WO1997016712A9 - Procede electrochimique pour une determination quantitative de proteines de liaison - Google Patents
Procede electrochimique pour une determination quantitative de proteines de liaisonInfo
- Publication number
- WO1997016712A9 WO1997016712A9 PCT/DE1996/001977 DE9601977W WO9716712A9 WO 1997016712 A9 WO1997016712 A9 WO 1997016712A9 DE 9601977 W DE9601977 W DE 9601977W WO 9716712 A9 WO9716712 A9 WO 9716712A9
- 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 45
- 108091008324 binding proteins Proteins 0.000 title claims abstract description 31
- 102000014914 Carrier Proteins Human genes 0.000 title abstract description 23
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 claims abstract description 19
- 239000010439 graphite Substances 0.000 claims abstract description 19
- 229910002804 graphite Inorganic materials 0.000 claims abstract description 19
- 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 5
- 108090000608 Phosphoric Monoester Hydrolases Proteins 0.000 claims abstract description 5
- 238000003776 cleavage reaction Methods 0.000 claims abstract description 4
- 230000007017 scission Effects 0.000 claims abstract description 4
- 239000008366 buffered solution Substances 0.000 claims abstract 2
- 238000005259 measurement Methods 0.000 claims description 15
- 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
- 229910052709 silver Inorganic materials 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
- 229910021607 Silver chloride Inorganic materials 0.000 claims description 3
- 239000004332 silver Substances 0.000 claims description 3
- HKZLPVFGJNLROG-UHFFFAOYSA-M silver monochloride Chemical compound [Cl-].[Ag+] HKZLPVFGJNLROG-UHFFFAOYSA-M 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 15
- 238000002965 ELISA Methods 0.000 description 4
- 101000799549 Homo sapiens Aspartate aminotransferase, mitochondrial Proteins 0.000 description 4
- PLIKAWJENQZMHA-UHFFFAOYSA-N 4-aminophenol Chemical compound NC1=CC=C(O)C=C1 PLIKAWJENQZMHA-UHFFFAOYSA-N 0.000 description 3
- BQCADISMDOOEFD-UHFFFAOYSA-N Silver Chemical compound [Ag] BQCADISMDOOEFD-UHFFFAOYSA-N 0.000 description 3
- 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
- 238000001035 drying Methods 0.000 description 2
- 238000001493 electron microscopy Methods 0.000 description 2
- 230000001900 immune effect Effects 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
- 102100035882 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
- 238000006243 chemical reaction Methods 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
- 230000001419 dependent effect Effects 0.000 description 1
- 238000009795 derivation Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000018109 developmental process Effects 0.000 description 1
- 230000002255 enzymatic effect Effects 0.000 description 1
- 238000006911 enzymatic reaction Methods 0.000 description 1
- 238000011156 evaluation Methods 0.000 description 1
- 239000011888 foil Substances 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 239000003550 marker Substances 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
- 108090000623 proteins and genes Proteins 0.000 description 1
- 238000010079 rubber tapping Methods 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- 238000003756 stirring Methods 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
Definitions
- the invention relates to a method for the immunological / enzymatic detection of binding proteins in aqueous media and to an apparatus for carrying out this method.
- the determination of binding proteins has a special relevance in clinical diagnostics.
- cardiac human fatty acid binding proteins H-FABP
- the determination of FABP has particular importance in diagnostics, since in the case of cardiac muscle damage cardiac FABP (H-FABP) emerges from the heart muscle and can be detected in the blood after a few hours. For this reason, FABP is to be regarded as a heart attack marker. A fast and accurate method for the detection of FABP is therefore of particular importance.
- ERSATZBLA ⁇ (REGEL26) Determination based on ELISA or RIA method.
- This method is based on an immobilization of capture antibodies on a membrane and a competitive reaction between FABP of the sample and added catalase labeled FABP.
- the disadvantage of this method of competition is the number of necessary process steps, the elaborate preparation of the antibody membranes, the very long analysis times, the insufficient sensitivity, the conditional use in pure plasma or blood, and the level of nonspecific measurements.
- the inventive method is thus based on a combination of three process steps.
- a special electrode namely a disposable graphite electrode with an antibody directed against the binding protein (capture antibody) is used.
- ERSATZBLA1T (REGEL26) body) immobilized.
- a sandwich of antibody, binding protein and conjugate is then produced on the electrode and finally the measurement is carried out with an electrode thus produced, by carrying out a sensitive amperometric detection of the cleavage product of the aromatic phosphate ester.
- Essential in the process according to the application is the use of a disposable graphite electrode and the immobilization of the antibody directly on the electrode surface.
- an electrode is available which can be used in a simple manner for the measuring method according to the invention.
- the antibody-immobilized electrode is e.g. be ⁇ with a BSA solution and dried, can be stored easily over a longer period. It has been shown that the thus prepared graphite electrodes are stable even over several weeks at 4 ° C. It is particularly preferred if a graphite layer deposited on a carrier material by screen printing is used as the disposable electrode.
- carrier material a laminating film is particularly preferred. The procedure is such that e.g.
