+

WO1997016712A9 - Procede electrochimique pour une determination quantitative de proteines de liaison - Google Patents

Procede electrochimique pour une determination quantitative de proteines de liaison

Info

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
Application number
PCT/DE1996/001977
Other languages
German (de)
English (en)
Other versions
WO1997016712A3 (fr
WO1997016712A2 (fr
Filing date
Publication date
Priority claimed from DE1995141033 external-priority patent/DE19541033C1/de
Application filed filed Critical
Publication of WO1997016712A2 publication Critical patent/WO1997016712A2/fr
Publication of WO1997016712A3 publication Critical patent/WO1997016712A3/fr
Publication of WO1997016712A9 publication Critical patent/WO1997016712A9/fr

Links

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.
PCT/DE1996/001977 1995-11-03 1996-10-15 Procede electrochimique pour une determination quantitative de proteines de liaison WO1997016712A2 (fr)

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)

* Cited by examiner, † Cited by third party
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)

* Cited by examiner, † Cited by third party
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

Similar Documents

Publication Publication Date Title
DE69427353T2 (de) Potentiometrischer biosensor und verfahren zu dessen gebrauch
DE69222272T2 (de) Biosensor und Verfahren zur quantitativen Analyse
DE69617464T2 (de) Teststreifen für einen elektochemischen biosensor
DE69025134T2 (de) Verfahren zur Herstellung eines Biosensors
DE68924026T3 (de) Biosensor und dessen herstellung.
DE69628588T2 (de) Elektrochemisches verfahren
DE69603416T2 (de) Elektrochemischer Immunobiosensor
DE3889724T2 (de) Immobilisierte enzymelektroden sowie eine methode zur reduzierung der alkoholempfindlichkeit.
DE10009503A1 (de) Verfahren zur Immobilisierung von Konjugaten in diagnostischen Tests
EP0790498A1 (fr) Capteurs électrochimiques à sélectivité améliorée et à sensibilité augmentée
DE3022426A1 (de) Elektrochemilumineszenz-immunoassay
DE3882723T2 (de) Verfahren zur Herstellung von Enzymelektroden.
DE10296998T5 (de) Enzymelektrode und Verfahren zum Herstellen derselben
DE69628243T2 (de) Planarer Bicarbonatsensor
DE4011428A1 (de) Messvorrichtung und verfahren zur automatischen bestimmung der konzentration von biokatalysatorsubstraten
EP1141713A1 (fr) Procede et dispositif pour determiner un analyte
DE602004007653T2 (de) Optischer Biosensor
DE69422781T2 (de) Elektrochemische metalluntersuchung
DE19540098C2 (de) Verfahren und Mehrkanalbiosensor zur Mehrkomponentenanalyse von Mischungen und/oder Gemischen
WO1997016712A9 (fr) Procede electrochimique pour une determination quantitative de proteines de liaison
DE19541033C1 (de) Elektrochemisches Verfahren zur quantitativen Bestimmung von Bindungsproteinen
EP0535578B1 (fr) Electrodes de conductivité à couche épaisse comme capteur biologique
DE4027728A1 (de) Immobilisierung von organischen makromolekuelen oder biopolymeren in einer polymermembran
DE4216980C2 (de) Immunosensorisches Nachweisverfahren
DE19632778A1 (de) Dickschichtsensor zur Bestimmung der Peroxidaseaktivität
点击 这是indexloc提供的php浏览器服务，不要输入任何密码和下载