WO1997016712A2 - Procede electrochimique pour une determination quantitative de proteines de liaison - Google Patents
Procede electrochimique pour une determination quantitative de proteines de liaison Download PDFInfo
- Publication number
- WO1997016712A2 WO1997016712A2 PCT/DE1996/001977 DE9601977W WO9716712A2 WO 1997016712 A2 WO1997016712 A2 WO 1997016712A2 DE 9601977 W DE9601977 W DE 9601977W WO 9716712 A2 WO9716712 A2 WO 9716712A2
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- electrode
- conjugate
- antibody
- binding protein
- aromatic phosphate
- Prior art date
Links
- 238000000034 method Methods 0.000 title claims abstract description 44
- 108091008324 binding proteins Proteins 0.000 title claims abstract description 30
- 102000014914 Carrier Proteins Human genes 0.000 title abstract description 22
- 239000010439 graphite Substances 0.000 claims abstract description 16
- 229910002804 graphite Inorganic materials 0.000 claims abstract description 16
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 claims abstract description 15
- 229910019142 PO4 Inorganic materials 0.000 claims abstract description 10
- 239000010452 phosphate Substances 0.000 claims abstract description 10
- -1 aromatic phosphate ester Chemical class 0.000 claims abstract description 8
- 102000004160 Phosphoric Monoester Hydrolases Human genes 0.000 claims abstract description 3
- 108090000608 Phosphoric Monoester Hydrolases Proteins 0.000 claims abstract description 3
- 238000003776 cleavage reaction Methods 0.000 claims abstract description 3
- 230000007017 scission Effects 0.000 claims abstract description 3
- 239000008366 buffered solution Substances 0.000 claims abstract 2
- 238000005259 measurement Methods 0.000 claims description 13
- 238000010030 laminating Methods 0.000 claims description 11
- 239000000872 buffer Substances 0.000 claims description 6
- 239000012876 carrier material Substances 0.000 claims description 6
- 238000007650 screen-printing Methods 0.000 claims description 5
- 229910021607 Silver chloride Inorganic materials 0.000 claims description 4
- 229910052709 silver Inorganic materials 0.000 claims description 4
- 239000004332 silver Substances 0.000 claims description 4
- HKZLPVFGJNLROG-UHFFFAOYSA-M silver monochloride Chemical compound [Cl-].[Ag+] HKZLPVFGJNLROG-UHFFFAOYSA-M 0.000 claims description 4
- 239000000243 solution Substances 0.000 claims description 4
- 102000002260 Alkaline Phosphatase Human genes 0.000 claims description 3
- 108020004774 Alkaline Phosphatase Proteins 0.000 claims description 3
- 239000000758 substrate Substances 0.000 claims description 3
- 238000002848 electrochemical method Methods 0.000 claims description 2
- 238000011534 incubation Methods 0.000 claims description 2
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 claims description 2
- 102000023732 binding proteins Human genes 0.000 claims 8
- 102000011026 Fatty Acid Binding Protein 3 Human genes 0.000 claims 1
- 108010062715 Fatty Acid Binding Protein 3 Proteins 0.000 claims 1
- 125000003118 aryl group Chemical group 0.000 claims 1
- 230000015572 biosynthetic process Effects 0.000 claims 1
- 229940127121 immunoconjugate Drugs 0.000 abstract 1
- 101100226596 Gallus gallus FABP gene Proteins 0.000 description 19
- 238000002965 ELISA Methods 0.000 description 4
- 101000799549 Homo sapiens Aspartate aminotransferase, mitochondrial Proteins 0.000 description 4
- 238000001514 detection method Methods 0.000 description 3
- 238000002360 preparation method Methods 0.000 description 3
- 239000000853 adhesive Substances 0.000 description 2
- 230000001070 adhesive effect Effects 0.000 description 2
- 239000008280 blood Substances 0.000 description 2
- 210000004369 blood Anatomy 0.000 description 2
- 238000011088 calibration curve Methods 0.000 description 2
- 230000000747 cardiac effect Effects 0.000 description 2
- 238000006243 chemical reaction Methods 0.000 description 2
- 238000001035 drying Methods 0.000 description 2
- 238000001493 electron microscopy Methods 0.000 description 2
- 239000011888 foil Substances 0.000 description 2
- 239000012528 membrane Substances 0.000 description 2
- 210000004165 myocardium Anatomy 0.000 description 2
- ZYPZVOKVDNSKLP-UHFFFAOYSA-N tris(4-aminophenyl) phosphate Chemical compound C1=CC(N)=CC=C1OP(=O)(OC=1C=CC(N)=CC=1)OC1=CC=C(N)C=C1 ZYPZVOKVDNSKLP-UHFFFAOYSA-N 0.000 description 2
- BVKZGUZCCUSVTD-UHFFFAOYSA-L Carbonate Chemical compound [O-]C([O-])=O BVKZGUZCCUSVTD-UHFFFAOYSA-L 0.000 description 1
- 102000016938 Catalase Human genes 0.000 description 1
- 108010053835 Catalase Proteins 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 208000029549 Muscle injury Diseases 0.000 description 1
