EP0679257A1 - Sonde marquee par une lipase - Google Patents

Sonde marquee par une lipase

Info

Publication number
EP0679257A1
EP0679257A1 EP94929543A EP94929543A EP0679257A1 EP 0679257 A1 EP0679257 A1 EP 0679257A1 EP 94929543 A EP94929543 A EP 94929543A EP 94929543 A EP94929543 A EP 94929543A EP 0679257 A1 EP0679257 A1 EP 0679257A1
Authority
EP
European Patent Office
Prior art keywords
lipase
enzyme
activity
solution
alkaline phosphatase
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Withdrawn
Application number
EP94929543A
Other languages
German (de)
English (en)
Inventor
Fritz Pittner
Thomas Schalkhammer
Bernhard Ecker
Eva Kynclova
Werner Wakolbinger
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.)
Roche Diagnostics GmbH
Original Assignee
Roche Diagnostics GmbH
Boehringer Mannheim 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 Roche Diagnostics GmbH, Boehringer Mannheim GmbH filed Critical Roche Diagnostics GmbH
Publication of EP0679257A1 publication Critical patent/EP0679257A1/fr
Withdrawn legal-status Critical Current

Links

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
    • G01N33/531—Production of immunochemical test materials
    • G01N33/532—Production of labelled immunochemicals
    • G01N33/535—Production of labelled immunochemicals with enzyme label or co-enzymes, co-factors, enzyme inhibitors or enzyme substrates
    • 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
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • G01N33/581—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with enzyme label (including co-enzymes, co-factors, enzyme inhibitors or substrates)

