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WO2000066770A2 - Procede de realisation d'une matrice en phase solide chimiostable et thermostable pour l'amplification par reaction en chaine de la polymerase (pcr) et les essais d'hybridation d'adn - Google Patents

Procede de realisation d'une matrice en phase solide chimiostable et thermostable pour l'amplification par reaction en chaine de la polymerase (pcr) et les essais d'hybridation d'adn Download PDF

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Publication number
WO2000066770A2
WO2000066770A2 PCT/DE2000/001309 DE0001309W WO0066770A2 WO 2000066770 A2 WO2000066770 A2 WO 2000066770A2 DE 0001309 W DE0001309 W DE 0001309W WO 0066770 A2 WO0066770 A2 WO 0066770A2
Authority
WO
WIPO (PCT)
Prior art keywords
solid phase
phase matrix
thermostable
pcr
chemically
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.)
Ceased
Application number
PCT/DE2000/001309
Other languages
German (de)
English (en)
Other versions
WO2000066770A3 (fr
Inventor
Pavel Strohner
Eberhard Schmidt
Wolfgang Klein
Joachim Schreiber
Renate Bienert
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
BIOTEZ BERLIN-BUCH GmbH
Bio Tez Berlin Buch GmbH
Original Assignee
BIOTEZ BERLIN-BUCH GmbH
Bio Tez Berlin Buch GmbH
Priority date (The priority date 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 date listed.)
Filing date
Publication date
Application filed by BIOTEZ BERLIN-BUCH GmbH, Bio Tez Berlin Buch GmbH filed Critical BIOTEZ BERLIN-BUCH GmbH
Publication of WO2000066770A2 publication Critical patent/WO2000066770A2/fr
Publication of WO2000066770A3 publication Critical patent/WO2000066770A3/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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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
    • G01N33/54393—Improving reaction conditions or stability, e.g. by coating or irradiation of surface, by reduction of non-specific binding, by promotion of specific binding
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6834—Enzymatic or biochemical coupling of nucleic acids to a solid phase
    • C12Q1/6837—Enzymatic or biochemical coupling of nucleic acids to a solid phase using probe arrays or probe chips

