DK173677B1 - Fremgangsmåde til ekstraktion af basalmembranproteiner fra væv fra mennesker og dyr - Google Patents
Fremgangsmåde til ekstraktion af basalmembranproteiner fra væv fra mennesker og dyr Download PDFInfo
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- DK173677B1 DK173677B1 DK198801356A DK135688A DK173677B1 DK 173677 B1 DK173677 B1 DK 173677B1 DK 198801356 A DK198801356 A DK 198801356A DK 135688 A DK135688 A DK 135688A DK 173677 B1 DK173677 B1 DK 173677B1
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- basement membrane
- membrane proteins
- proteins
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- 108010052285 Membrane Proteins Proteins 0.000 title claims abstract description 26
- 241001465754 Metazoa Species 0.000 title claims abstract description 13
- 210000002469 basement membrane Anatomy 0.000 title claims description 25
- 210000001519 tissue Anatomy 0.000 title claims description 19
- 238000000034 method Methods 0.000 title claims description 15
- 239000002738 chelating agent Substances 0.000 claims abstract description 3
- 239000000872 buffer Substances 0.000 claims description 8
- 239000000137 peptide hydrolase inhibitor Substances 0.000 claims description 7
- 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 description 4
- 229940124158 Protease/peptidase inhibitor Drugs 0.000 claims description 4
- 239000002253 acid Substances 0.000 claims description 4
- DEFVIWRASFVYLL-UHFFFAOYSA-N ethylene glycol bis(2-aminoethyl)tetraacetic acid Chemical compound OC(=O)CN(CC(O)=O)CCOCCOCCN(CC(O)=O)CC(O)=O DEFVIWRASFVYLL-UHFFFAOYSA-N 0.000 claims description 2
- 210000004379 membrane Anatomy 0.000 claims 1
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- 102000004169 proteins and genes Human genes 0.000 abstract description 12
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- 239000011780 sodium chloride Substances 0.000 description 7
- 102000018697 Membrane Proteins Human genes 0.000 description 6
- 239000000427 antigen Substances 0.000 description 6
- 102000036639 antigens Human genes 0.000 description 6
- 108091007433 antigens Proteins 0.000 description 6
- 239000000284 extract Substances 0.000 description 6
- YBYRMVIVWMBXKQ-UHFFFAOYSA-N phenylmethanesulfonyl fluoride Chemical compound FS(=O)(=O)CC1=CC=CC=C1 YBYRMVIVWMBXKQ-UHFFFAOYSA-N 0.000 description 6
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- 229920002307 Dextran Polymers 0.000 description 3
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 description 3
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- 238000002955 isolation Methods 0.000 description 3
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- 208000009331 Experimental Sarcoma Diseases 0.000 description 2
- HDFGOPSGAURCEO-UHFFFAOYSA-N N-ethylmaleimide Chemical compound CCN1C(=O)C=CC1=O HDFGOPSGAURCEO-UHFFFAOYSA-N 0.000 description 2
- 239000007983 Tris buffer Substances 0.000 description 2
- 238000007796 conventional method Methods 0.000 description 2
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- PJJJBBJSCAKJQF-UHFFFAOYSA-N guanidinium chloride Chemical compound [Cl-].NC(N)=[NH2+] PJJJBBJSCAKJQF-UHFFFAOYSA-N 0.000 description 2
- 230000003993 interaction Effects 0.000 description 2
- 150000002500 ions Chemical class 0.000 description 2
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- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 1
- QTBSBXVTEAMEQO-UHFFFAOYSA-M Acetate Chemical compound CC([O-])=O QTBSBXVTEAMEQO-UHFFFAOYSA-M 0.000 description 1
- 102000004506 Blood Proteins Human genes 0.000 description 1
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- 238000002238 CM-cellulose chromatography Methods 0.000 description 1
- KRKNYBCHXYNGOX-UHFFFAOYSA-K Citrate Chemical compound [O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O KRKNYBCHXYNGOX-UHFFFAOYSA-K 0.000 description 1
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- 241000282412 Homo Species 0.000 description 1
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- 108091005804 Peptidases Proteins 0.000 description 1
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- VREFGVBLTWBCJP-UHFFFAOYSA-N alprazolam Chemical compound C12=CC(Cl)=CC=C2N2C(C)=NN=C2CN=C1C1=CC=CC=C1 VREFGVBLTWBCJP-UHFFFAOYSA-N 0.000 description 1
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- VDQQXEISLMTGAB-UHFFFAOYSA-N chloramine T Chemical compound [Na+].CC1=CC=C(S(=O)(=O)[N-]Cl)C=C1 VDQQXEISLMTGAB-UHFFFAOYSA-N 0.000 description 1
- 238000003776 cleavage reaction Methods 0.000 description 1