Definitions

  • the invention relates to a sample or test substance labeled with lipase or the use of lipase for the determination of biological material.
  • the invention is therefore based on the object of creating a sample or test substance labeled with enzyme, which is distinguished by significantly improved stability and a substantially broadened range of applications and in this way makes substances accessible for analytical determination which have previously been labeled with enzyme or Test substances were in no way accessible.
  • the solution to this problem consists essentially in that a plant lipase, its isoenzymes or structural analogs with an amino acid homology of at least 70% and lipase activity are used as the enzyme.
  • Candida rugosa which has proven particularly suitable as a source and is also described in the literature as Candida Cylindracea, contains a lipase, the structure of which is largely determined. The cloning and analysis of the lipase sequence is described in particular in Gene 124: 1 (February 14, 1993, pages 45-55). The enzyme is given here as a 57-kDa protein with 534 amino acids, 5 isoenzymes with an amino acid homology of 80% having already been structurally elucidated.
  • a lipase from plants especially fungi.
  • a lipase from Candida rugosa DSM 2031 is particularly preferred.
  • the use of this lipase results in a significant improvement in the product properties compared to all previously known enzymes, but also compared to other known lipases, in particular animal lipases.
  • the thermostability achieved in this way creates the prerequisite for being able to carry out such tests in a reasonable time.
  • Another advantage of the lipase proposed according to the invention is that it does not in any way require metal ions for its activity.
  • metal ions In the case of alkaline phosphatase, magnesium and zinc ions are prerequisites for the enzymatic activity, and precisely these metal ions would activate nucleases, for example, which subsequently prevent the use of alkaline phosphatase for gene probes.
  • complex substances such as EDTA are often used. EDTA not only complexes metal ions and thus no longer allows the use of alkaline phosphatase, rather large amounts of EDTA also tend to denature proteins.
  • the lipase proposed according to the invention has not only distinguished itself by significantly higher thermal stability than known enzymes, but is also notable for complete stability against EDTA. Another clear superiority of the lipases which can be used according to the invention over known enzymes is that urea or corresponding derivatives in large quantities do not in any way impair the function of the lipase. When using lipase, EDTA can be used to protect SH groups, which was not possible with previous enzyme-labeled samples or test substances.
  • the lipases which can be used according to the invention are also distinguished by a significantly improved compatibility with organic solvents.
  • the lipases proposed according to the invention can also be used in toluene, as a result of which the analytical range of use of the samples or test substances according to the invention is significantly increased.
  • the high stability permits the analytical usability of the samples and test substances according to the invention in a pH range between 2.5 and 9, the insensitivity to ureas allowing the detection of highly hydrophobic substances.
  • lipase conjugates can be dried and, if necessary, lyophilized at room temperature or even air-dried for years without loss of activity.
  • turnover rate of the same order of magnitude with a simultaneous improved sensitivity, which is due to the improved detectability of the lipase.
  • lipase can still be determined precisely in a range up to about 5 pg, up to a range of 10 ng in addition to purely qualitative analysis a largely simple estimate of the amount is possible.
  • the lipase can be determined in a customary manner, for which purpose esters of phenols or their derivatives can be used as substrates.
  • the color reaction achieved in this way allows an immediate and simple evaluation. Appropriate methods are known to the person skilled in the art.
  • the sample or test substance is characterized in that the lipase with biotin, avidin,chthin, protein A, protein G, antibody-binding proteins, antibodies, antigens, viral protein antigens, bacterial surface proteins, digoxygenin, DNA, RNA, oligonucleotides or synthetic analogues of metal colloids or plastic microparticles ( ⁇ 5 ⁇ m) is conjugated.
  • conjugates have reactive groups and, in particular, biorecognitive groups for a wide range of applications, which thus open up an extremely large area of use for such labeled samples or test substances.
  • the invention relates to the use of lipase from plants, in particular from Candida rugosa DSM 2031, their isoenzymes or structural analogs with an amino acid homology of at least 70% and lipase activity for the preparation of analytical samples or test substances, the lipase conjugating to biorecognitive groups is used and such test substances are particularly suitable for bioassays, test strips, biosensors or as gene probes.
  • Classic oligonucleotide samples can be used as gene probes, which allow selective hybridization due to the favorable thermostability of the lipase using stringent conditions.
  • Fig. 1 shows the thermostability of the lipase of the Candida species. It is evident that the activity is almost constant over time at a pH of 5 at temperatures of 50 ° C. and that it still retains over 75% of the original activity even after more than an hour at a pH of 7 . Even at temperatures of 60 ° C and a pH of 5, values in the order of 80% of the original activity are retained after one hour.