- a Leit-C special adhesive for the electron microscopy is mixed with a screen-printing oil and applied to a laminating film by a stencil or by a screen with a corresponding electrode shape.
- the reference electrode can also be produced by e.g. a Sil ber / silver chloride paste applied to another laminating or together with the graphite electrode
- ERSATZBLA ⁇ (REGEL26) is applied to a film. It is also possible to weld the electrode leads to a second laminating foil for stiffening.
- a single-use graphite electrode as described above can now be used in various ways for the process 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 formed.
- a conjugate means a phosphatase-labeled antibody to the binding protein.
- the binding protein can also be mixed with the conjugate before application to the electrode, and then the mixture thus produced can be applied to the electrode.
- a third particularly preferred process variant now provides that the antibody-coated disposable graphite electrode is first coated with conjugate.
- the procedure is such that the conjugate is applied to the electrode and dried.
- the electrode prepared in this way is then only to be moved with the sample shortly before the measurement.
- Die ⁇ se last variant has the advantage that the mixing of conjugate and binding protein ent falls before the measurement and thus a pipetting step is saved.
- the electrodes coated with conjugate and antibody are otherwise stable for several weeks.
- the implementation of the measurement is possible in a very simple manner.
- the binding protein in dependence on the concentration of binding protein binds in the alkaline phosphatase binding protein antibody and the immobilized binding protein antibody to the binding protein.
- the enzyme fixed in proportion to the binding protein concentration, then generates the electrochemically reacted cleavage product upon addition of aromatic phosphate ester.
- H-FABP is used as the binding protein. It has proven to be advantageous in this case if alkaline phosphatase is used in the conjugate and p-amino phenyl phosphate is used in the case of the aromatic phosphate ester.
- the invention further relates to a device for carrying out the method.
- the device is characterized in particular by the fact that at least one row with two to six containers which can be filled with buffers and substrate is provided in a housing.
- the device is designed so that the electrodes can be introduced into the vessels via a suitable device. Characterized in that at least two vessels are provided, both a reference electrode and a measuring electrode can be measured simultaneously.
- ERSATZBLA ⁇ (REGEL26)
- Fig. 1 shows the calibration curves of FABP in plasma and a comparison with the ELISA method
- 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 wire with a corresponding 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 a subsequent body.
- a second laminating film is additionally placed over the web in order to achieve stiffening. The head of the electrode remains free here.
- the second laminating film is also arranged above the head, in which case a corresponding opening (for example perforation) of the second laminating film is present in the region of the electrode head 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 a 30 mm long bar for the derivation and the tapping of the current.
- the reference electrode is either applied in the same form with silver / Silberchloridpa ⁇ ste on a second film or together with the graphite electrode on a film.
- the capture antibody (1 ⁇ g) is applied to the graphite electrode as described above, and adsorbed for about 15 minutes. Subsequently, the surface is treated for about 15 minutes with a 2% BSA solution.
- the electrode can now be used for a measurement or stored after drying at 4 ° C.
- a coating with conjugate and subsequent drying can be carried out. The last variant eliminates the need to mix the conjugate and sample prior to measurement and saves a pipetting step.
- the conjugate and antibody coated electrodes are also stable for several weeks.
- the electrodes coated with conjugate are concerned, the patient's 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 potentiostat 5 and a stirring 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.
- 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 successively 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 of p-aminophenyl phosphate.
- the amperometric current signal develops in about 10 to 20 seconds at 300 mV against silver / silver chloride and is converted according to the calibration curve into the concentration of FABP.
- the immunological and enzymatic reaction is reproduced in the following equations Ia to Id. O
- Fig. 4 shows the results obtained in the measurement with respect to calibration and comparison with the ELISA method. Fig. 4 makes it clear that with the measuring method according to the invention results can be achieved in a simple and fast manner, which are comparable to those of the ELISA method.
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 WO1997016712A2 (fr) | 1997-05-09 |
WO1997016712A3 WO1997016712A3 (fr) | 1997-06-12 |
WO1997016712A9 true 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) |
Families Citing this family (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP1136819A3 (fr) * | 1997-04-24 | 2001-11-28 | Daikin Industries, Ltd. | Microplaque avec beaucoup de cellules, chaque cellule comprenant deux électrodes dans le fond |
DE19725190A1 (de) * | 1997-06-14 | 1998-12-17 | Innova Gmbh | Vorrichtungen mit integrierten Elektroden aus elektrisch leitfähigen Kunststoffen |
US6682648B1 (en) | 1997-08-12 | 2004-01-27 | University Of Southern California | Electrochemical reporter system for detecting analytical immunoassay and molecular biology procedures |
CA2300268A1 (fr) * | 1997-08-12 | 1999-02-18 | Robert D. Macphee | Systeme reporteur electrochimique permettant de detecter les dosages immunologiques analytiques et procedures de biologie moleculaire |
DE19836109A1 (de) * | 1998-08-10 | 2000-03-02 | Biotul Bio Instr Gmbh | Vorrichtung und Verfahren zur grenzflächennahen Mischung von Proben in Biosensorsystemen |
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 |
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
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