- 238000004082 amperometric method Methods 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 239000012736 aqueous medium Substances 0.000 description 1
- 230000000903 blocking effect Effects 0.000 description 1
- 239000007853 buffer solution Substances 0.000 description 1
- 239000011248 coating agent Substances 0.000 description 1
- 238000000576 coating method Methods 0.000 description 1
- 230000002860 competitive effect Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000018109 developmental process Effects 0.000 description 1
- 238000007599 discharging Methods 0.000 description 1
- 230000002255 enzymatic effect Effects 0.000 description 1
- 238000011156 evaluation Methods 0.000 description 1
- 230000001900 immune effect Effects 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- 238000000691 measurement method Methods 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 208000010125 myocardial infarction Diseases 0.000 description 1
- 238000010079 rubber tapping Methods 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- 238000001179 sorption measurement Methods 0.000 description 1
- 239000000126 substance Substances 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
- G01N33/54366—Apparatus specially adapted for solid-phase testing
- G01N33/54373—Apparatus specially adapted for solid-phase testing involving physiochemical end-point determination, e.g. wave-guides, FETS, gratings
- G01N33/5438—Electrodes
Definitions
- the invention relates to a method for the immunological / enzymatic detection of binding proteins in aqueous media and a device for carrying out this method.
- the determination of binding proteins is of particular relevance in clinical diagnostics.
- the determination of cardiac human fatty acid binding proteins (H-FABP) may be mentioned here as an example.
- the determination of FABP is of particular importance in diagnostics since cardiac FABP (H-FABP) emerges from the heart muscle in the case of cardiac muscle damage and can be detected in the blood after a few hours. For this reason, FABP can also be regarded as a heart attack marker. A fast and precise method for the detection of FABP is therefore of particular importance.
- Classic methods of FABP Determination are based on ELISA or RIA methods. In addition to these classic methods, Spener, F. et al. , 1992, Dechema Biotechnol. Conf. an amperometric method for determining FABP is known.
- This method is based on an immobilization of capture antibodies on a membrane and a competition reaction between FABP of the sample and added catalase-labeled FABP.
- the disadvantage of this competitive process is the number of necessary process steps, the complex preparation of the antibody membranes, the very long analysis times, the insufficient sensitivity, the limited use in pure plasma or blood and the level of the non-specific measured values.
- the method according to the invention is therefore based on a combination of three method steps.
- a special electrode namely a single-use graphite electrode with an antibody directed against the binding protein (catcher anti body) immobilized.
- a sandwich of antibody, binding protein and conjugate is then produced on the electrode and the measurement is ultimately carried out with an electrode produced in this way by performing a sensitive amperometric detection of the cleavage product of the aromatic phosphate ester.
- the procedure is such that, for example, a Leit-C special adhesive for electron microscopy is mixed with a screen printing oil and applied to a laminating film through a stencil or through a sieve with an appropriate electrode shape.
- the reference electrode can also be produced in a corresponding manner, for example by applying a silver / silver chloride paste to a further laminating film or together with the graphite electrode is applied to a film. It is also possible to weld the electrode leads to a second laminating film for stiffening.
- a disposable graphite electrode as described above can now be used in various ways for the method according to the invention.
- the binding protein and the conjugate can be pipetted onto the graphite electrode, so that the sandwich of antibody, binding protein and conjugate is then formed.
- a conjugate is understood to mean a phosphatase-labeled antibody against the binding protein.
- the binding protein can also be mixed with the conjugate before being applied to the electrode and the mixture thus prepared can then be applied to the electrode.
- a third particularly preferred method variant now provides that the single-graphite electrode coated with antibody is first coated with conjugate.
- the procedure here is that the conjugate is applied to the electrode and dried.