  • Fig. 2 shows the detection limits for color reactions of lipase and alkaline phosphatase. It can be seen that with alkaline phosphatase the detection limit is largely reached at around 250 pg, whereas in the original a point could be detected as a color reaction at 5 pg in the original. With lipase, the diameter of the dots remains largely correlated with the actual amount up to a range of 10 ng, whereas a simple quantitative estimate for alkaline phosphatase from around 500 pg is no longer easily possible.
  • Fig. 3 the use of lipase for an oligonucleotide gene probe is illustrated schematically in the manner of a flow chart.
  • a structural gene 2 is immobilized on a microtiter plate 1 via a biotin-avidin coupling.
  • An oligonucleotide probe 4 is connected to lipase 3, whereby in the case of an analyte 5 the lipase is fixed on the microtiter plate if this analyte contains both the gene which is fixed on the microtiter plate 1 and the oligonucleotide 4 recombined.
  • the hybridization reaction results in immobilization of lipase 3 on the Mirotiter plate 1.
  • the qualitative detection is subsequently achieved by conventional methods, for example by a color reaction of the lipase with a corresponding one Substrate as reagent.
  • Fig. 4 shows a comparison of the optimal pH values of lipase, peroxidase and alkaline phosphatase.
  • Fig. 5 shows a comparison of dye precipitation assays for lipase and alkaline phosphatase.
  • Horseradish peroxidase was detected spectrophotometrically at 23 ° C by monitoring the rate of oxidation of ABTS (1.49 mM) in the presence of hydrogen peroxide (0.045%) in 0.1 M citrate / phosphate buffer, pH 4.5, at a wavelength of 420 ⁇ m.
  • 50 ⁇ l of an enzyme solution was mixed with 500 ⁇ l of the substrate solution mentioned above and the kinetics of the enzymatic reaction at a wavelength of 420 nm were monitored over a period of one minute.
  • the concentration of the oxidized substrate was calculated using the extinction coefficient for ABTS.
  • the concentration of the enzyme solution was adjusted so that a linear course of the extinction curve was ensured under working conditions.
  • Alkaline phosphatase was detected spectrophotometrically at 23 ° C by following the hydrolysis of 0.1 mM p-nitrophenyl phosphate in 0.1 M TRIS, 0.1 M NaCl and 50 mM MgCl2, pH 8.5, at a wavelength of 405 nm [Lazdunski, C. and Lazdunski, M. (1966) BBA 113, 551-566].
  • Lipase was detected spectrophotometrically at 23 ° C by monitoring the hydrolysis of 0.1 mM p-nitrophenyl butyrate in 0.1 M phosphate buffer, pH 7.0, at a wavelength of 405 nm.
  • Lipase (with a protein concentration of 0.09 mg / ml; determined using the Bradford method) was incubated for 1 to 60 min with a 20 to 44% (v / v) effector solution (e.g. tetrahydrofuran, 2-methylpentanediol , 2-isopropanol ”) in 0.1 M acetate buffer, 0.1% BSA, pH 5.0 at room temperature.
  • the enzyme solution was then diluted to a measurable concentration with 0.1 M acetate buffer, pH 5.0, 0.1% of BSA, or 0.2% (v / v) of Tween-20 in 0.1 M acetate buffer, pH 5.0, 0.1% of BSA.
  • the samples were stored at 4 ° C. The activity was determined using the pNPB assay.
  • HMB hydroquinone monobutyrate
  • the activity was related to the total protein concentration of the crude enzymatic preparation.
  • the lipase activity was determined at different pH values using the hydroquinone assay.
  • the enzyme lipase has a very broad pH optimum (between pH 3 - 8) and is therefore suitable for coupled enzyme assays (e.g. with peroxidase).
  • Horseradish peroxidase was determined with 0.1 M phosphate buffer, pH 7.5, 0.05% in BSA.
  • Alkaline phosphatase was diluted with 50 mM TRIS buffer, pH 7.6, 1 mM in MgCl2 and 0.1 mM in ZnCl2, 0.05% in BSA.
  • Lipase was diluted with 0.1 M acetate buffer, pH 5.0, 0.05% in BSA.
  • the comparison of the thermal stability of the above-mentioned enzymes was carried out at a protein concentration of 0.2 mg / ml by incubation at 50 ° C. for a period of 120 min.
  • Alkaline phosphatase was incubated in 50 mM TRIS, 1 mM MgCl2 and 0.1 mM ZnCl2, pH 7.6.
  • Lipase was diluted with 0.1 M acetate buffer, pH 5.0, 0.5% in BSA.
  • HRP Horseradish peroxidase
  • Alkaline phosphatase was diluted with 50 mM TRIS, 1 mM MgCl2 and 0.1 mM ZnCl2, 0.05% BSA, pH 7.6.
  • HRP Peroxidase
  • Alkaline phosphatase was diluted with 50 mM TRIS (pH 7.6), 1 mM MgCl2 and 0.1 mM ZnCl2, 0.05% BSA.
  • Lipase was diluted with 0.1 M acetate buffer (pH 5.0), 0.05% BSA.
  • thiol groups in lipase was carried out in 0.1 M EDTA, 01 M phosphate buffer, pH 7.5 at a final protein concentration of 3 mg / ml using a ten-fold excess of 2-iminothiolane.
  • heterobifunctional crosslinkers which have at least two different reactive groups allow sequential conjugations and minimize undesired polymerization or self-conjugation. These crosslinkers were tested as follows:
  • the protein concentration of the lipase fraction was determined by means of the Bradford test and was 3 mg / ml. The lipase showed no appreciable loss of enzyme activity.
  • the carboxyl groups of the lipase were modified in the presence of polyethylene glycol, l-ethyl-3- (3-dimethylaminopropyl) carbodiimide (EDC) and N-hydroxysuccinimide (NHS). Description of the method:
  • a stock solution of EDC / NHS was prepared by mixing an aqueous solution of EDC with a solution of NHS in dimethylformamide (DMF) in a molar ratio of 15: 1. This stick solution was used immediately.
