HRP20100285T1 - Visoko protočno ispitivanje aktivnosti lp-pla2 - Google Patents
Visoko protočno ispitivanje aktivnosti lp-pla2 Download PDFInfo
- Publication number
- HRP20100285T1 HRP20100285T1 HR20100285T HRP20100285T HRP20100285T1 HR P20100285 T1 HRP20100285 T1 HR P20100285T1 HR 20100285 T HR20100285 T HR 20100285T HR P20100285 T HRP20100285 T HR P20100285T HR P20100285 T1 HRP20100285 T1 HR P20100285T1
- Authority
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- Prior art keywords
- paf
- solution
- complexing molecule
- contacting
- pla2
- Prior art date
Links
- 230000000694 effects Effects 0.000 title claims abstract 6
- 238000012203 high throughput assay Methods 0.000 title 1
- 238000000034 method Methods 0.000 claims abstract 19
- 102000016752 1-Alkyl-2-acetylglycerophosphocholine Esterase Human genes 0.000 claims abstract 6
- 108010024976 Asparaginase Proteins 0.000 claims abstract 6
- 241001465754 Metazoa Species 0.000 claims abstract 2
- 239000000243 solution Substances 0.000 claims 12
- 238000006243 chemical reaction Methods 0.000 claims 6
- HVAUUPRFYPCOCA-AREMUKBSSA-N 2-O-acetyl-1-O-hexadecyl-sn-glycero-3-phosphocholine Chemical compound CCCCCCCCCCCCCCCCOC[C@@H](OC(C)=O)COP([O-])(=O)OCC[N+](C)(C)C HVAUUPRFYPCOCA-AREMUKBSSA-N 0.000 claims 5
- 108091003079 Bovine Serum Albumin Proteins 0.000 claims 5
- 108010003541 Platelet Activating Factor Proteins 0.000 claims 5
- 229940098773 bovine serum albumin Drugs 0.000 claims 5
- 230000000536 complexating effect Effects 0.000 claims 5
- 238000002360 preparation method Methods 0.000 claims 5
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims 4
- 239000008366 buffered solution Substances 0.000 claims 3
- YNJBWRMUSHSURL-UHFFFAOYSA-N trichloroacetic acid Chemical compound OC(=O)C(Cl)(Cl)Cl YNJBWRMUSHSURL-UHFFFAOYSA-N 0.000 claims 3
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 claims 2
- 238000011166 aliquoting Methods 0.000 claims 2
- 238000001556 precipitation Methods 0.000 claims 2
- 239000011780 sodium chloride Substances 0.000 claims 2
- 239000006228 supernatant Substances 0.000 claims 2
- 210000001519 tissue Anatomy 0.000 claims 2
- JKMHFZQWWAIEOD-UHFFFAOYSA-N 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid Chemical compound OCC[NH+]1CCN(CCS([O-])(=O)=O)CC1 JKMHFZQWWAIEOD-UHFFFAOYSA-N 0.000 claims 1
- 239000007995 HEPES buffer Substances 0.000 claims 1
- 108091006905 Human Serum Albumin Proteins 0.000 claims 1
- 102000008100 Human Serum Albumin Human genes 0.000 claims 1
- 102000004895 Lipoproteins Human genes 0.000 claims 1
- 108090001030 Lipoproteins Proteins 0.000 claims 1
- 102000015439 Phospholipases Human genes 0.000 claims 1
- 108010064785 Phospholipases Proteins 0.000 claims 1
- 239000013592 cell lysate Substances 0.000 claims 1
- 238000005119 centrifugation Methods 0.000 claims 1
- 239000006166 lysate Substances 0.000 claims 1
- 239000003358 phospholipase A2 inhibitor Substances 0.000 claims 1
- 210000002381 plasma Anatomy 0.000 claims 1
- 239000002244 precipitate Substances 0.000 claims 1
- 238000003345 scintillation counting Methods 0.000 claims 1
- 210000002966 serum Anatomy 0.000 claims 1
- 210000002700 urine Anatomy 0.000 claims 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
- G01N33/573—Immunoassay; Biospecific binding assay; Materials therefor for enzymes or isoenzymes
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
- C12N9/18—Carboxylic ester hydrolases (3.1.1)
- C12N9/20—Triglyceride splitting, e.g. by means of lipase
-
- 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
- C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
- C12Q1/44—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase involving esterase
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/90—Enzymes; Proenzymes
