗原決定基具有物質に、この抗原決定基具有
物質の一の抗原決定基に対する抗体と水に不溶性
の高分子物質に作用しうる酵素との結合物及び他
の抗原決定基に対する抗体を接触せしめて反応さ
せ、 さらに、前記の高分子物質に前記の結合物を接
触せしめて酵素反応させ、酵素活性を測定するこ
とを特徴とする 抗原決定基具有物質の測定方法。 2 他の抗原決定基に対する抗体が水溶性高分子
が結合されたものである特許請求の範囲第1項記
載の測定方法。 3 他の抗原決定基に対する抗体にこの抗体に対
する抗体をさらに接触せしめることを特徴とする
特許請求の範囲第1項又は第2項記載の測定方
法。[Scope of Claims] 1. A method for measuring a substance containing two or more antigenic determinants contained in a specimen, wherein the antigenic determinant-containing substance is coated with an antibody against one antigenic determinant of the antigenic determinant-containing substance. A conjugate with an enzyme capable of acting on a water-insoluble polymeric substance is brought into contact with an antibody against another antigenic determinant, and the conjugate is brought into contact with the polymeric substance to cause an enzymatic reaction. , a method for measuring an antigenic determinant-containing substance, characterized by measuring enzyme activity. 2. The measuring method according to claim 1, wherein the antibody against the other antigenic determinant is bound to a water-soluble polymer. 3. The measuring method according to claim 1 or 2, characterized in that the antibody directed against this antibody is further brought into contact with an antibody directed against another antigenic determinant.
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP20345384A JPS6180050A (en) | 1984-09-28 | 1984-09-28 | Measurement of substance possessing antigen determining group by utilizing enzyme |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP20345384A JPS6180050A (en) | 1984-09-28 | 1984-09-28 | Measurement of substance possessing antigen determining group by utilizing enzyme |
Publications (2)
Publication Number | Publication Date |
---|---|
JPS6180050A JPS6180050A (en) | 1986-04-23 |
JPH0340832B2 true JPH0340832B2 (en) | 1991-06-20 |
Family
ID=16474368
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
JP20345384A Granted JPS6180050A (en) | 1984-09-28 | 1984-09-28 | Measurement of substance possessing antigen determining group by utilizing enzyme |
Country Status (1)
Country | Link |
---|---|
JP (1) | JPS6180050A (en) |
Families Citing this family (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JPH0814580B2 (en) * | 1987-10-26 | 1996-02-14 | 富士レビオ株式会社 | Dry immunoassay element |
Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JPS56133661A (en) * | 1980-02-22 | 1981-10-19 | Aa Tooma Hansu | Competing uniform determination of ligand |
JPS59210365A (en) * | 1983-05-02 | 1984-11-29 | マイルス・ラボラトリ−ズ・インコ−ポレ−テツド | Uniform group immunity test method and reagent group used for said method, test kit and testing tool |
-
1984
- 1984-09-28 JP JP20345384A patent/JPS6180050A/en active Granted
Patent Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JPS56133661A (en) * | 1980-02-22 | 1981-10-19 | Aa Tooma Hansu | Competing uniform determination of ligand |
JPS59210365A (en) * | 1983-05-02 | 1984-11-29 | マイルス・ラボラトリ−ズ・インコ−ポレ−テツド | Uniform group immunity test method and reagent group used for said method, test kit and testing tool |
Also Published As
Publication number | Publication date |
---|---|
JPS6180050A (en) | 1986-04-23 |
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