- the electrode prepared in this way is then only to be added to the sample shortly before the measurement.
- This last variant has the advantage that the mixing of conjugate and binding protein is omitted before the measurement and thus a pipetting step is saved.
- the electrodes coated with conjugate and antibody are otherwise stable for several weeks.
- the measurement can be carried out in a very simple manner.
- the binding protein antibody binds in the alkaline phosphatase and the immobilized binding protein antibody binds to the binding protein.
- the enzyme which is fixed in proportion to the concentration of binding protein, then generates the electrochemically convertible product to be converted when aromatic phosphate ester is added.
- H-FABP H-FABP
- alkaline phosphatase is used for the conjugate and p-aminophenyl phosphate is used for the aromatic phosphate ester.
- the invention further relates to an apparatus for performing the method.
- the device is particularly characterized in that at least one row with two to six vessels which can be filled with buffers and substrate is provided in a housing.
- the device is designed such that the electrodes can be inserted into the vessels via a suitable device. Because at least two vessels are provided, both a reference electrode and a measuring electrode can be measured simultaneously.
- Fig. 4 shows the device in plan view.
- a Leit-C special adhesive for electron microscopy (Neubauer chemicals) is mixed with a screen printing oil and applied to a laminating film through a stencil or through a sieve with an appropriate electrode shape.
- the electrode 1 shows three different embodiments of how such an electrode can be constructed.
- the electrode 1 consists of a laminating film and an applied graphite layer in the form of a web and an adjoining body.
- a second laminating film is additionally placed over the web to achieve stiffening. The head of the electrode remains free here.
- the second laminating film is also arranged above the head, in which case there is a corresponding opening (eg perforation) in the second laminating film in the region of the electrode head in order to make contact with the electrode.
- the Electrode usually has a diameter of 5 mm and an approximately 30 mm long web for discharging and tapping the current.
- the reference electrode is either applied in the same form with silver / silver chloride paste on a second foil or together with the graphite electrode on a foil.
- the catcher antibody (1 ⁇ g) is applied to the graphite electrode, as described above, and immobilized by adsorption for about 15 minutes.
- the surface is then treated with a 2% BSA solution for about 15 minutes.
- the electrode can now be used for a measurement or can be stored at 4 ° C after drying.
- a coating with conjugate and subsequent drying can be carried out. With the last variant, there is no need to mix the conjugate and sample before the measurement and saves a pipetting step.
- the electrodes coated with conjugate and antibody are also stable for several weeks.
- conjugate can be added to the patient's plasma beforehand. The incubation period is 15 min. If the electrodes are coated with conjugate, the patient plasma can also be applied directly. Then the electrode becomes short Rinsed with TBS buffer, the electrodes are then prepared for the measurement.
- the measurement is carried out with a device according to FIGS. 2 and 3.
- Fig. 2 shows in cross section schematically the structure of the device.
- the device consists of a housing 4, in which potentiostats 5 and a stirrer motor 6 are installed.
- four vessels 14 to 17 for receiving the buffer solution and the substrate are arranged in a row 12 (measuring buffer well).
- the electrodes 7, 8, 9, 10 can be inserted into these vessels 14 to 17.
- the electrodes are connected via a line 11 to the potentiostat 5, which in turn is connected to a PC for evaluation.
- Fig. 3 shows the device described in Fig. 2 in plan view.
- the device according to the invention it is also possible with the device according to the invention to immerse the electrodes 7 to 10 in succession in different measuring buffer wells 13, 18, 19, 20, 21. This can be done by automatic control.
- the electrodes are immersed in a 0.1 M carbonate buffer pH 9.4 0.1 M KCl 1 ⁇ g / ml p-aminophenyl phosphate.
- the amperometric current signal develops in about 10 to 20 seconds at 300 mV against silver / silver chloride and is converted into the concentration of FABP according to the calibration curve.
- the reaction which proceeds immunologically and enzymatically is shown in the following equations la to ld.
- FIG. 4 shows the results obtained in the measurement in relation to the calibration and the comparison with the ELISA method. Fig. 4 makes it clear that the measurement method according to the invention achieves results in a simple and quick manner which are comparable with those of the ELISA method.