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- 238000011065 in-situ storage Methods 0.000 description 1
- 238000010255 intramuscular injection Methods 0.000 description 1
- 239000007927 intramuscular injection Substances 0.000 description 1
- ZCYVEMRRCGMTRW-YPZZEJLDSA-N iodine-125 Chemical compound [125I] ZCYVEMRRCGMTRW-YPZZEJLDSA-N 0.000 description 1
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- 239000007929 subcutaneous injection Substances 0.000 description 1
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 1
- 210000002700 urine Anatomy 0.000 description 1
Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/78—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin or cold insoluble globulin [CIG]
-
- 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
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6887—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids from muscle, cartilage or connective tissue
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
- Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10S261/00—Gas and liquid contact apparatus
- Y10S261/88—Aroma dispensers
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
- Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10S435/00—Chemistry: molecular biology and microbiology
- Y10S435/803—Physical recovery methods, e.g. chromatography, grinding
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
- Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10S435/00—Chemistry: molecular biology and microbiology
- Y10S435/82—Subcellular parts of microorganisms
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
- Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10S435/00—Chemistry: molecular biology and microbiology
- Y10S435/961—Chemistry: molecular biology and microbiology including a step of forming, releasing, or exposing the antigen or forming the hapten-immunogenic carrier complex or the antigen per se
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
- Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10S435/00—Chemistry: molecular biology and microbiology
- Y10S435/962—Prevention or removal of interfering materials or reactants or other treatment to enhance results, e.g. determining or preventing nonspecific binding
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
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- Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10S436/00—Chemistry: analytical and immunological testing
- Y10S436/811—Test for named disease, body condition or organ function
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
- Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10S436/00—Chemistry: analytical and immunological testing
- Y10S436/825—Pretreatment for removal of interfering factors from sample
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
- Y10T—TECHNICAL SUBJECTS COVERED BY FORMER US CLASSIFICATION
- Y10T436/00—Chemistry: analytical and immunological testing
- Y10T436/25—Chemistry: analytical and immunological testing including sample preparation
- Y10T436/25375—Liberation or purification of sample or separation of material from a sample [e.g., filtering, centrifuging, etc.]
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- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Biomedical Technology (AREA)
- Molecular Biology (AREA)
- Medicinal Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Organic Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Hematology (AREA)
- Immunology (AREA)
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- Gastroenterology & Hepatology (AREA)
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- Manufacture Of Macromolecular Shaped Articles (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
- External Artificial Organs (AREA)
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Description
DK 173677 B1
Den foreliggende opfindelse angår en fremgangsmåde til ekstraktion af basalmem-branproteiner fra væv fra mennesker eller dyr.
Laminin, nidogen og BM40 er glycoproteiner, som tilskrives en nøglerolle ved organi-5 seringen af basalmembraner og ved vekselvirkningen af celler med basalmembraner (M. Paulsson et al., Eur. J. Biochem. 161. 455-464 (1986), R. Timpl. M. Dziadek, Int.
Rev. Exp. Pathol. 29, 1-112 (1986)). Således understøtter laminin udvoksningen af neuriter fra neuronerne af perifere nerver (D. Edgar et al., EMBO J. 3, 1463-1467 (1984)) og deres regenerering in situ (Madison et al. Exp. Neurol. 88, 767-772 (1985)).
