EP0040224A4 - IMPROVED PROCESS FOR IMMUNOLOGICAL ANALYSIS OF NON-HOMOGENOUS ENZYMES. - Google Patents
IMPROVED PROCESS FOR IMMUNOLOGICAL ANALYSIS OF NON-HOMOGENOUS ENZYMES.Info
- Publication number
- EP0040224A4 EP0040224A4 EP19800902222 EP80902222A EP0040224A4 EP 0040224 A4 EP0040224 A4 EP 0040224A4 EP 19800902222 EP19800902222 EP 19800902222 EP 80902222 A EP80902222 A EP 80902222A EP 0040224 A4 EP0040224 A4 EP 0040224A4
- Authority
- EP
- European Patent Office
- Prior art keywords
- enzyme
- analogue
- ligand
- antibody
- liberated
- 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.)
- Withdrawn
Links
- 102000004190 Enzymes Human genes 0.000 title claims abstract description 40
- 108090000790 Enzymes Proteins 0.000 title claims abstract description 40
- 238000000034 method Methods 0.000 title claims abstract description 17
- 238000003018 immunoassay Methods 0.000 title claims abstract description 11
- 239000003446 ligand Substances 0.000 claims abstract description 46
- 229940064790 dilantin Drugs 0.000 claims description 15
- CXOFVDLJLONNDW-UHFFFAOYSA-N Phenytoin Chemical compound N1C(=O)NC(=O)C1(C=1C=CC=CC=1)C1=CC=CC=C1 CXOFVDLJLONNDW-UHFFFAOYSA-N 0.000 claims description 10
- 239000002245 particle Substances 0.000 claims description 6
- 239000011324 bead Substances 0.000 claims description 5
- 102000005936 beta-Galactosidase Human genes 0.000 claims description 4
- 108010005774 beta-Galactosidase Proteins 0.000 claims description 4
- 239000011521 glass Substances 0.000 claims description 3
- 150000004676 glycans Chemical class 0.000 claims description 3
- 229920001282 polysaccharide Polymers 0.000 claims description 3
- 239000005017 polysaccharide Substances 0.000 claims description 3
- 230000001580 bacterial effect Effects 0.000 claims description 2
- 239000007787 solid Substances 0.000 claims 2
- 239000000758 substrate Substances 0.000 claims 2
- 239000007790 solid phase Substances 0.000 description 6
- 238000003556 assay Methods 0.000 description 5
- 229920002684 Sepharose Polymers 0.000 description 4
- 238000000926 separation method Methods 0.000 description 4
- 230000000694 effects Effects 0.000 description 3
- 210000002966 serum Anatomy 0.000 description 3
- 239000000126 substance Substances 0.000 description 3
- -1 Methyl dilantin Chemical compound 0.000 description 2
- 238000011088 calibration curve Methods 0.000 description 2
- 229940079593 drug Drugs 0.000 description 2
- 239000003814 drug Substances 0.000 description 2
- 238000004108 freeze drying Methods 0.000 description 2
- 238000005259 measurement Methods 0.000 description 2
- 239000012071 phase Substances 0.000 description 2
- 229960002695 phenobarbital Drugs 0.000 description 2
- ZFXYFBGIUFBOJW-UHFFFAOYSA-N theophylline Chemical compound O=C1N(C)C(=O)N(C)C2=C1NC=N2 ZFXYFBGIUFBOJW-UHFFFAOYSA-N 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- VZSBFDREVUPFLX-LBPRGKRZSA-N (2s)-2-amino-3-[4-(3-bromo-4-hydroxyphenoxy)-3,5-diiodophenyl]propanoic acid Chemical compound IC1=CC(C[C@H](N)C(O)=O)=CC(I)=C1OC1=CC=C(O)C(Br)=C1 VZSBFDREVUPFLX-LBPRGKRZSA-N 0.000 description 1
- KKEBXNMGHUCPEZ-UHFFFAOYSA-N 4-phenyl-1-(2-sulfanylethyl)imidazolidin-2-one Chemical compound N1C(=O)N(CCS)CC1C1=CC=CC=C1 KKEBXNMGHUCPEZ-UHFFFAOYSA-N 0.000 description 1
- 241000487918 Acacia argyrodendron Species 0.000 description 1
- 108010032595 Antibody Binding Sites Proteins 0.000 description 1
- 241000283707 Capra Species 0.000 description 1
- XUIIKFGFIJCVMT-GFCCVEGCSA-N D-thyroxine Chemical compound IC1=CC(C[C@@H](N)C(O)=O)=CC(I)=C1OC1=CC(I)=C(O)C(I)=C1 XUIIKFGFIJCVMT-GFCCVEGCSA-N 0.000 description 1
- AUYYCJSJGJYCDS-LBPRGKRZSA-N Thyrolar Chemical compound IC1=CC(C[C@H](N)C(O)=O)=CC(I)=C1OC1=CC=C(O)C(I)=C1 AUYYCJSJGJYCDS-LBPRGKRZSA-N 0.000 description 1
- 239000000427 antigen Substances 0.000 description 1
- 102000036639 antigens Human genes 0.000 description 1
- 108091007433 antigens Proteins 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- 235000011222 chang cao shi Nutrition 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 238000011161 development Methods 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