- G01N2333/914—Hydrolases (3)
- G01N2333/916—Hydrolases (3) acting on ester bonds (3.1), e.g. phosphatases (3.1.3), phospholipases C or phospholipases D (3.1.4)
- G01N2333/918—Carboxylic ester hydrolases (3.1.1)
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- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Organic Chemistry (AREA)
- Molecular Biology (AREA)
- Immunology (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Microbiology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biomedical Technology (AREA)
- Genetics & Genomics (AREA)
- Biotechnology (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Analytical Chemistry (AREA)
- Physics & Mathematics (AREA)
- General Engineering & Computer Science (AREA)
- Medicinal Chemistry (AREA)
- Hematology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Urology & Nephrology (AREA)
- Cell Biology (AREA)
- Biophysics (AREA)
- Pathology (AREA)
- General Physics & Mathematics (AREA)
- Food Science & Technology (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Pyrane Compounds (AREA)
- Investigating Or Analysing Biological Materials (AREA)
- Color Electrophotography (AREA)
- Nitrogen And Oxygen Or Sulfur-Condensed Heterocyclic Ring Systems (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Heterocyclic Carbon Compounds Containing A Hetero Ring Having Oxygen Or Sulfur (AREA)
Abstract
Postupak visoke protočnosti za određivanje aktivnosti enzima fosfolipaze povezane s lipoproteinom A2 (Lp-PLA2) u većem broju uzoraka, naznačen time što sadržava sljedeće korake: (i) pripravljanje otopine koja sadržava označeni faktor za aktiviranje trombocita (PAF); (ii) uzimanje alikvota svakog od množine uzoraka u jažice mikrotitracijske ploče ili mikrocentrifugalne epruvete; (iii) dovođenje u kontakt svakog od većeg broja tkivnih uzoraka s otopinom iz koraka priprave; (iv) dovođenje u kontakt svake od otopina, iz koraka prvog dovođenja u kontakt, s molekulom koja stvara kompleks s PAF, da se formira kompleks PAF-kompleksirajuća molekula; (v) dovođenje u kontakt spomenutog kompleksa PAF-kompleksirajuća molekula s otopinom za taloženje koja sadržava trikloroctenu kiselinu (TCA), da nastane talog i supernatant; (vi) uklanjanje spomenutog kompleksa PAF-kompleksirajuća molekula centrifugiranjem spomenute mikrotitracijske ploče ili mikrocentrifugalne epruvete pri najmanje 6.000g najmanje 5 minuta pri manje od 10°C; i (vii) određivanje aktivnosti Lp-PLA2, pritom najmanje jedan uzorak je izuzet iz životinje kojoj je bio primijenjen inhibitor Lp-PLA2. Patent sadrži još 17 patentnih zahtjeva.
Claims (18)
1. Postupak visoke protočnosti za određivanje aktivnosti enzima fosfolipaze povezane s lipoproteinom A2 (Lp-PLA2) u većem broju uzoraka, naznačen time što sadržava sljedeće korake:
(i) pripravljanje otopine koja sadržava označeni faktor za aktiviranje trombocita (PAF);
(ii) uzimanje alikvota svakog od množine uzoraka u jažice mikrotitracijske ploče ili mikrocentrifugalne epruvete;
(iii) dovođenje u kontakt svakog od većeg broja tkivnih uzoraka s otopinom iz koraka priprave;
(iv) dovođenje u kontakt svake od otopina, iz koraka prvog dovođenja u kontakt, s molekulom koja stvara kompleks s PAF, da se formira kompleks PAF-kompleksirajuća molekula;
(v) dovođenje u kontakt spomenutog kompleksa PAF-kompleksirajuća molekula s otopinom za taloženje koja sadržava trikloroctenu kiselinu (TCA), da nastane talog i supernatant;
(vi) uklanjanje spomenutog kompleksa PAF-kompleksirajuća molekula centrifugiranjem spomenute mikrotitracijske ploče ili mikrocentrifugalne epruvete pri najmanje 6.000g najmanje 5 minuta pri manje od 10°C; i
(vii) određivanje aktivnosti Lp-PLA2,
pritom najmanje jedan uzorak je izuzet iz životinje kojoj je bio primijenjen inhibitor Lp-PLA2.