10
En bredere anvendelse af disse proteiner har hidtil fremfor alt været forhindret af, at der ikke har været nogen egnede fremgangsmåder til rådighed, med hvilke disse proteiner kunne isoleres fra humant væv på en skånsom måde, Den af Risteli og Timpl (Biochem. J. 193. 749-755 (1981)) beskrevne fremgangsmåde behandler isoleringen 15 af det proteaseresistente laminin-fragment P1 fra humanplacenta. Dette fragment indeholder ikke mere det for vekselvirkningen med nerver og tumorceller vigtige domæne 8. Ved hjælp af den af Timpl. et al. (J. Biol. Chem. 254. 9933-9937 (1979)) beskrevne fremgangsmåde til isolering af laminin fra tumorvæv kan der ikke opløseliggøres noget laminin fra humanplacenta, og fra tumorvæv fås der ifølge denne frem-20 gangsmåde kun lave udbytter.
Ifølge den af Ohno et al. (Biochem. Biophys, Res. Com. 112. 1091-1098 (1983)) og Wewer et al. (J. Biol. Chem. 258, 12654-12660 (1983)) beskrevne fremgangsmåde behøver man enten denaturerende betingelser eller proteolyse for at ekstrahere signi-25 fikante mængder laminin eller laminin-brudstykker fra vævet. Også nidogel eller BM40 har hidtil kun kunnet isoleres under denaturerende betingelser (Dziadek et al., Eur. J. Biochem. 161. 455-464 (1985), Paulsson et al., Eur. J. Biochem. 156, 467-478 (1986)).
30 Ekstraktion af humant laminin med 1 M NaCI i nærværelse af EDTA og en detergent, såsom Triton X-100, er kendt fra Connective Tissue Research 14:31-40 (1985).
Det er nu muligt at ekstrahere basalmembranproteiner på skånsom måde med fremgangsmåden ifølge opfindelsen. Overraskende kan man ekstrahere intakte basal-35 membranproteiner fra vandig opløsning i nærværelse af en chelatdannende organisk 2 DK 173677 B1 polysyre, og det i større mængder end hidtil muligt, uden brug af denaturerende midler eller detergenter.
Opfindelsen angår således en fremgangsmåde til ekstraktion af basalmembranprotei-5 ner fra væv fra mennesker eller dyr, hvilken fremgangsmåde er ejendommelig ved, at den omfatter følgende trin: a) forekstraktion af basalmembranholdigt væv fra mennesker eller dyr i nærværelse af en proteaseinhibitor i en fysiologisk puffer, som bevarer den naturlige konfor-mation af basalmembranproteineme, hvorved den fysiologiske puffer ikke inde- 10 holder en detergent, og b) ekstraktion af basalmembranproteineme fra det forekstraherede væv i nærværelse af en proteaseinhibitor og en chelatdannende organisk polysyre i en puffer, som bevarer den naturlige konformation af basalmembranproteineme.
15 I det følgende forklares opfindelsen detaljeret, især i de foretrukne udførelsesformer.
Ved hjælp af fremgangsmåden ifølge opfindelsen kan der ekstraheres alle menneske-og dyrevævsarter, i hvilke der befinder sig basalmembranproteiner, f.eks. humanplacenta, menneske- eller dyretumorvæv eller nyre. Der gennemføres først en forekstrak-20 tion. Vævet homogeniseres og centrifugeres i en for basalmembranproteiner fysiologisk puffer i nærværelse af proteaseinhibitorer. Ved denne behandling fjernes fortrinsvis letopløselige proteiner, såsom serumproteiner og cytoplasmaproteiner, fra vævet. Vævsremanensen optages derpå en gang til i en fysiologisk puffer og homogeniseres og ekstraheres i nærværelse af proteaseinhibitorer og chelatdannende organiske po-25 lysyrer, f.eks. EDTA, EGTA eller citrat. Ekstraktionen gennemføres under tilsætning af 1-500 mmol af det chelatdannende middel, fortrinsvis 10-50 mmol, hensigtsmæssigt under afkøling ved 0-15eC, fortrinsvis 4-10eC. En ekstraktion er også mulig ved højere temperaturer, men ledsages så af forstyrrende nedbrydningsreaktioner. Ekstrakterne indeholder de ønskede basalmembranproteiner. Særligt foretrukket isoleres der ifølge 30 opfindelsen BM40, nidogen og lamidin, til dels som ikke-kovalent kompleks.