- 238000006073 displacement reaction Methods 0.000 description 1
- 238000001952 enzyme assay Methods 0.000 description 1
- 206010015037 epilepsy Diseases 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- MYWUZJCMWCOHBA-VIFPVBQESA-N methamphetamine Chemical compound CN[C@@H](C)CC1=CC=CC=C1 MYWUZJCMWCOHBA-VIFPVBQESA-N 0.000 description 1
- 125000002496 methyl group Chemical group [H]C([H])([H])* 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- DDBREPKUVSBGFI-UHFFFAOYSA-N phenobarbital Chemical compound C=1C=CC=CC=1C1(CC)C(=O)NC(=O)NC1=O DDBREPKUVSBGFI-UHFFFAOYSA-N 0.000 description 1
- 239000002953 phosphate buffered saline Substances 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 229920006395 saturated elastomer Polymers 0.000 description 1
- 239000000243 solution Substances 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 229960000278 theophylline Drugs 0.000 description 1
- SRVJKTDHMYAMHA-WUXMJOGZSA-N thioacetazone Chemical compound CC(=O)NC1=CC=C(\C=N\NC(N)=S)C=C1 SRVJKTDHMYAMHA-WUXMJOGZSA-N 0.000 description 1
- 229940034208 thyroxine Drugs 0.000 description 1
- XUIIKFGFIJCVMT-UHFFFAOYSA-N thyroxine-binding globulin Natural products IC1=CC(CC([NH3+])C([O-])=O)=CC(I)=C1OC1=CC(I)=C(O)C(I)=C1 XUIIKFGFIJCVMT-UHFFFAOYSA-N 0.000 description 1
- 229940035722 triiodothyronine 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
- G01N33/536—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase
- G01N33/537—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase with separation of immune complex from unbound antigen or antibody
Definitions
- AN IMPROVED METHOD OF NON HOMOGENOUS ENZYME IMMUNOASSAY This invention relates to an improved method of the technique known as non homogenous enzyme immunoassa , described, for example, in “Quantitative Enzyme Immunoassay", Ed. E. Engvall and A.J. Pesce, Blackwood Scientific Publications (Scand. J. Immunol. 8, Suppl. 7, 1978) .
- non homogenous enzyme immunoassay is based on the competition for the active site of an anti ⁇ body between a ligand and a covalently coupled derivative of that same ligand to an enzyme. With separation of the bound and free enzyme ligand phases, enzyme assay of either fraction can then be made. To aid in the separation of the phases, it is often convenient to bind the antibody to an insoluble particle such as the wall of the reaction vessel itself, or a polysaccharide particle. Plotting of the percent bound against the ligand concentration, results in a typical immunoassay curve.
- the enzyme immunoassay system is also capable of simple modification to provide for the measurement of serum antibody levels by making enzyme-anti- body derivatives and using a solid phase ligand to assist in the separation step.
- the antibody characteristics required for the development of a successful non homogenous enzyme immunoassay for a particular ligand are those of high specificity for the ligand, as well as a high apparent affinity constant (as 1/mol) for the ligand.
- an antibody demonstrating a high specificity and a high affinity constant towards a particular ligand will also bind substances which are closely similar in chemical structure to the ligand itself. Such substances are herein referred to as "analogues of the ligand”.
- the affinity constant of the antibody observed with respect to the analogue may differ markedly from that observed for the antibody with the ligand itself. It is the object of
- the present invention provides a method of non homogenous enzyme immunoassay including the steps of: (i) binding an analogue of a ligand to an enzyme to form an enzyme-ligand analogue complex; (ii) contacting the enzyme-ligand analogue complex with antibody for the ligand so as to bind the complex to the antibody;
- a suitable analogue of the ligand is selected such that it is bound specifically by the antibody, though to a much lower affinity than is the case for the original ligand itself.
- Such an analogue is then covalently joined to an appropriat enzyme by known methods of chemistry.