2. Postupak prema zahtjevu 1, naznačen time što najmanje jedan uzorak je odabran iz skupine koju sačinjavaju serum, stanični lizat, tkivni lizat, urin, ili krvna plazma.
3. Postupak prema bilo kojem prethodnom zahtjevu, naznačen time što označeni PAF je radio-označen.
4. Postupak prema bilo kojem prethodnom zahtjevu, naznačen time što ukupna koncentracija PAF u otopini u koraku priprave je najmanje 20 µM.
5. Postupak prema bilo kojem prethodnom zahtjevu, naznačen time što otopina koja sadržava označeni i ne-označeni PAF je puferirana otopina.
6. Postupak prema zahtjevu 5, naznačen time što puferirana otopina sadržava HEPES, natrij klorid (NaCl), i etilendiamintetraoctenu kiselinu (EDTA).
7. Postupak prema bilo kojem prethodnom zahtjevu, naznačen time što otopina iz koraka priprave i uzorak se miješaju najmanje 5 sekundi i inkubiraju pri 21 °C najmanje 1 minutu.
8. Postupak prema bilo kojem prethodnom zahtjevu, naznačen time što kompleksirajuća molekula iz koraka dovođenja u kontakt je egzogeni goveđi serumski albumin (BSA).
9. Postupak prema zahtjevu 8, naznačen time što BSA ima temperaturu manju od 10 °C.
10. Postupak prema zahtjevu 8, naznačen time što BSA ima temperaturu od 4 °C.
11. Postupak prema zahtjevu 8 ili 9, naznačen time što BSA ima koncentraciju od 50 mg/mL.
12. Postupak prema bilo kojem prethodnom zahtjevu, naznačen time što kompleksirajuća molekula iz koraka dovođenja u kontakt je endogeni ljudski serumski albumin.
13. Postupak prema bilo kojem prethodnom zahtjevu, naznačen time što otopina za taloženje koja sadržava TCA ima temperaturu manju od 10 °C.
14. Postupak prema bilo kojem prethodnom zahtjevu, naznačen time što se centrifugiranje provodi pri 4 °C 15 minuta.
15. Postupak prema bilo kojem prethodnom zahtjevu, naznačen time što se aktivnost Lp-PLA2 mjeri brojanjem scintilacije.
16. Postupak prema zahtjevu 1, naznačen time što dalje sadržava alikvotiranje puferirane otopine u najmanje dva spremnika za upotrebu kao reakcije ukupnog broja ili slijepe reakcije.
17. Postupak prema zahtjevu 16, naznačen time što spremnici su mikrocentrifugalne epruvete ili jažice mikrotitracijske ploče.