De enkelte proteiner isoleres fra ekstrakten ved moiekylsigtechromatografi på egnede søjler, f.eks. dextran, især allyldextran tværbundet med Ν,Ν'-methylenbisacrylamid, eller agarose. Finrensningen af basalmembranproteineme sker alt efter proteinet på 35 fortrinsvis svagt basiske ionbyttere med efterfølgende moiekylsigtechromatografi eller yderligere rensning på agarose- eller "Sepharose'-søjler.
3 DK 173677 B1 På denne måde kan der isoleres mængder af intakte basalmembranproteiner, som ikke kan opnås med de traditionelle isoleringsmetoder.
5 De isolerede rensede basalmembranproteiner kan derpå anvendes som antigen til fremstilling af højspecifikke antisera eller antistoffer. Dette sker på gængs måde ved subcutan eller intramuskulær injektion til forsøgsdyr, fortrinsvis kaniner. Hensigtsmæssigt arbejdes der derved i nærværelse af komplet Freund’s adjuvans. Der kan anvendes de i disse tilfælde gængse antigendoser. Den foretrukne dosis til kaniner er på 10 0,5-1 mg pr. dyr. Det dannede antiserum udvindes derpå på den for fagmanden kendte måde og kan anvendes som sådant Det er også muligt at rense de i serumet tilstedeværende antistoffer mere i forvejen. En foretrukken metode derved er affinitets-chromatografi.
15 Det ved hjælp af de rensede basalmembranproteiner fremstillede højspecifikke antiserum muliggør bestemmelse af basalmembranproteiner i legemsvæsker, f.eks. serum, urin osv., og vævsekstrakter under anvendelse af de i og for sig kendte immunologiske påvisningsmetoder.
20 Der kan ved disse bestemmelser anvendes både de kendte radio-immun-assay-(RIA)-varianter og enzym-immun-assay-varianterne og analoge fremgangsmåder.
Den til den immunologiske bestemmelse nødvendige markering af antigenet kan ske ved de til proteinmarkering kendte metoder. Markeringen sker fortrinsvis ved radioak-25 tiv, enzym- eller fluorescensmarkering. Når der er tale om radioaktiv markering anvendes der som radionuklid fortrinsvis iod 125. Markeringen kan derpå ske ifølge den kendte chloramin T-metode (Int. Arch. Allergy 29,185, 1966).
Ved disse påvisningsreaktioner konkurrerer det markerede basalmembranprotein på 30 kendt måde om antistoffet, således at mængden af markeret antigen i det dannede antigen-antistof-kompleks er så meget des ringere, jo mere ikke-markerede antigener, der er indeholdt i prøven, der skal bestemmes. Man kan enten anvende markeringen af komplekset, eksempelvis radioaktiviteten eller enzymaktiviteten, eller markeringen af den ovenstående væske efter fraskillelse af antigen-antistof-komplekset for, ved 35 hjælp af en kalibreringskurve, som fremstilles ved hjælp af prøver med et kendt ind- 4 DK 173677 B1 hold af basalmembranprotein, at konstatere mængderne af antigen indeholdt i prøven, der skal undersøges. Fortrinsvis bestemmes markeringen i komplekset.
Fortrinsvis gennemføres fraskillelsen af det med det specifikke antiserum dannede 5 antigen-antistof-kompleks ved hjælp af et andet antistof. Dertil anvendes fortrinsvis et antistof, der er rettet mod immunglobulin G af den tii udvindingen af det specifikke antiserum anvendte dyreart. Fraskillelsen af antigen-antistof-komplekset fra opløsningen kan ske ved hertil gængse metoder, såsom udfældning, centrifugering eller frafil-trering. Alternativt hertil bindes antiserumet hhv. det andet antistof til en fast bærer.