Definitions

  • the invention relates to a method for producing a chemically and thermally stable support coated with crosslinked streptavidin and / or avidin, the layer (V 2) consisting of crosslinked streptavidin and / or avidin via a crosslinked and biotinylated Velcro substance (VI) for which preferably immunoglobulin gamma (IgG) or bovine gamma globulin is used, in strongly basic buffers with pH values> 11.0 or in a strongly acidic medium with pH values ⁇ 3.0, is bound to the surface of the solid support material.
  • the invention further relates to the coated carriers themselves and their use in solid-phase hybridization techniques and solid-phase PCR.
  • the invention was therefore based on the object of developing supports with coatings which permit use even at higher temperatures (_> 60 ° C. to 100 ° C.) and which also have both thermostability and chemostability at these temperatures.
  • thermostable solid phase matrix if a biotinylated crosslinking product V 1 with a molecular weight> 500 KD is bound to the surface of the carrier material as a Velcro substance underlayer either in a basic buffer at pH> 11.0 or in a strongly acidic medium at pH ⁇ 3.0 , and then reacted with crosslinked streptavidin (also recombinant streptavidin), crosslinked avidin or streptavidin-avidin crosslinking products.
  • the invention accordingly relates to a process for producing a stable solid phase matrix containing streptavidin and / or avidin, in which the crosslinking products of streptavidin, avidin or mixtures thereof are bound to the surface of the solid support material via the biotinylated Velcro protein V 1.
  • the process is characterized in that the crosslinking product V 1, preferably a protein, in particular IgG or bovine gamma globulin, is biotinylated after it has been crosslinked using reagents known per se and then with a thermostable, preferably uncharged carrier material in a strongly basic medium at a pH value> 11 or in a strongly acidic environment at a pH ⁇ 3.0.
  • Carnonate buffer solutions are preferably used as the strongly basic buffer solutions and citrate buffer solutions are preferably used as the strongly acidic environment.
  • the preferably uncharged carrier surface consists of plastic, preferably of polycarbonate or polypropylene.
  • the carrier is a coated solid phase on a thermostable plastic surface, preferably made of polycarbonate, polypropylene, silicate, oxidic or else metallic and semiconductor surfaces, as well as combinations of the carrier materials mentioned.
  • the invention furthermore relates to the stable solid carriers thus prepared, coated with streptavidin and / or avidin per se, which consist of a thermostable, generally uncharged carrier material, a crosslinked and biotinylated protein, preferably IgG or bovine gamma globulin, as an underlayer and crosslinked streptavidin and / or avidin as the upper class.
  • streptavidin and / or avidin per se consist of a thermostable, generally uncharged carrier material, a crosslinked and biotinylated protein, preferably IgG or bovine gamma globulin, as an underlayer and crosslinked streptavidin and / or avidin as the upper class.
  • the new carrier produced according to the invention provides a coated solid phase on a thermostable plastic surface, preferably made of polycarbonate, polypropylene, silicate, oxidic or else metallic and Semiconductor surfaces and combinations of the carrier materials mentioned and has the following advantageous properties:
  • the solid support described is therefore advantageously suitable for carrying out molecular biological solid-phase reactions and solid-phase hybridization techniques, in particular for hybridization at temperatures> 60 ° C., which was not possible with the previously known streptavidin coatings, and for the first time enables all possible variants of solid-phase reactions in which higher temperatures are required, such as DNA displacement assay, hot start PCR, allele-specific PCR with labeled primers or primer extension assays with labeled dideoxy nucleotides and the like. a ..
  • ⁇ -globulin 120 mg are dissolved in 13 ml of 0.1 M phosphate buffer (pH 7.5). 7.5 mg of disuccinimidyl suberate (DSS, PIERCE), dissolved in 620 ⁇ l of dioxane, are added dropwise to the ⁇ -globulin solution over the course of 25 min with stirring. After a reaction time of 4 hours with stirring at room temperature, 6.0 mg of sulfo-NHS-LC-biotin (F. PIERCE) are introduced into the reaction solution in solid form, the biotinylation reagent dissolving immediately. A previous separation by gel filtration can be carried out, but is not necessary, and the reaction is continued immediately. This second synthesis step is also carried out with stirring at room temperature. The reaction time is initially 2.5 hours. The reaction mixture is stored in the refrigerator overnight.
  • the mixture is worked up by dialysis, first four times against water, then against 0.05 M PBS (pH 7.4) / 0.05% sodium azide.
  • the largely clear retentate is expediently pressed through a 0.6 ⁇ m Sartorius filter before the spectrophotometric measurement.
  • microtiter plate made of thermostable polycarbonate
  • 50 - 250 ⁇ l of highly cross-linked biotinylated ⁇ -globulin (cattle) according to Example 1 in a concentration of 10-20 ⁇ g / ml in 0.1 M carbonate buffer are added to each well (pH> 11) pipetted and let stand at 25 ° C for 12 hours.
  • the wells are washed with distilled water with the addition of 0.9% NaCl for 12 hours at 25 ° C. with a solution of the highly crosslinked streptavidin (V 2) in 0.05 / 0.15 M PBS buffer (pH 7.2) treated.
  • V 2 highly crosslinked streptavidin
  • the layer is washed with Denhardt's solution in 0.1 M Mc Ilvaine. Buffer (pH 6.0) preserved.
  • the coated MTP (with desiccant) can be sealed in plastic bags and refrigerated be stored.
  • the example describes the determination of the biotin binding capacity of thermally stable coated MTP after different temperature loads.
  • a competitive enzyme immunoassay using biotin (biotinylated compounds) and biotinylated horseradish peroxidase (biotin-POD) is used as the determination method for the biotin binding capacity of the streptavidin coating.
  • the binding yield is determined by measuring the difference in the liquid free phase.
  • the biotin binding capacity of the carrier layer can be calculated from the difference between the total value (enzyme activity of the biotin-POD used) and the unbound enzyme activity in the protruding free phase of the assay.
  • the wells are filled with a PCR buffer mixture (0.01 M Tris / HCl, 0.15 M NaCl, 0.002 M MgCl 2 ; pH 7.9) and exposed to fixed temperatures for a fixed period:
  • the chemothermal stability is determined by loading at 25 ° C with 0.15 M NaOH, at 65 ° C with 0.1 M NH 4 OH and at 85 ° C with PCR buffer.
  • the chemothermal load on the streptavidin layers is ended by washing the wells with distilled water with the addition of 0.9% NaCl and 0.1% Tween 20 at room temperature and the binding capacity still available for biotin or biotinylated compounds with the aid of a competitive biotin EIA certainly.
  • reaction mixtures come from various concentrated solutions of d ( + ) biotin or biotinylated compound (biot oligonucleotide, biot IgG) and a solution of a constant concentration of biotin POD in 0.005 M PBS buffer (pH 7 , 4 / 0.1% BSA). These reaction mixtures (50 - 200 ⁇ l) should react with the coated wells of the chemothermally loaded MTP for at least 2 hours at 25 ° C.
  • the biotin-POD content is determined photometrically in the liquid phase using an enzyme immunoassay standard curve of the extinctions of the substrate reaction with o-phenylenediamine (OPD).
  • enzyme immunoassay standard curves can be drawn up, in which the resulting B / T values are shown as a function of the biotin concentrations used, as shown in the figure below.
  • the biotin binding capacity of the surface layer can be determined with the aid of a curve fit using the Hill model. The differences in the biotin binding capacity between the surfaces coated with thermostable streptavidin according to the invention and the surfaces coated with streptavidin / avidin according to the standard are shown in the following tables:

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  • Health & Medical Sciences (AREA)
  • Immunology (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Chemical & Material Sciences (AREA)
  • Hematology (AREA)
  • Urology & Nephrology (AREA)
  • Biomedical Technology (AREA)
  • Molecular Biology (AREA)
  • Microbiology (AREA)
  • Analytical Chemistry (AREA)
  • Biotechnology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Food Science & Technology (AREA)
  • Medicinal Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Cell Biology (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)

Abstract

L'invention concerne un procédé de réalisation d'une matrice en phase solide chimiostable et thermostable, au moyen d'un matériau support thermostable inerte, sur lequel est appliqué un produit de réticulation V 1 biotinylé présentant un poids moléculaire ≥ 500 KD, de préférence de l'immunoglobuline gamma ou de la gammaglobuline de boeuf, comme substance agrippante, dans des tampons fortement basiques présentant un pH ≥ 11,0, ou bien dans un milieu fortement acide présentant un pH ≤ 3,0. Après adsorption sur le support, de la streptavidine et/ou de l'avidine réticulées, en tant que produit de réticulation V2 de poids moléculaire élevé, sont appliquées sur le pont biotinyque. La matrice ainsi réalisée est chimiostable et thermostable et peut donc être utilisée de façon combinée pour une amplification par PCR et des essais d'hybridation.
PCT/DE2000/001309 1999-05-03 2000-04-28 Procede de realisation d'une matrice en phase solide chimiostable et thermostable pour l'amplification par reaction en chaine de la polymerase (pcr) et les essais d'hybridation d'adn Ceased WO2000066770A2 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
DE19921099 1999-05-03
DE19921099.3 1999-05-03

Publications (2)

Publication Number Publication Date
WO2000066770A2 true WO2000066770A2 (fr) 2000-11-09
WO2000066770A3 WO2000066770A3 (fr) 2001-08-02

Family

ID=7907316

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/DE2000/001309 Ceased WO2000066770A2 (fr) 1999-05-03 2000-04-28 Procede de realisation d'une matrice en phase solide chimiostable et thermostable pour l'amplification par reaction en chaine de la polymerase (pcr) et les essais d'hybridation d'adn

Country Status (2)

Country Link
DE (1) DE10020885A1 (fr)
WO (1) WO2000066770A2 (fr)

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE10237691B4 (de) * 2002-08-15 2010-01-28 Biotez Berlin-Buch Gmbh Biochemisch-Technologisches Zentrum Verfahren zum Nachweis von Einzelnukleotid-Polymorphismen (SNP) in Genen des Arzneimittelmetabolismus und Testkit zur Durchführung des Verfahrens
EP1561823A1 (fr) * 2004-02-04 2005-08-10 Biotez Berlin-Buch GmbH Procédé pour la détection des polymorphismes de nucléotide simple (SNP) des gênes du métabolisme des médicaments et dispositif pour l'utilisation correspondante

Family Cites Families (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE3806431A1 (de) * 1988-02-29 1989-09-07 Boehringer Mannheim Gmbh Verfahren zur herstellung einer festphasenmatrix
ATE131617T1 (de) * 1988-10-17 1995-12-15 Molecular Devices Corp Haptenderivatisierte aufnahmemembran und diagnostische tests, die eine solche membran verwenden
FR2724461B1 (fr) * 1994-09-09 1996-12-20 Prolabo Sa Microsphere de latex biotinylee, procede de preparation d'une telle microsphere et utilisation en tant qu'agent de detection biologique
DE19724787A1 (de) * 1997-06-06 1998-12-10 Biotez Berlin Buch Gmbh Bioche Streptavidin/Avidin beschichtete Oberflächen
US20020022217A1 (en) * 1997-06-24 2002-02-21 Chandran R. Sabanayagam High density streptavidin supports

Also Published As

Publication number Publication date
WO2000066770A3 (fr) 2001-08-02
DE10020885A1 (de) 2001-01-18

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