- preparations of the antib are linked by appropriate means to an insoluble particle which may be the surface of the inner wall of the small tube or may be a small insoluble particle, such as a polysaccharide or a glass bead.
- an insoluble particle which may be the surface of the inner wall of the small tube or may be a small insoluble particle, such as a polysaccharide or a glass bead.
- Such a "formed solid phase” antibody is required to retain its function as an antibody.
- the enzyme-labelled analogue is then brought into contact with the solid phase antibody, such that all the binding sites of the solid phase antibody are occupied by the enzyme-labelled analogue. Such an.
- enzyme-labelled analogue may then be displaced from the binding site of the solid phase antibody by addition under appropriate conditions of the original ligand to which the antibody has been derived, or a different analogue having a higher affinity for the antibody than the enzyme-labelled analogue.
- liberated enzyme analogue By separation of the liberated enzyme analogue from the enzyme analogue remaining bound to the solid phases , it is found that the quantity of liberated enzyme analogue, as measured by whatever means, bears an analytical relation ⁇ ship to the quantity of antigen or other ligand which causes the displacement.
- the amount of enzyme analogue liberated by known concentration of ligand in appropriate standards, the measurement of an unknown concen ⁇ tration of the ligand in a sample can be effected.
- known amounts of the enzyme-labelled analogue bound to the antibody can be dispensed into vials and subjected to the process known as freeze-drying.
- freeze-drying By adding a required volume of water to the vial to reconstitute the active enzyme-analogue-antibody-complex, the assay can then be performed directly.
- appropriate precautions to maintain stability of the reagent it would be possible to utilise a previously defined calibration curve from which the amount of ligand in a sample can be calculated without the necessity of so performing a calibration curve at that time. This method thus has application in times of emergency when speed
- an assay for the anti- epilepsy drug dilantin, in the patients taking this drug is described.
- Antibodies were raised to dilantin in goats by appropriate means and the antibodies demonstrated an affinity for dilantin of 10 1/mole, while having an affini for meth l-dilantin of 10 1/mole.
- Methyl dilantin was the covalently joined to bacterial beta-galactosidase using water soluble carbo-dii ide, and the antibody covalently bound to sepharose beads. Aliquots of the sepharose antibo methyl dilantin-beta-galactosidase complexes were formed an all available antibody binding sites saturated.
- Tube No. Dilantin Cont b-Galactosidase activity (Mg/L) (A405/min/ml)
- ligand or ligand analogue such as the systems phenobarbitone (meta-nitro phenobarbitone) theophylline (l-methyl-3-carboxy butyl xanthine) thyroxine (3,3' 5 - triiodo-5 '-bromo thyronine) and triiodothyronine (3,5 - diiodo-3'-bromo thyronine).
- phenobarbitone metal-nitro phenobarbitone
- theophylline l-methyl-3-carboxy butyl xanthine
- thyroxine l-methyl-3-carboxy butyl xanthine
- thyroxine l-methyl-3-carboxy butyl xanthine
- thyroxine l-methyl-3-carboxy butyl xanthine
- thyroxine l-methyl-3-carboxy butyl x
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)
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| AU1374/79 | 1979-11-19 | ||
| AUPE137479 | 1979-11-19 |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| EP0040224A1 EP0040224A1 (en) | 1981-11-25 |
| EP0040224A4 true EP0040224A4 (en) | 1982-09-09 |
Family
ID=3768341
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| EP19800902222 Withdrawn EP0040224A4 (en) | 1979-11-19 | 1980-11-19 | IMPROVED PROCESS FOR IMMUNOLOGICAL ANALYSIS OF NON-HOMOGENOUS ENZYMES. |
Country Status (3)
| Country | Link |
|---|---|
| EP (1) | EP0040224A4 (enrdf_load_stackoverflow) |
| JP (1) | JPS56501583A (enrdf_load_stackoverflow) |
| WO (1) | WO1981001414A1 (enrdf_load_stackoverflow) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| DE3626468A1 (de) * | 1986-08-05 | 1988-02-11 | Hoechst Ag | Verfahren und testkit zur bestimmung freier wirkstoffe in biologischen fluessigkeiten |
Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| FR2184371A5 (enrdf_load_stackoverflow) * | 1972-05-11 | 1973-12-21 | Akzo Nv | |
| GB1401298A (en) * | 1971-05-14 | 1975-07-16 | Syntex Energy Res | Ligands bonded to enzymes |
| US3966764A (en) * | 1972-07-10 | 1976-06-29 | Syva Company | Ligand determination of spin labeled compounds by receptor displacement-amphetamine analogs |
| FR2348493A1 (fr) * | 1976-04-15 | 1977-11-10 | Technicon Instr | Immunotest pour la diphenyl-hydantoine |
| GB1575610A (en) * | 1976-04-15 | 1980-09-24 | Technicon Instr | Immunoassay for diphenylhydantoin |
| EP0026103A1 (en) * | 1979-09-24 | 1981-04-01 | AMERSHAM INTERNATIONAL plc | A method for determining the free portion of substances in biological fluids |
Family Cites Families (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US3673410A (en) * | 1969-06-25 | 1972-06-27 | John H Waite | Method of examination of cell samples using a radioactively tagged dye |
| US3852157A (en) * | 1971-05-14 | 1974-12-03 | Syva Corp | Compounds for enzyme amplification assay |
| US3817837A (en) * | 1971-05-14 | 1974-06-18 | Syva Corp | Enzyme amplification assay |
| US3905871A (en) * | 1971-05-14 | 1975-09-16 | Syva Co | Lactam conjugates to enzymes |
| DE2128670B2 (de) * | 1971-06-09 | 1977-06-30 | Merck Patent Gmbh, 6100 Darmstadt | Immunologische isoenzym-bestimmungsmethode |
| US3975237A (en) * | 1972-11-06 | 1976-08-17 | Syva Company | Compounds for enzyme amplification assay - - ecgonine analogs |
| US3966556A (en) * | 1972-11-06 | 1976-06-29 | Syva Company | Compounds for enzyme amplification assay methadone analogs |
| IN142734B (enrdf_load_stackoverflow) * | 1975-04-28 | 1977-08-20 | Miles Lab | |
| JPS5925183B2 (ja) * | 1978-07-05 | 1984-06-15 | ダイナボット株式会社 | エラスタ−ゼ−１の測定方法 |
| US4273866A (en) * | 1979-02-05 | 1981-06-16 | Abbott Laboratories | Ligand analog-irreversible enzyme inhibitor conjugates and methods for use |
-
1980
- 1980-11-19 JP JP50264580A patent/JPS56501583A/ja active Pending
- 1980-11-19 EP EP19800902222 patent/EP0040224A4/en not_active Withdrawn
- 1980-11-19 WO PCT/AU1980/000090 patent/WO1981001414A1/en not_active Application Discontinuation
Patent Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| GB1401298A (en) * | 1971-05-14 | 1975-07-16 | Syntex Energy Res | Ligands bonded to enzymes |
| GB1401297A (en) * | 1971-05-14 | 1975-07-16 | Syntex Energy Res | Enzyme aplification assay |
| FR2184371A5 (enrdf_load_stackoverflow) * | 1972-05-11 | 1973-12-21 | Akzo Nv | |
| GB1433783A (en) * | 1972-05-11 | 1976-04-28 | Akzo Nv | Process for the detection and determination of haptens |
| US3966764A (en) * | 1972-07-10 | 1976-06-29 | Syva Company | Ligand determination of spin labeled compounds by receptor displacement-amphetamine analogs |
| FR2348493A1 (fr) * | 1976-04-15 | 1977-11-10 | Technicon Instr | Immunotest pour la diphenyl-hydantoine |
| GB1575610A (en) * | 1976-04-15 | 1980-09-24 | Technicon Instr | Immunoassay for diphenylhydantoin |
| EP0026103A1 (en) * | 1979-09-24 | 1981-04-01 | AMERSHAM INTERNATIONAL plc | A method for determining the free portion of substances in biological fluids |
Non-Patent Citations (1)
| Title |
|---|
| See also references of WO8101414A1 * |
Also Published As
| Publication number | Publication date |
|---|---|
| EP0040224A1 (en) | 1981-11-25 |
| WO1981001414A1 (en) | 1981-05-28 |
| JPS56501583A (enrdf_load_stackoverflow) | 1981-10-29 |
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Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PUAI | Public reference made under article 153(3) epc to a published international application that has entered the european phase |
Free format text: ORIGINAL CODE: 0009012 |
|
| 17P | Request for examination filed |
Effective date: 19810810 |
|
| AK | Designated contracting states |
Designated state(s): DE FR GB NL SE |
|
| STAA | Information on the status of an ep patent application or granted ep patent |
Free format text: STATUS: THE APPLICATION IS DEEMED TO BE WITHDRAWN |
|
| 18D | Application deemed to be withdrawn |
Effective date: 19831129 |
|
| RIN1 | Information on inventor provided before grant (corrected) |
Inventor name: DUFFY, PATRICK |