10 Efter fraskillelse af antigen-antistof-komplekset bestemmes derpå den i komplekset indeholdte hhv. den i opløsningen tilbageværende radioaktivitet eller anden markering.
Enzym-immun-assay-varianten til bestemmelse af basalmembranprotein kan også gennemføres således, at antistoffet mod immunglobulin G af den pågældende dyreart, 15 f.eks. fra kaniner, er enzymmarkeret. Ved denne art af enzym-immun-assay bestemmes den efter dannelse af antigen-antistof-komplekset tilbageværende del af anti-basalmembranprotein-serumet, idet man binder denne til bærerbundet basalmembranprotein og dernæst omsætter med enzymmarkeret antistof mod immunglobulin G fra kaniner. Mængden af bundet enzymmarkeret antistof bestemmes dernæst ved må-20 ling af enzymreaktionen og er indirekte proportional med den ukendte mængde af ba-salmembranproteinet i prøven.
i det følgende eksempel forklares opfindelsen nærmere.
25 Eksempel
Isolering af basalmembranproteinerne laminin, nidogen og BM40 EHS-Tumorceller injiceres subcutant i C57B1-mus (Orkin et al., J. Exp. Med. 145. 204 (1977)). Efter 10-14 dage isoleres tumoren, og det derefter rensede vævsmateriale 30 opbevares ved -20°C. 75 g af den frosne tumor homogeniseres i 1 minut i 1,5 liter 0,15 M NaCI, 0,05 M tris-HCI (pH-værdi 7,4) (TBS) med 0,05 mM phenylmethansulfonylflu-orid (PMSF) og N-ethylmaleimid (NEM). Efter centrifugering ved 8000 o/m i et tidsrum på 20 minutter bortkastes den ovenstående væske, og remanensen homogeniseres en gang til i 375 ml af den ovennævnte puffer under tilsætning af 10 mM EDTA og 35 omrøres i 1 time i kølerum. Ekstrakten udvindes efter den ovennævnte centrifugering 5 DK 173677 B1 og videreanvendes enten straks eller opbevares ved -20”C. Udbyttet og renheden af det ønskede produkt kan forhøjes yderligere, når ekstraktionstrinene gentages.
En portion af TBS-EDTA-ekstrakten sættes til en agarose- ("Biogel A5m") eller en 5 "Sepharose C1-6B"-søjle (3 x 135 cm), som er bragt i ligevægt i TBS med 2 mM ED-TA, 0,5 mM PMSF og 0,5 mM NEM. Derved sker der en opspaltning i 3-4 fraktioneringsområder. Materialet, der først elueres med udelukkelsesvolumenet, indeholder laminin-nidogen-kompleks og frit laminin, de efterfølgende fraktioner indeholder frit nidogen i intakt (150 kD) og delvis nedbrudt form (130, 100 kD). Dernæst elueres BM-10 40.
Rensningen af laminet sker først på en svagt basisk ionbytter (DEAE-cellulose) med 2 , M urinstof i 0,05 M tris/HCI (pH-værdi 8,6) under tilsætning af proteaseinhibitorer. Der elueres ved hjælp af en lineær NaCI-gradient (0-0,4 M). Ved dette trin fjernes forstyr-15 rende urenheder af proteoglycan og nucleinsyrer. Ved chromatografi på "Sepharose C1-4B" i 2 M guanidinchlorid (pH-værdi 7,4) befries laminin sluttelig for resterende nidogen og mindre proteiner.
Rensningen af nidogen sker først på DEAE-cellulose og "Sepharose CI4-B" som be-20 skrevet ved laminin. Yderligere rensningstrin omfatter, når det er nødvendigt, chromatografi på CM-cellulose med 2 M urinstof, 0,02 M Na-acetat (pH-værdi 4,8) og tværbundet allyldextran ("Sephacryi S200") med 0,2 M NH4HC03.
Den yderligere rensning af BM40 sker ligeledes på DEAE-cellulose med 2 M urinstof,
25 0,05 M tris (pH-værdi 8,6) ved eluering ved hjælp af en lineær NaCI-gradient (0-0,4 M
NaCI). BM40 elueres ved 0,2-0,25 M NaCI og renses endegyldigt ved koncentrering over "Sephacryi S200" med 0,2 M NH4HC03.