18. Postupak prema zahtjevu 17, naznačen time što dalje sadržava dovođenje u kontakt najmanje jedne slijepe reakcije s otprilike jednakim volumenom i koncentracijom otopine iz koraka priprave i kompleksirajuće molekule i dovođenje u kontakt najmanje dva alikvota reakcije ukupnog broja s otprilike jednakim volumenom otopine iz koraka priprave i zamjenske otopine za kompleksirajuću molekulu, pritom zamjenska otopina ne sadržava kompleksirajuću molekulu; alikvotiranje dijela supernatanta svakog uzorka, slijepe reakcije, i reakcije ukupnog broja u odvojene spremnike; i dovođenje u kontakt scintilacijskog koktela sa svakim alikvotom.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US47377703P | 2003-05-28 | 2003-05-28 | |
| PCT/US2004/016716 WO2005001416A2 (en) | 2003-05-28 | 2004-05-27 | High throughput assay of lp-pla2 activity |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| HRP20100285T1 true HRP20100285T1 (hr) | 2010-06-30 |
Family
ID=33551459
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| HR20100285T HRP20100285T1 (hr) | 2003-05-28 | 2010-05-21 | Visoko protočno ispitivanje aktivnosti lp-pla2 |
Country Status (13)
| Country | Link |
|---|---|
| US (1) | US7531316B2 (hr) |
| EP (2) | EP1633864B1 (hr) |
| JP (2) | JP4892350B2 (hr) |
| AT (1) | ATE463562T1 (hr) |
| CY (1) | CY1110138T1 (hr) |
| DE (1) | DE602004026439D1 (hr) |
| DK (1) | DK1633864T3 (hr) |
| ES (1) | ES2341772T3 (hr) |
| HR (1) | HRP20100285T1 (hr) |
| PL (1) | PL1633864T3 (hr) |
| PT (1) | PT1633864E (hr) |
| SI (1) | SI1633864T1 (hr) |
| WO (1) | WO2005001416A2 (hr) |
Families Citing this family (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US7531316B2 (en) | 2003-05-28 | 2009-05-12 | Glaxo Group Limited | High throughput assay of Lp-PLA2 activity |
| EP2280282A1 (en) | 2004-02-03 | 2011-02-02 | Diadexus, Inc. | Methods of detecting Lp-PLA2 activity |
| WO2005113797A2 (en) | 2004-04-16 | 2005-12-01 | Glaxo Group Limited | METHODS FOR DETECTING Lp-PLA2 ACTIVITY AND INHIBITION OF Lp-PLA2 ACTIVITY |
| US20150017671A1 (en) | 2004-04-16 | 2015-01-15 | Yaping Shou | Methods for detecting lp-pla2 activity and inhibition of lp-pla2 activity |
| US20140283157A1 (en) | 2013-03-15 | 2014-09-18 | Diadexus, Inc. | Lipoprotein-associated phospholipase a2 antibody compositions and methods of use |
| WO2016127319A1 (zh) * | 2015-02-10 | 2016-08-18 | 深圳市新产业生物医学工程股份有限公司 | 用于检测脂蛋白相关磷脂酶a2的试剂盒及其制备和应用 |
| CN104820097A (zh) * | 2015-05-22 | 2015-08-05 | 北京协和洛克生物技术有限责任公司 | 一种定量检测样本中脂蛋白磷脂酶a2浓度的液态芯片试剂盒及其制备方法 |
| US10900063B2 (en) | 2016-06-22 | 2021-01-26 | Asahi Kasei Pharma Corporation | Measurement of Lp-PLA2 activity |
Family Cites Families (15)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP3204681B2 (ja) | 1991-05-24 | 2001-09-04 | 株式会社シノテスト | 新規なエーテル型チオリン脂質化合物およびその製造法 |
| EP0658205B1 (en) | 1993-06-25 | 2000-03-15 | Smithkline Beecham Plc | Lipoprotein associated phospholipase a2, inhibitors thereof and use of the same in diagnosis and therapy |
| JPH0759597A (ja) | 1993-08-23 | 1995-03-07 | Shinotesuto:Kk | 血小板活性化因子アセチルヒドロラーゼ活性測定方法 |
| US6146625A (en) | 1993-10-06 | 2000-11-14 | Icos Corporation | Platelet-activating factor acetylhydrolase |
| EP1634953A3 (en) | 1993-10-06 | 2006-06-28 | ICOS Corporation | Platelet-activating factor acethylhydrolase |