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)
- Investigating Or Analysing Biological Materials (AREA)
- Peptides Or Proteins (AREA)
Abstract
L'invention concerne un procédé électrochimique pour la détermination quantitative de protéines de liaison, la protéine de liaison étant une composante d'un système anticorps/conjugué appliqué sur une électrode. Ledit procédé est caractérisé en ce que: a) l'anticorps est immobilisé sur une électrode graphite jetable, b) la protéine de liaison et un conjugué contenant une phosphatase et des anticorps sont appliqués sur l'électrode de façon à se lier pour former un composé sandwich anticorps-protéine de liaison-conjugué et c) cette électrode est mélangée à une solution tampon d'un ester phosphaté aromatique après quoi le produit de fission de l'ester phosphaté aromatique est mesuré ampérométriquement.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| DE19541033.5 | 1995-11-03 | ||
| DE1995141033 DE19541033C1 (de) | 1995-11-03 | 1995-11-03 | Elektrochemisches Verfahren zur quantitativen Bestimmung von Bindungsproteinen |
Publications (3)
| Publication Number | Publication Date |
|---|---|
| WO1997016712A2 true WO1997016712A2 (fr) | 1997-05-09 |
| WO1997016712A3 WO1997016712A3 (fr) | 1997-06-12 |
| WO1997016712A9 WO1997016712A9 (fr) | 1997-08-14 |
Family
ID=7776559
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| PCT/DE1996/001977 WO1997016712A2 (fr) | 1995-11-03 | 1996-10-15 | Procede electrochimique pour une determination quantitative de proteines de liaison |
Country Status (2)
| Country | Link |
|---|---|
| DE (1) | DE19541033C1 (fr) |
| WO (1) | WO1997016712A2 (fr) |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1999007879A1 (fr) * | 1997-08-12 | 1999-02-18 | University Of Southern California | Systeme reporteur electrochimique permettant de detecter les dosages immunologiques analytiques et procedures de biologie moleculaire |
| EP0978722A4 (fr) * | 1997-04-24 | 2000-08-09 | Daikin Ind Ltd | Capteur |
| US6682648B1 (en) | 1997-08-12 | 2004-01-27 | University Of Southern California | Electrochemical reporter system for detecting analytical immunoassay and molecular biology procedures |
| CN107389758A (zh) * | 2017-07-31 | 2017-11-24 | 重庆微奥云生物技术有限公司 | 一种酶检测系统及检测质量控制方法 |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| DE19725190A1 (de) * | 1997-06-14 | 1998-12-17 | Innova Gmbh | Vorrichtungen mit integrierten Elektroden aus elektrisch leitfähigen Kunststoffen |
| DE19836109A1 (de) * | 1998-08-10 | 2000-03-02 | Biotul Bio Instr Gmbh | Vorrichtung und Verfahren zur grenzflächennahen Mischung von Proben in Biosensorsystemen |
Family Cites Families (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4997526A (en) * | 1985-03-19 | 1991-03-05 | Eic Laboratories, Inc. | Assaying for a biologically active component |
| IL78034A (en) * | 1986-03-04 | 1991-08-16 | Univ Ramot | Biosensors comprising antibodies bonded to glassy carbon electrode for immunoassays |
| IL82131A0 (en) * | 1987-04-07 | 1987-10-30 | Univ Ramot | Coulometric assay system |
| US5391272A (en) * | 1992-03-06 | 1995-02-21 | Andcare, Inc. | Electrochemical immunoassay methods |
-
1995
- 1995-11-03 DE DE1995141033 patent/DE19541033C1/de not_active Expired - Fee Related
-
1996
- 1996-10-15 WO PCT/DE1996/001977 patent/WO1997016712A2/fr active Application Filing
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0978722A4 (fr) * | 1997-04-24 | 2000-08-09 | Daikin Ind Ltd | Capteur |
| WO1999007879A1 (fr) * | 1997-08-12 | 1999-02-18 | University Of Southern California | Systeme reporteur electrochimique permettant de detecter les dosages immunologiques analytiques et procedures de biologie moleculaire |
| US6682648B1 (en) | 1997-08-12 | 2004-01-27 | University Of Southern California | Electrochemical reporter system for detecting analytical immunoassay and molecular biology procedures |
| CN107389758A (zh) * | 2017-07-31 | 2017-11-24 | 重庆微奥云生物技术有限公司 | 一种酶检测系统及检测质量控制方法 |
Also Published As
| Publication number | Publication date |
|---|---|
| WO1997016712A3 (fr) | 1997-06-12 |
| DE19541033C1 (de) | 1997-06-26 |
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