Tabellen viser eksempelvis, at der med den skånsomme ekstraktion kan ekstraheres 30 mængder af intakte basafmembranproteiner, f.eks. EHS-tumor, som ikke kan opnås med traditionelle fremgangsmåder.
6 DK 173677 B1
Tabel
Fundet protein i mg pr. g fugtig vægt
Laminin Nidogen BM40 _____ __ ___ __ ________ __ _ __ 0,5 M NaCI 0,39 <0,05 ikke bestemt 6 M guanidinchiorid 0,83 0,30 <0,01
Claims (3)
1. Fremgangsmåde til ekstraktion af basalmembranproteiner fra væv fra mennesker eller dyr, kendetegnet ved, at den omfatter følgende trin: 5 a) forekstraktion af basatmembranholdigt væv fra mennesker eller dyr i nærværelse af en proteaseinhibitor i en fysiologisk puffer, som bevarer den naturlige konfor-mation af basalmembranproteineme, hvorved den fysiologiske puffer ikke indeholder en detergent, og b) ekstraktion af basalmembranproteineme fra det forekstraherede væv i nærvæ-10 relse af en proteaseinhibitor og en chelatdannende organisk polysyre i en puffer, som bevarer den naturlige konformation af basalmembranproteineme.
2. Fremgangsmåde ifølge krav 1, kendetegnet ved, at der ekstraheres under tilsætning af EDTA eller EGTA. 15
3. Fremgangsmåde ifølge et eller flere af kravene 1-2, kendetegnet ved, at der ekstraheres under tilsætning af 1-500 mM af den chelatdannende organiske polysyre. 1 Fremgangsmåde ifølge krav 3, kendetegnet ved, at der ekstraheres under tilsæt-20 ning af 10-50 mM af det chelatdannende middel.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| DE19873708198 DE3708198A1 (de) | 1987-03-13 | 1987-03-13 | Verfahren zur isolierung von basalmembranproteinen aus menschlichen und tierischen geweben |
| DE3708198 | 1987-03-13 |
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| Publication Number | Publication Date |
|---|---|
| DK135688D0 DK135688D0 (da) | 1988-03-11 |
| DK135688A DK135688A (da) | 1988-09-14 |
| DK173677B1 true DK173677B1 (da) | 2001-06-11 |
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| DK198801356A DK173677B1 (da) | 1987-03-13 | 1988-03-11 | Fremgangsmåde til ekstraktion af basalmembranproteiner fra væv fra mennesker og dyr |
Country Status (11)
| Country | Link |
|---|---|
| US (1) | US5147782A (da) |
| EP (1) | EP0282018B1 (da) |
| JP (1) | JP2532559B2 (da) |
| AT (1) | ATE124706T1 (da) |
| DE (2) | DE3708198A1 (da) |
| DK (1) | DK173677B1 (da) |
| ES (1) | ES2074983T3 (da) |
| FI (1) | FI99114C (da) |
| IE (1) | IE67563B1 (da) |
| NO (1) | NO173336C (da) |
| PT (1) | PT86944B (da) |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| US5512657A (en) * | 1988-12-12 | 1996-04-30 | Bainbridge Sciences, Inc. | Detection of complexes which include basement membrane components as diagnostic of cancer and other diseases |
| US5541076A (en) * | 1988-12-12 | 1996-07-30 | Bard Diagnostic Sciences, Inc. | Methods for determining the invasiveness of a bladder tumor |
| WO1992019970A1 (en) * | 1991-04-26 | 1992-11-12 | Emanuel Calenoff | Methods to detect and treat diseases caused by bacterial allergens |
| NZ524868A (en) | 2003-03-21 | 2005-09-30 | Velvet Antler Res New Zealand | Process for purifying low molecular weight proteins from tissue in situ |
| NZ526157A (en) * | 2003-05-27 | 2006-01-27 | Velvet Antler Res New Zealand | Method for the extraction of deer antler and use of the extract therefrom |