  • DMF dimethylformamide
  • Lipase (384 ⁇ g; 6.7 nmol) was mixed with a solution of DiaminoPEG (12.2 mg, 6.4 ⁇ mol).
  • a mixture of EDC / NHS (EDC: 0.15 mg, 780 nmol; NHS: 0.006 mg, 52 nmol) was then added and the mixture was incubated for 16 hours at 4 ° C. in the dark (pH of the reaction mixture was 6 ,8th).
  • the final protein concentration was 5 mg / ml.
  • the reaction was stopped with 500 mM acetate buffer, pH 5 and the excess DiaminoPEG was separated by electrodialysis against 50 mM acetate buffer, pH 5.0.
  • the reaction products were analyzed on a native polyacrylamide gel and detected with staining with indolylacetate / nitro-blue-tetrazolium salt (NBT).
  • NBT indolylacetate / nitro-blue-tetrazolium salt
  • a stock solution of EDC / NHS was prepared by mixing an aqueous solution of EDC with a solution of NHS in dimethylformamide (DMF) in a molar ratio of 15: 1. This stock solution was used immediately.
  • DMF dimethylformamide
  • Lipase (384 ⁇ g; 6.7 nmol) was mixed with a solution of DiaminoPEG (12.2 mg, 6.4 ⁇ mol).
  • a mixture of EDC / NHS (EDC: 0.61 mg, 3.2 ⁇ mol; NHS: 0.024 mg, 0.21 ⁇ mol) was then added and the mixture was incubated for 16 hours at 4 ° C. in the dark (pH the reaction mixture was 6.8).
  • the final protein concentration was 5 mg / ml.
  • the reaction was stopped with 500 mM acetate buffer, pH 5, and the excess of diaminoPEG was electrodialysed against 50 mM acetate buffer, pH 5.0. severed.
  • the reaction products were analyzed on a native polyacrylamide gel and detected with staining with indolylacetate / nitro-blue-tetrazolium salt (NBT).
  • NBT indolylacetate / nitro-blue-tetrazolium salt
  • a stock solution of EDC / NHS was prepared by mixing an aqueous solution of EDC with a solution of NHS in dimethylformamide (DMF) in a molar ratio of 15: 1. This stock solution was used immediately.
  • DMF dimethylformamide
  • Lipase (384 ⁇ g; 6.7 nmol) was mixed with a solution of DiaminoPEG (12.2 mg, 6.4 ⁇ mol).
  • a mixture of EDC / NHS (EDC: 0.61 mg, 3.2 ⁇ mol; NHS: 0.024 mg, 0.21 ⁇ mol) was then added and the mixture was incubated for 16 hours at 4 ° C. in the dark (pH the reaction mixture was 6.8).
  • the final protein concentration was 5 mg / ml.
  • the reaction was stopped with 500 mM acetate buffer, pH 5 and the excess DiaminoPEG was separated by electrodialysis against 50 mM acetate buffer, pH 5.0.
  • the reaction products were analyzed on a native polyacrylamide gel and detected with staining with indolylacetate / nitro-blue-tetrazolium salt (NBT).
  • NBT indolylacetate / nitro-blue-tetrazolium salt
  • Modification of lipase (modified with polyethylene glycol) with SMCC The modification of PEG-lipase was carried out in 20 mM phosphate buffer, 150 mM NaCl, 1 mM EDTA, pH 7.4 at a final protein concentration of 10 mg / ml using a 20-fold molar excess of SMCC (in relation to five amino groups to be modified). After incubation for 30 min at room temperature and in the dark, the excess crosslinker was separated on a Sephadex 10 column, which had previously been equilibrated with 20 mM phosphate buffer + 2 mM EDTA (pH 6.5).
  • the desired fractions of lipase activity were concentrated using Centricon 30 tubes at a room temperature of 4 ° C. and then lyophilized.
  • the modified lipase was stored at -20 ° C and used directly for coupling with oligonucleotides.
  • the oligonucleotides were synthesized using the phosphoramidite method.
  • the terminal sulfhydryl group at the 5 'end was introduced with C6-thiol modifiers during the last synthesis step.
  • the trityl group was split off using silver nitrate and stored in DTT at ⁇ 20 ° C. [working instructions from Clontech].
  • the reaction was carried out first at room temperature for one hour and then at 4 ° C. for 16 hours to complete the reaction.
  • the unbound proteins were removed by centrifugation at 1600 g for 15 minutes.
  • the supernatant was removed with a pipette and the precipitate was dissolved in 1000 ul 10% polyethylene glycol 20000 (PEG 20000) (Polyscience).
  • PEG 20000 polyethylene glycol 20000
  • Each solution step required 50 ⁇ l 10% SDS and the use of ultrasound (Bandelin SONOREX Sper RK510II) over a few minutes. This procedure was repeated three times.
  • the precipitate was dissolved in 50 ul 1% PEG 20000 and 200 ul PBST-3% BSA.
  • the supernatants and the dissolved precipitates were examined for lipase activity and antigen activity. Only the first supernatant showed lipase activity and thus represents unbound proteins. After the remaining centrifugation steps, no enzyme activity could be found in the supernatants. However, as expected, the dissolved precipitates showed lipase activity.
  • Substrate stock solution 50 mg indolylacetate in 1 ml 100% dimethylformamide (DMF); Stock solution of nitro-blue-tetrazolium chloride (NBT); 50 mg NBT dissolved in 1 ml 70% dimethylformamide.
  • the substrate solution was prepared by mixing 66 ul NBT stock solution in 10 ml 0.1 M phosphate buffer, pH 6.5. The mixture was used immediately.
  • Substrate stock solution 50 mg 5-bromo-4-chloro-3-indolyl phosphate (BCIP) dissolved in 1 ml 100% dimethylformamide (DMF); NBT stock solution: 50 mg NBT dissolved in 1 ml 70% dimethylformamide (DMF).
  • the substrate solution was prepared by mixing 33 ul substrate stock solution and 66 ul NBT stock solution in 10 ml 0.1 M substrate buffer (0.1 M TRIS, 0.1 M NaCl, 50 mM MgCl 2 , pH 8.5). The mixture was used immediately.
  • the lipase used in this case was not optimally purified. Therefore, a better detection limit can be achieved with pre-cleaned and pre-treated lipase.