| JPH1017600A (ja) | 1996-06-28 | 1998-01-20 | Suntory Ltd | 血小板活性化因子アセチルヒドロラーゼおよびその遺伝子 |
| JP4220603B2 (ja) | 1998-12-02 | 2009-02-04 | アルフレッサファーマ株式会社 | 血小板活性化因子アセチルヒドロラーゼ活性の測定方法 |
| US20020102231A1 (en) | 1999-05-07 | 2002-08-01 | Icos Corporation | Therapeutic uses of PAF-AH products in diabetes |
| FR2815857B1 (fr) | 2000-10-30 | 2003-02-14 | Oreal | Composition, notamment cosmetique, renfermant un retinoide et une silicone benzotriazole |
| ATE269342T1 (de) | 2001-12-07 | 2004-07-15 | Sirs Lab Gmbh | Verbindungen zur bestimmung der aktivität von phospholipase a2 |
| US20030134082A1 (en) * | 2001-12-21 | 2003-07-17 | Morin Brian G. | Carpet comprising a low-shrink backing of polypropylene tape fibers |
| US20070077614A1 (en) | 2003-04-01 | 2007-04-05 | Wolfert Robert L | Uses of lp-pla2 in combination to assess coronary risk |
| US7531316B2 (en) | 2003-05-28 | 2009-05-12 | Glaxo Group Limited | High throughput assay of Lp-PLA2 activity |
| EP2280282A1 (en) | 2004-02-03 | 2011-02-02 | Diadexus, Inc. | Methods of detecting Lp-PLA2 activity |
| WO2005113797A2 (en) * | 2004-04-16 | 2005-12-01 | Glaxo Group Limited | METHODS FOR DETECTING Lp-PLA2 ACTIVITY AND INHIBITION OF Lp-PLA2 ACTIVITY |
-
2004
- 2004-05-27 US US10/557,540 patent/US7531316B2/en not_active Expired - Fee Related
- 2004-05-27 JP JP2006533451A patent/JP4892350B2/ja not_active Expired - Fee Related
- 2004-05-27 PT PT04753533T patent/PT1633864E/pt unknown
- 2004-05-27 DK DK04753533.1T patent/DK1633864T3/da active
- 2004-05-27 EP EP04753533A patent/EP1633864B1/en not_active Expired - Lifetime
- 2004-05-27 DE DE602004026439T patent/DE602004026439D1/de not_active Expired - Lifetime
- 2004-05-27 ES ES04753533T patent/ES2341772T3/es not_active Expired - Lifetime
- 2004-05-27 EP EP10158845A patent/EP2253702A1/en not_active Withdrawn
- 2004-05-27 AT AT04753533T patent/ATE463562T1/de active
- 2004-05-27 SI SI200431433T patent/SI1633864T1/sl unknown
- 2004-05-27 PL PL04753533T patent/PL1633864T3/pl unknown
- 2004-05-27 WO PCT/US2004/016716 patent/WO2005001416A2/en active Application Filing
-
2010
- 2010-05-21 HR HR20100285T patent/HRP20100285T1/hr unknown
- 2010-06-21 CY CY20101100572T patent/CY1110138T1/el unknown
-
2011
- 2011-09-22 JP JP2011207479A patent/JP5637959B2/ja not_active Expired - Fee Related
Also Published As
| Publication number | Publication date |
|---|---|
| PL1633864T3 (pl) | 2010-09-30 |
| US7531316B2 (en) | 2009-05-12 |
| EP1633864A2 (en) | 2006-03-15 |
| EP1633864A4 (en) | 2006-06-14 |
| CY1110138T1 (el) | 2015-01-14 |
| US20070065892A1 (en) | 2007-03-22 |
| EP1633864B1 (en) | 2010-04-07 |
| DE602004026439D1 (de) | 2010-05-20 |
| EP2253702A1 (en) | 2010-11-24 |
| PT1633864E (pt) | 2010-06-24 |
| WO2005001416A3 (en) | 2005-03-17 |
| ATE463562T1 (de) | 2010-04-15 |
| JP2007502131A (ja) | 2007-02-08 |
| ES2341772T3 (es) | 2010-06-28 |
| JP5637959B2 (ja) | 2014-12-10 |
| JP4892350B2 (ja) | 2012-03-07 |
| DK1633864T3 (da) | 2010-07-12 |
| SI1633864T1 (sl) | 2010-07-30 |
| WO2005001416A2 (en) | 2005-01-06 |
| JP2012050443A (ja) | 2012-03-15 |
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| Glick | Trends in quantification in histochemistry and cytochemistry | |
| Brown | Validation of a Deployable Proteomic Assay for the Serological Screening of Sexual Assault Samples |