| US20070190165A1 (en) * | 2005-10-21 | 2007-08-16 | Brey Eric M | Tissue-specific basement membrane gels |
| US10076535B2 (en) | 2012-04-27 | 2018-09-18 | The United States Of America, As Represented By The Secretary, Department Of Health And Human Services | Use of CPG oligonucleotides co-formulated with an antibiotic to accelerate wound healing |
| WO2024130365A1 (en) * | 2022-12-21 | 2024-06-27 | Cristália Produtos Químicos Farmacêuticos Ltda | Laminin extraction, purification and polymerization processes, use, polylaminin and kit |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| DE2923583A1 (de) * | 1979-06-11 | 1980-12-18 | Max Planck Gesellschaft | Verfahren zur immunologischen bestimmung von basalmembranmaterial und hierfuer geeignete neue basalmembranfragmente |
| US4628027A (en) * | 1982-05-19 | 1986-12-09 | Molecular Engineering Associates, Ltd. | Vitro diagnostic methods using monoclonal antibodies against connective tissue proteins |
| DE3511199A1 (de) * | 1985-03-28 | 1986-10-02 | Hoechst Ag, 6230 Frankfurt | Verfahren zur immunologischen bestimmung von heparansulfat-proteoglykan, verfahren zur herstellung bzw. reinigung von dafuer geeignetem heparansulfat-proteoglykan aus basalmembran-haltigen geweben |
| JPH01155757A (ja) * | 1987-12-11 | 1989-06-19 | Nec Corp | 可変不在転送解除方式 |
-
1987
- 1987-03-13 DE DE19873708198 patent/DE3708198A1/de not_active Withdrawn
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1988
- 1988-03-09 AT AT88103668T patent/ATE124706T1/de not_active IP Right Cessation
- 1988-03-09 ES ES88103668T patent/ES2074983T3/es not_active Expired - Lifetime
- 1988-03-09 DE DE3854096T patent/DE3854096D1/de not_active Expired - Lifetime
- 1988-03-09 EP EP88103668A patent/EP0282018B1/de not_active Expired - Lifetime
- 1988-03-10 FI FI881122A patent/FI99114C/fi not_active IP Right Cessation
- 1988-03-10 PT PT86944A patent/PT86944B/pt not_active IP Right Cessation
- 1988-03-11 NO NO881111A patent/NO173336C/no not_active IP Right Cessation
- 1988-03-11 IE IE72988A patent/IE67563B1/en not_active IP Right Cessation
- 1988-03-11 US US07/167,177 patent/US5147782A/en not_active Expired - Lifetime
- 1988-03-11 DK DK198801356A patent/DK173677B1/da not_active IP Right Cessation
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Also Published As
| Publication number | Publication date |
|---|---|
| FI881122A (fi) | 1988-09-14 |
| DE3854096D1 (de) | 1995-08-10 |
| IE880729L (en) | 1988-09-13 |
| IE67563B1 (en) | 1996-04-17 |
| NO881111D0 (no) | 1988-03-11 |
| FI99114C (fi) | 1997-10-10 |
| US5147782A (en) | 1992-09-15 |
| FI99114B (fi) | 1997-06-30 |
| DE3708198A1 (de) | 1988-09-22 |
| ATE124706T1 (de) | 1995-07-15 |
| NO173336C (no) | 1993-12-01 |
| PT86944A (pt) | 1988-04-01 |
| NO881111L (no) | 1988-09-14 |
| EP0282018A2 (de) | 1988-09-14 |
| FI881122A0 (fi) | 1988-03-10 |
| EP0282018A3 (en) | 1990-01-24 |
| PT86944B (pt) | 1992-05-29 |
| JPS63243098A (ja) | 1988-10-07 |
| DK135688A (da) | 1988-09-14 |
| NO173336B (no) | 1993-08-23 |
| ES2074983T3 (es) | 1995-10-01 |
| DK135688D0 (da) | 1988-03-11 |
| EP0282018B1 (de) | 1995-07-05 |
| JP2532559B2 (ja) | 1996-09-11 |
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