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  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Immunology (AREA)
  • Engineering & Computer Science (AREA)
  • Molecular Biology (AREA)
  • Biomedical Technology (AREA)
  • Chemical & Material Sciences (AREA)
  • Hematology (AREA)
  • Urology & Nephrology (AREA)
  • Cell Biology (AREA)
  • Biochemistry (AREA)
  • Biotechnology (AREA)
  • Food Science & Technology (AREA)
  • Medicinal Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • Microbiology (AREA)
  • General Health & Medical Sciences (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Enzymes And Modification Thereof (AREA)
  • Investigating Or Analysing Biological Materials (AREA)

Abstract

Afin d'améliorer la stabilité thermique et chimique d'une sonde marquée par une enzyme, on utilise comme enzyme de la lipase, de préférence extraite de Candida rugosa, et dont les isoenzymes ou analogues structuraux sont homologues d'acides aminés pour au moins 70 % et ont une activité de lipase.
EP94929543A 1993-10-15 1994-10-13 Sonde marquee par une lipase Withdrawn EP0679257A1 (fr)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
AT2071/93 1993-10-15
AT207193A AT400036B (de) 1993-10-15 1993-10-15 Mit enzym markierte probe oder testsubstanz
PCT/EP1994/003379 WO1995010775A1 (fr) 1993-10-15 1994-10-13 Sonde marquee par une lipase

Publications (1)

Publication Number Publication Date
EP0679257A1 true EP0679257A1 (fr) 1995-11-02

Family

ID=3527156

Family Applications (1)

Application Number Title Priority Date Filing Date
EP94929543A Withdrawn EP0679257A1 (fr) 1993-10-15 1994-10-13 Sonde marquee par une lipase

Country Status (6)

Country Link
EP (1) EP0679257A1 (fr)
JP (1) JPH07509618A (fr)
AT (1) AT400036B (fr)
AU (1) AU671392B2 (fr)
CA (1) CA2151731A1 (fr)
WO (1) WO1995010775A1 (fr)

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20050074828A1 (en) * 2003-07-16 2005-04-07 Dimagno Theodore John Uae of lipase for high-density lipoprotein cholesterol detection

Family Cites Families (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DK601984D0 (da) * 1984-12-14 1984-12-14 Hansens Lab Fremgangsmaade til fremstilling af genprodukter
EP0350808B1 (fr) * 1988-07-11 1995-11-08 Roche Diagnostics GmbH Méthode pour la détection de substances avec activité hydrolase
US5093256A (en) * 1989-02-22 1992-03-03 Shen Gwo Jenn Essentially purified, thermostable and alkalophilic lipase from bacillus sp. a30-1 atcc 53841
US5108916A (en) * 1989-06-05 1992-04-28 Rhone-Poulenc Rorer, S.A. Process for stereoselectively hydrolyzing, transesterifying or esterifying with immobilized isozyme of lipase from candida rugosa
US5248618A (en) * 1991-06-05 1993-09-28 Life Technologies, Inc. Methods for generating light with chemiluminescent dioxetanes activated by anchimeric assisted cleavage

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO9510775A1 *

Also Published As

Publication number Publication date
WO1995010775A1 (fr) 1995-04-20
JPH07509618A (ja) 1995-10-26
AT400036B (de) 1995-09-25
AU671392B2 (en) 1996-08-22
CA2151731A1 (fr) 1995-04-20
AU7855794A (en) 1995-05-04
ATA207193A (de) 1995-01-15

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